International Review of Cytology, A Survey of Cell Biology, Vol. 188

VOLUME 188

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik

1952-1988 1952-1 984 19671984-1 992 1993-1 995

EDITORIAL ADVISORY BOARD Aimee Bakken Eve Ida Barak Rosa Beddington Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Laurence Etkin Hiroo Fukuda Elizabeth D. Hay P. Mark Hogarth Anthony P.Mahowald

M. Melkonian Keith E. Mostov Andreas Oksche Vladimir R. Pantic L. Evans Roth Jozef St.Schell Manfred Schliwa Robert A. Smith Wilfred D. Stein Ralph M. Steinman M. Tazawa Donald P. Weeks Robin Wright Alexander L. Yudin

Edited by

Kwang W. Jeon Department of Biochemistry University of Tennessee Knoxville, Tennessee

VOLUME 188

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Front cover photograph: Human epidermal keratinocytes grown on microcarriers. (For more details, see Chapter 1, Figure 1.)

This book is printed on acid-free paper. @ Copyright 0 1999 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923). for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1999 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0074-7696199 $30.00

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Academic Press 24-28 Oval Road, London NWl 7DX, UK http:llwww.hbuk.co.uWapl International Standard Book Number: 0-12-364592- I PRINTED IN THE UNllED STATESOF AMERICA 99 0 0 0 1 02 03 04 EB 9 8 7 6

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Contributors .............................................................

ix

Microfibrils from the Arterial Subendothelium 1.

Frangoise Fauvel-Lafeve Introduction ........................................................... Structure of Subendothelial Microfibrils ...................................... Microfibrils and Elastinogenesis ........................................... The Thrombogenicity of the Subendothelium .................................

I1. 111. IV . V . The Thrombogenicity of Type VI Collagen ................................... Vl . The Thrombogenicity of Elastin-Associated Microfibrils ......................... VII. Conclusions and Perspectives ............................................ References ...........................................................

1 2 19 19 23 25 29 30

Cultivation and Transplantation of Epidermal Keratinocytes J. V . Terskikh and A . V. Vasiliev 1. II. 111 . IV. V.

Introduction ........................................................... Culture of Keratinocytes ................................................. Behavior of Keratinocytes in Culture ........................................ Transplantation of Cultured Keratinocytes ................................... Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ...........................................................

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41 43 47 54 63

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CONTENTS

Retinoids and Maminalian Development G. M. Morriss-Kay and S. J. Ward I. Introduction ........................................................... II . The Retinoic Acid Signaling Pathway in Embryogenesis ........................ 111 . Synthesis and Catabolism of Retinoic Acid in Embryonic Tissues ................. IV. Nuclear Aspects of Retinoic Acid Signal Transduction .......................... V . Retinoic Acid in Craniofacial Development ................................... VI Retinoic Acid and Limb Development ....................................... VII. Retinoic Acid in Other Developing Systems .................................. VIII. Conclusions ........................................................... References ...........................................................

.

73 77 85 97 99 106 110 116 119

Gene Expression in the Epididymis 1. II. 111. IV. V.

C. Kirchhoff Introduction ........................................................... Ontogenesis of Epididymal Gene Expression Pattem .......................... Gene Expression in the Adult Epididymis .................................... Factors Regulating Gene Expression in the Adult Epididymis .................... Concluding Remarks .................................................... References ...........................................................

133 135 153 169 184 186

Roles of Reactive Oxygen Species: Signaling and Regulation of Cellular Functions 1. A. Gamaley and 1. V . Klyobin I. II. 111 . IV. V. VI. VII. VIII.

Introduction ........................................................... Mechanisms Inducing Generation of ROS by Cells ............................ ROS Homeostasis in Animal Cells ......................................... Cytotoxic Effects of ROS ................................................ Regulation of Cell Functions by ROS ....................................... ROS as Signal Molecules ................................................ ROS in Plants ......................................................... Concluding Remarks .................................................... References ...........................................................

203 204 211 214 220 223 235 237 238

CONTENTS

vii

High-Density Lipoprotein: Multipotent Effects on Cells of the Vasculature 1. II. 111 . IV. V. VI. VII. VIII. IX.

Gillian W . Cockerill and Stephen Reed Introduction ........................................................... In Wtro Effects of HDL on Endothelial Cells .................................. Effects of HDL on Leukocyte Activation ..................................... Effects of HDL on Cell Transmigration ...................................... HDL. Thrombosis. and Platelet Function .................................... Effect of HDL on Vasoactive Molecules ..................................... Acute-Phase Response and HDL .......................................... HDL Receptors and Cell Signal Transduction ................................ Concluding Remarks .................................................... References ...........................................................

Index .....................................................................

257 262 267 271 273 276 279 281 286 287 299

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CONTRIBUTORS

Numbers in parentheses indicate the pages on which the author's contributions begin.

Gillian W. Cockerill (257),Department of Cardiovascular Medicine, National Heart and Lung Institute, Imperial College School of Medicine, HammersmithHospital Campus, London W12 ONN, United Kingdom FranGoise Fauvel-Lefeve(1), Unite353 INSERM, lnstitut d'Hematologie, H6pital SaintLouis, 75475 Paris Cedex 10, France I. A. Gamaley (203),Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia Christiane Kirchhoff (133),IHF Institute forHormone and Fertili!y Research, D-22529 Hamburg, Germany

I. V. Klyubin (203),Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia Gillian M. Morriss-Kay (73),Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom Stephen C. Reed (257),School of Biosciences, University of Westminstec London WIM US, United Kingdom V. V. Terskikh (41), Instituteof DevelopmentalBiology, RussianAcademy of Sciences, Moscow 117334, Russia A. V. Vasiliev (41), Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117334, Russia Simon J. Ward (73),Department of Biomedical Science, University of Shefield, Sheffield, United Kingdom

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Microfibrils from the Arterial Subendothelium Frangoise Fauvel-Lafeve Unit6 353 INSERM, Institut d'HCmatologie, HGpital Saint-Louis,75475 Pans Cedex 10, France

Microfibrillar structures of the subendothelium are represented by either type VI collagen or elastin-associated microfibrils which are also referred to as fibrillin-containing microfibrils. These structures are present throughout the subendothelium irrespective of the presence of elastin. The localization, structure, and protein composition of microfibrils are reviewed. The arterial subendothelium is thrombogenic despite its very low content in fibrillar collagens. This thrombogenicity is linked to the microfibrillar structures, essentially to type VI collagen and to thrombospondin-containing microfibrils. Their respective ability to bind the von Willebrand factor and to activate blood platelets is discussed. KEY WORDS: Microfibrils,Thrombospondin, Fibrillins, Type VI collagen, Platelets, von Willebrand factor. o 1999 Academic Press

1. Introduction The arterial subendothelium is formed by the endothelial cell basement membrane and the extracellular matrix located between the basement membrane and the internal elastic lamina. The thickness of the subendothelium is variable according to its localization within the vascular tree. In capillaries, endothelial cells are in close contact with the fenestrated elastin lamina, while in aorta, the subendothelium could reach a size of 1 pm (Wight, 1996). In man, the subendothelium thickens with increasing age, a process known as diffuse intimal thickening, and an accelerated intimal thickening indicates a predisposition for the development of atherosclerotic lesions (Mayne, 1987;Stary er al., 1992).Due to its location just beneath endothelial cells which form a barrier between the blood and vascular tissues, components of the subendothelium play an important role in all physiological and Inrernational Review of Cytology,VoL

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Copyright 8 1999 by Academic Press. All rights of reproduction in any form reserved.

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pathological phenomena taking place after an endothelial cell injury or during the circulating cell diapedesis. This vascular wall layer is thus implicated in hemostasis and thrombosis (Hawiger, 1994), arterosclerosis (Ross, 1995),inflammation (Gimbrone, 1995),and tumor cell metastatic dissemination (Crissman et al., 1988). The subendothelium is composed of numerous macromolecules (Kefalides, 1994). In addition to the basement membrane components (type IV collagen, laminin, nidogen, and heparan sulfate proteoglycans), the subendothelium contains large amounts of proteoglycans, fibronectin, and fibrillar structures formed by fibrillar type I11 collagen (Gay et al., 1975) and microfibrils. Microfibrils were first described as a histological entity (Low, 1961). These structures are particularly insoluble and thus much less is known about them than collagens. Since the discovery of type VI collagen (Engval et al., 1986) and fibrillin (Sakai et al., 1986), microfibrillar structures of the subendothelium have been separated into two groups: type VI collagen and elastin-linkedmicrofibrils. In fact, the term microfibrils is now restricted to 10-nm fibrils regardless of whether they are associated with elastin or not. Our results (Fauvel-Lafkve et aZ., 1996) and studies from Zhang et al. (1995) show that noncollagenous microfibrils are also heterogeneous. In this review, the structure and physiological roles of subendothelial microfibrillar structures (microfibrilsand type VI collagen) will be discussed with a special regard to their role in the thrombogenicity of the subendothelium.

II. Structure of Subendothelial Microfibrils A. Ultrastructure In early electron microscopic studies, fine extracellular filaments were described in many tissues. In stained tissue sections they were seen free in the ground substance or linked to collagen fibers or to basement membrane and associated to elastin. Microfibrilswere first defined by Low (1961) as thin filaments with diameters of <20 nm nonpresenting the 63 nm periodic striation of collagen. This definition was further completed by Ross and Bornstein (1969), who showed that elastin and elastin-associatedmicrofibrilshave different susceptibilities to enzyme digestion and that microfibrils must be resistant to highly purified collagenase. In the vessel wall as in other tissues, many structures conform to this definition. Several studies have shown that filaments with a diameter of

SUBENDOTHELIAL MICROFIBRILS

3

<5 nm and a typical morphology in stained preparations are proteoglycans as confirmed by their susceptibility to digestion by specific enzymes (Frederickson and Low, 1971; Mayer et al., 1981). These structures are no longer considered microfibrils. In the vessel wall, the two microfibrillar structures present in the subendothelium are type VI collagen and microfibrils linked to elastin or oxytalan fibers. 1. Elastin-Associated Microfibrils Elastin microfibrils form filaments of 12-20 nm presenting a 170-nm periodic striation. Microfibrils have an affinity for cationic stains such as uranyl lead (Greenlee et al., 1966) and ruthenium red (Fahrenbach et aL, 1966; Yu and Lai, 1970). Labeling with tannic acid or an anti-fibrillin antibody revealed their arrangement along the vascular tree: They are associated to elastin and also present in the vicinity of endothelial cells as shown in Figure 1. These microfibrils appear to connect other extracellular matrix components to each other and to the cells (Arbeille et al., 1991). Microfibrils associated with the elastic lamina are lengthwise arranged: In the vessel cross sections, microfibril sections are observed around the elastin lamina and in the pockets formed by elastin folding. The staining of microfibrils by ruthenium red, a hexavalent cation specific for polyanions such as glycoproteins and proteoglycans, or by lectins (ricinus cornrnunis and concanavalin A) demonstrated the glycoprotein structure of microfibrils (Birembaut et aL, 1982). At high resolution, the microfibrils are not homogenous in structure. In cross sections they appear as a translucent core about 4 nm in diameter surrounded by an outer electron-dense shell about 3 nm in thickness. In longitudinal section, microfibrils are composed of a beaded chain with a bead periodicity of 50-55 nm, suggesting a protein heterogeneity (Rosenbloom et al., 1993). In addition to elastin-associated microfibrils, similar structures are observed in elastin-free tissues. These microfibrils were termed “oxytalan fibers” by Fuilmer and Lillie (1958). They appear to have a common distribution in tissues which are subjected to mechanical stress. In the subendothelium Gerrety and Cliff (1972) described in young rat aorta-anchoring filaments that connect endothelial cells to the elastin lamina. These filaments were also observed by Yohro and Burnstock (1973) in the coronary vessel subendothelium. Davis (1994) showed that anchoring filaments in mouse aorta contain fibrillin and fibronectin. In porcine aorta, we have observed microfibrils connecting the endothelial cells to elastin or to collagen (Arbeille et aL, 1991). In this tissue as well as in skin, placenta, or human umbilical arteries, elastin-associated microfibrils and anchoring filaments were found to contain thrombospondin associated to fibrillin (Ar-

4

FRANWISE FAUVEL-LAFEVE

FIG. 1 Microfibrils in the subendothelium. Microfibrils (arrows) are present beneath the endothelial cell (CE) and link the cell to the elastin lamina (E). Scale bar = 500 nm.

beille et al., 1991; Fauvel-Lafhve et aL, 1996). AU these studies suggest that microfibrils represent a family of structurally identical microfilaments with common properties but different biochemical compositions. 2. Type VI Collagen

Type VI collagen has a microfibrillar structure with a diameter of 6-10 nm (Fig. 2) that is resistant to collagenase and extractable by chaotropic salts such as guanidiniumchloride (Kielty et al., 1991).This collagen appears in all vascular layers between type I and type I11 collagen (Murata and Matoyama, 1990; Rauterberg et al., 1993). Type VI collagen fibrils are not entirely

SUBENDOTHELIAL MICROFIBRILS

5

FIG. 2 Anti-type VI collagen antibodies in the umbilical arteries subendothelium decorate fine fibrils (arrows) with a mean diameter of 5 or 6 nm completely distinct from the typical microfibrillar structures (arrowheads) presenting 12-nm fibrils reticulated at the level of 40nm aggregates. Scale bar = 100 nm.

composed of collagen but are often bound to the proteoglycan decorin (Bidanset et al., 1992). Type VI collagen links collagen fibers to other noncollagenous structures. Type VI collagen is not present in elastin microfibrils (Mayne, 1987; Sear et aZ., 1981). In the subendothelium from human umbilical arteries, we have observed the presence of type VI collagen along interstitial collagen fibers and microfibrils were not labeled by an anti-type VI collagen antibody (Fauvel-Laf&veet aZ., 1996). Type VI collagen forms a second fibrillar network possibly functionning as a scaffold to support or provide an organizational network to the larger collagen fibrils.

B. Extraction of Microfibrils Because of their insolubility and complexity, the chemical characterization of microfibrils progresses slowly. Studies on microfibrils were first performed on bovine nuchal ligament of fetal calves. This tissue is highly elastic

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FRANGOISE FAUVEL-LAFEVE

and contains microfibrils primarily found in association with elastin. Ross and Bornstein (1969) performed a detailed systematic study of enzyme susceptibilities and extractabilities of the two components of the elastic fiber in the fetal calf nuchal ligament. This work served as a basis for all later studies involved in the isolation and characterization of microfibrils. They extracted the tissues with 5 M guanidinium chloride, treated them with collagenase, and reextracted them with guanidinium chloride in the presence of a reducing agent to cleave disulfide bonds. An electron microscopic study of the tissues was performed after each extraction step. The authors showed that the microfibrillar components of the nuchal ligament were resistant to collagenase digestion and to extraction with guanidinium chloride in the absence of a reducing agent. The arterial subendothelium contains loose microfibrils bundles devoid of elastin (Cleary and Gibson, 1983) and few collagen fibers; thus, the treatment with guanidinium chloride in the absence of collagenase or a reducing agent was sufficient to remove the microfibrils as shown by electron microscopy of a guanidimiun chloridetreated aorta in which the elasin lamina and particularly its pocket appeared devoid of microfibrils (Fauvel et al., 1983). Several precautions should be taken in order to optimize the extraction and to preserve the protein structure. This implies a rapid cooling of the tissues, working at low temperature and using a cocktail of protease inhibitors at each step of the process. The tissues must be disrupted by low shear methods such as crushing in liquid nitrogen, and soluble components derived from blood and tissues should be removed by washings with 0.15 M sodium chloride. According to the procedure used for the extractions, the guanidinium chloride extract should contain proteins such as those from or linked to elastin-associatedmicrofibrils (Cleary and Gibson, 1983) and type VI collagen (Gibson and Cleary, 1985; Ayad et al., 1985).

C. Type VI Collagen Microfibrils Chung et al. (1976) isolated an unusual disulfide-bound collagenous aggregate from pepsin digests of aortic intima. This collagen was termed “intima collagen” and “short chain collagen” before it was classed as type VI collagen (Furthmayr et al., 1983; Jander et al., 1983). The molecule is composed of three genetically distinct a chains: al(VI), cr2(VI), and a3(VI). The electron microscopicvisualization of rotary shadowed molecules showed a characteristic dumbbell shape consisting of two globules separated by a 105-nm-long triple helical domain (Fig. 3). The monomers form antiparallel dimers which aggregate laterally to yield unstaggered tetramers. The aggregate is stabilized by a large number of disul-

7

SUBENDOTHELIAL MICROFIBRILS

Monomer Dimer

Tetramer

Beaded filament

FIG. 3 The organization of type VI collagen fibers. Type VI monomers composed by the three a (VI) chains form antiparallel dimers which aggregate laterally in tetramers. These tetramers associate end to end in beaded filaments.

fide bonds (Burgeson and Nimmi, 1992). These tetramers associate end to end to form a network of beaded filaments (Engvall ef al., 1986). Type VI collagen can also be extracted by a 6 M guanidinium chloride treatment (Gibson and Cleary, 1985). The resulting material, first named CL glycoprotein, showed the type VI collagen characteristics: a high cystein content and a ratio of hydroxylysine to hydroxyproline of about 1:1. An antibody directed against the CL protein exhibited a strong immunological reactivity with type VI collagen isolated by pepsin from the placenta. Kielty ef al. (1991) extracted type VI collagen in native form by hydrolyzing fetal calf nuchal ligament with bacterial collagenase under conditions in which fibrillar collagens were digested but type VI collagen was not degraded; this type of collagen is resistant to bacterial collagenase in the absence of a reducing agent (Timpl and Engel, 1987). This treatment also extracted fibrillin into its native form but no direct interaction between fibrillin and collagen was demonstrated by electron microscopy. The function of type VI collagen is not known but the ultrastructure of the network suggests that it forms an independent fibrous system important for the development of type I or type I11 fibers (Burgeson and Nimni, 1992). Type VI collagen is a substrate for cell attachment and migration

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FRAN$OISE FAUVEL-LAFEVE

(Aumailley et al., 1989; Kielty et al., 1992). These properties are linked to the presence of 13 RGD sequences in the triple helice which are involved in the binding to cells (Kuo et al., 1995). The role of type VI collagen in the thrombogenicity of the subendothelium will be discussed later. D. Proteins from Elastin-Associated Microfibrils

Due to their insolubility, the nature of elastin-associated microfibrils was unknown until the development of cell culture and gene cloning which now allows the characterization and the purification of microfibril structural proteins or proteins linked to these structures. The most important are fibrillins 1 and 2 (Sakai et al., 1986; Zhang et al., 1994) and a number of additional proteins have been identified in microfibrils in developing tissues. These include the microfibril-associated glycoproteins (MAGPs) 1 and 2 (Gibson et al,, 1986, 1991, 1996); emilin, a specific microfibrillar protein from chick tissues (Bressan et al., 1993); fibulin-2 (Reinhardt et aZ., 1996); the latent transforming growth factor-p (TGF-P) binding proteins; LTBP1 and -2 (Taipale et aZ., 1996; Gibson et al., 1995) and M P 78/70 (P-IG-H3) (Gibson et al., 1989, 1996); and two glycoproteins with molecular weight of 36 and 32 kDa (Kobayashi et al., 1989; Horrigan et al., 1992). We have shown that thrombospondin is a component of microfibrils in the subendothelium and in other tissues (Arbeille et al., 1991;Fauvel-Laf6veet al., 1996). Fibronectin has been described as a component of microfibrils especially in blood vessels (Krauhs, 1983;Davis, 1994). Plasma proteins such as vitronectin (Dahlback et al., 1989) and amyloid P component (Breathnach et aZ., 1981) have been observed on elastin-linked microfibrils but they are not structural components of microfibrils and cannot be considered associated proteins since they are age-related deposited proteins.

1. Fibrillins Sakai et al. (1986), using monoclonal antibodies directed against proteins from pepsin-treated placenta, immunoprecipitated a 350-kDa protein from the culture medium of human skin fibroblasts which they named fibrillin. The same antibody recognized elastin-associatedmicrofibrils in human skin as well as 12-nm microfibrils in nonelastic tissues, and fibrillin was later described as a structural component of microfibrils (Maddox et al., 1989; Sakai et al., 1991). Figure 4 shows a labeling with an anti-fibrilin antiboby of microfibrils adjacent to the basement membrane of endothelial cells. This form of fibrillin is known as fibrillin-1 because two different fibrillin genes were identified by Lee et al. (1991). The structure and the expression of a new form of fibrillin, fibrillin-2, was described by Zhang el al. (1994).

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FIG. 4 Immunolocaliiation of fibrillin in the subendothelium from human umbilical cord arteries. The anti-fibrillinantibodieslabel microfibrils (arrows) present just beneath the basement membrane (BM) of endothelial cells (EC). Scale bar = 200 nm.

Fibrillin-1 is coded by the 110-kb FBNl gene, whose genomic organization was described by Pereira et aZ. (1993). The gene appears to be composed of 65 exons coding for each protein domain. cDNA (10 kb) was cloned in two steps: its 3’ part (Malsen et al., 1991; Lee et al., 1991) and its 5’ part (Corson et al., 1993). FBNl codes for a complex protein of 2871 amino acids composed of numerous repeated domains (Pereira et aZ., 1993). The protein possessed three domains without homology with other proteins: these domains are the N- and C-terminal parts and the proline-rich area. The central part of the molecule is formed by 56 cystein-rich repeats (Fig. 5), 47 of which are EGF-like repeats. This series of EGF motifs is interrupted by a sequence of nine cysteine residues unique to fibrillin and eight examples of eight-cystein motifs. Seven of these motifs contain the CCC tripeptide sequence homologous to the modules described in the TGF-pl-binding protein named the TGF-BP repeat (Kanzaki et aZ., 1990). The EGF-like repeats which are also found in other proteins correspond to extended arm-like regions indicating a rod-like shape for the fibrillin

FIBRILLIN-1

FIBRILLIN-2

R

FIBRILLIN-LIKE PROTEIN

R

R

TGF-I31 BINDING PROTEIN

1

cystein-freeregion

@

I

4-cystein motif

8-cystein motif

I

mP4-cystein sequence

hybrid motif

EGF- Like motif lacking calcium binding consensus

1

0

potential N-glycosylation site

9-cysteinmotif

1

R RGD sequence

0

EGF-like motif with calcium binding consensus

mp-unknom

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11

molecule which correlates with the fibrillin shape observed by rotary shadowing (Sakai et al., 1991). Forty-three of the EGF-like motifs contain a consensus sequence for hydroxylation of a specific asparagine or aspartate residue and for calcium binding (Corson et al., 1993; Pereira et al., 1993). The calcium bound in the EGF repeats could be necessary for specific protein-protein interactions (Handford et al., 1991). Fibrillin binds calcium in vitro (Corson et al., 1993) and the morphology of fibrillin-containing microfibrils is disrupted by calcium-chelating reagents such as EDTA and EGTA. This effect can be due to the rupture of calcium bridges between adjacent EGF-like repeats involved in lateral packing and alignment of fibrillin molecules (Kielty and Shuttelworth, 1993). Intermolecular disulfide bridges are also important for the stabilization of fibrillin into microfibrils since reducing agents are needed for the purification of the proteins from microfibrils (Gibson et al., 1989). Moreover, fibrillin forms disulfide-bound aggregates in vitro (Sakai, 1990).Four cysteins in the amino-terminalregion, two cysteins in the carboxy-terminal region, and the nine-cystein motif could be engaged in the crosslinks. An RGD sequence is present in the center of the fibrillin molecule and could be a binding site for nuchal ligament cells via the integrin a& (Pfaff et al., 1996; Sakamoto et al., 1996). The fibrillin molecule contains 15 potential sites of N-glycosylation, indicating that the molecule is highly glycosylated (Pereira et al., 1993). Electron microscopy after rotary shadowing of fibrillin-containing microfibrils isolated from cell cultures reveals a beaded structure with a bead periodicity of 50-55 nm. This periodicity could be due to the arrangement of pairs of fibrillin monomers. A model based on the EGF-like repeat succession in the molecule has been proposed by Handford et al. (1995): Two fibrillin monomers would dimerize antiparallely and two dimers would associate to form a four-helix structure. The association of fibrillin with the other microfibrillar components is not known. These interactions could form at the level of the beads. Recent studies show that calcium determines the fibrillin-1shape (Reinhardt et al., 1997a) and stabilizes it against proteolytic degradation (Reinhardt et al., 1997b). The fibrillin-2 molecule is similar in size to fibrillin-1 (2889 amino acids) and contains the same basic domains and cystein-repeat motifs (Fig. 5) (Zhang et al., 1994). Fibrillin-2 is 80% homologous to fibrillin-1. It differs in its N- and C-terminal domains and in a short region interrupting cystein-

FIG. 5 Domain structure of fibrillins and related proteins (FLP and LTBPs). The molecules are formed by cystein-richrepeats,mostly EGF-likerepeatsinterruptedby nine-cysteinresidue (fibrillin) and eight-cystein residue motifs (adapted with permission from Cleary and Gibson, 1996).

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FRANCOISE FAUVEL-LAFEVE

repeat motifs. This region is proline rich in fibrillin 1 and glycine rich in fibrillin-2, indicating a higher flexibility of this molecule. In the vessel wall, anti-fibrillin-1 antibodies react with microfibrils in the three layers of the fetal aorta, whereas anti-fibrillin-2 antibodies only stain the media where elastic fibers are most abundant (Zhang et aZ., 1994). These observations correlate well with the general role given to fibrillins: Fibrillin1 is implicated in the tissue organization and located in an area submitted to mechanical stress, whereas fibrillin-2 is involved in the organization of tropoelastin molecules to form elastin. Following FBNl and FBN2 gene cloning, several studies showed that mutations in these genes are associated with different connective tissue diseases known as fibrillinopathies. Mutations in the FBNl gene located at chromosome 15 on 15q21.1 (Magenis et aZ., 1991) are associated with Marfan syndrome (Dietz et aZ., 1991; Lee et aZ., 1991). To date, about 76 mutations of this gene have been described (Collod et aZ., 1995); they are associated with incomplete forms of the Marfan disease, the ShprintzenGoldberg syndrome (Sood et aZ., 1996), and the Weill-Marchesani syndrome (Cisler et aL, 1995). The mutations of the FBN2 gene located on chromosome 5 between 923 and 931 (Lee et d.,1991) are linked to two exceptional diseases: the congenital contractural arachnodactyly (Putnam et aL, 1995) and a new autosomal-dominant syndrome described by Bay et aZ. (1995) showing the skeletal and joint features of the Marfan syndrome without cardiovascularor ocular abnormalities.This confirms the predominant role of fibrillin-1 in the structure of the vessel wall. Two other genes, MFAPl (Yeh et aZ., 1994) and MFAP3 (Abrams et aZ., 1995), were respectively found at the same location as (15q15-q21) or near to (5q32-q33.2) FBNl and FBN2 loci and were suggested as candidate genes for heritable diseases affecting microfibrils. A recent study (Liu et aZ., 1997) shows that the MFAPl locus is physically close to the FBNl gene but is not mutated in Marfan syndrome. 2. Microfibril-AssociatedProteins

Microfibril-associated proteins with molecular weights of 340, 78, 70, 31, and 25 kDa were extracted from fetal bovine nuchal ligament using reductive saline instead of reductive guanidinium hydrochloride buffer (Gibson et al., 1988). The 340-kDa protein was further identified as the bovine form of fibrillin (Gibson et aZ., 1996). The 31-kDa protein described as MAGP-1 is synthesized by a 10-kbgene mapped to human chromosome 1p36-p35 (Faraco et aZ., 1993).The protein consist of 183 amino acids and has a molecular weight of 21 kDa. The difference in weight of 31 kDa previously determined by polyacrylamide gel electrophoresis is due to an abnormal migration on the gel (Cleary and

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13

Gibson, 1996). The sequence analysis shows that the protein is formed by three domains consisting of a small globular amino-terminal domain rich in glutamine and acidic amino acids connected to a larger disulfidebond (13 cysteine residues) globular carboxy-terminal domain by an extended proline-rich region (Gibson et aZ., 1991; Bashir et aZ., 1994). MAGP-1 contains sulfated tyrosines and a polyglutamine region implied in transglutaminase-catalyzed cross-linking (Gibson et aZ., 1991; TomasiniJohansson et al., 1993). MAGP-1 was shown to act as a substrate for liver transglutaminase, resulting in large cross-linked aggregates (BrownAugsburger er aZ., 1994). These authors showed that MAGP-1 bound to the carboxy-terminal domain of tropoelastin and Henderson et aZ. (1996) located MAGP-1 on the beads on the beaded-filament structure of fibrillincontaining microfibrils. MAGP-1 binds to the a3(VI) chain of type VI collagen, indicating that MAGP-1 may mediate a molecular interaction between type VI collagen microfibrils and fibrillin-containing microfibrils, structures which are often found in close proximity in the extracellular matrix (Finnis and Gibson, 1997). The 25-kDa polypeptide has a similar structure to that of MAGP-1, indicating that the two proteins form a family of microfibrillar proteins. This polypeptide was therefore named MAGP-2 (Gibson et aL, 1996). The gene for human MAGP-2 is located on chromosome 12 in the region 12~12.3-12~13.1. The protein contains 173 amino acids, is rich in serine and threonine residues, and possesses an RGD motif. MAGP-2 differs from MAGP-1 by the lack in proline-, glutamine-, and tyrosine-rich sequences and by the hydrophobic C-terminal sequence characteristic of MAGP-1. These structural differences suggest distinct functionsfor these two proteins. Antibodies directed against MAGP-2 specifically bind to elastin-associated microfibrils. MAGP-2 and fibrillin-2 are absent from microfibrils of the ciliary zonule and the connective tissue around kidney tubules, suggesting an association of MAGP-2 to fibrillin-2 containing microfibrils. The 78 and 70 kDa proteins are structurally similar to each other but distinct from other microfibrillar proteins (Cleary and Gibson, 1996). The peptide sequence presents an extensive homology to P-IG-H3, a protein expressed in an adenocarcinoma cell line following treatment with TGF/3 (Skonier etal., 1992).This protein was also found in normal skin (LeBaron et aZ., 1995), cartilage (Hashimoto et aZ., 1997), and arterosclerotic and restenotic human vascular lesions, especially in areas of dense fibrous connective tissue in the intima (O’Brien et al., 1996). 3. Fibdin-2

Fibulin-2 was predicted from sequence analysis of cDNA clones obtained from a mouse fibroblast library and showed a 43% sequence identity with

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fibulin-1 or BM 90 (Pan et al., 1993). The protein is composed of 1195 amino acids and the C-terminal region is formed by three anaphylatoxinrelated segments and 11 EGF-like repeats, 10 of which have a consensus motif for calcium binding. Recombinant fibulin-2 was produced and rotary shadowing vizualizes the protein as three 40- to 45-nm long rods which are connected at one end in a globe-like structure. Immunofluorescence localization shows that fibulin-2 is present in basement membrane and in microfibrillar structures formed by fibronectin (Sasaki et al., 1996) and fibrillin (Reinhardt et al., 1996). Immunofluorescence in tissues demonstrates a colocalization of fibulin-2 and fibrilin-1 in skin, perichondrium, blood vessel intima, and kidney glomerulus, but fibulin-2 was not present in oculary ciliary zonule, tendon, and connective tissues around kidney tubules and lung alveoli which contain fibrillin. In basement membranes, fibulin-2 binds to nidogen, laminin, type IV collagen, and perlecan (Sasaki et al., 1995; Utani et aL, 1997), and in the matrix it binds to the N-terminal globule of type VI collagen a3 chain but not to other collagen type a chains (Sasaki et al., 1995). The interactions between fibulin-2 and nidogen or fibrillin are calcium dependent and thus probably modulated by the EGFlike domains (Reinhardt et al,, 1996). 4. Latent TGF-m-Binding Proteins TGF-Ps are cytokines involved in the control of cell growth and morphology. Cells in culture produce TGF-Ps in a latent form, the activity being masked by its propeptide (LAP) which is cleaved during the secretory pathway but remains associated by noncovalent interactions (Miyazono et al., 1991).Additional high-molecular-weightproteins, latent TGF-/3binding proteins (LTBPs), associate with secreted latent TGF-/3. LTBPsl-4 have been identified which associate with LAP by disulfide bond(s) (Saharinen et al., 1996; Yin et al., 1995; Moren et al., 1994; Miyazono et al., 1988; Giltay et al., 1997). LTBPs are highly homologuous in their domain structure to fibrillins 1and 2 (Zhang et al., 1995; Sakai et al., 1986). Like fibrillins, LTBP 1 and 2 contain EGF-like motifs with calcium binding consensus and eightcystein repeats (Fig. 5; Gibson et al., 1995), and these two proteins have been located in 10-nm microfibrils (Taipale et al., 1996; Gibson et al., 1995). This suggests that LTBPs can associate with fibrillin-containing microfibrils or are capable of replacing fibrillin as a structural microfibrillar component. However, it is unclear whether microfibrils are composed of single or multiple members of the fibrillinLTBP family (Taipale et aL, 1996). In the vessel wall, latent TGF-/3 is found in the subendothelial matrix (Taipale et al., 1995) and it has been proposed that the subendothelial matrix could serve as a storage site for latent TGF-/3. A dissolution of the endothelial cell basement membrane could generate soluble latent TGF-

SUBENDOTHELIAL MICROFIBRILS

15

0,which could be activated at the surface of smooth muscle cells (Sato et al., 1993).

5. Associated Microfibril Protein Associated microfibril protein (AMP) was identified by screening the whole chick embryo cDNA library with polyclonal antibodies to bovine ocular zonule (Horrigan et al., 1992). AMP is a 32-kDa protein which can be extracted from the elastin microfibrils of bovine and chick elastic tissues with a solution of 6 M urea in the presence of a reducing agent. 6. Fibrillin-like Protein

Using a monoclonal antibody to fibrillin-1, Gibson et al. (1993) identified and cloned a 290- to 310-kDa fibrillin-like protein (FLP) which contained the typical six- and eight-cysteine repeats of fibrillins and ressembled the TGF-@binding protein (Fig. 5). Antibodies directed against FLP recognize microfibrils in nuchal ligament tissues and FLP was extracted from this tissue together with fibrillin-1 and MAGP. The gene coding for FLP is located on the human 14q24.3 chromosome, which is also the locus for the TGF-03 gene (Barton et aL, 1988) and thus FLP could be a binding protein for this molecule (Cleary and Gibson, 1996). 7. MAGP-36 Kobayashi et al. (1989) isolated a 36-kDa microfibril-associatedglycoprotein from porcine aorta which was further characterized as a calcium-binding protein (Kobayashi et al., 1994). However, the localization of this protein has been shown to be restricted to the aortic adventitia.

8. Thrombospondh In our early studies on subendothelial microfibrils, bovine aortas were treated by guanidinium chloride in the absence of a reducing agent and without digestion by collagenase (Fauvel et al., 1983).Ultrastructural studies showed that the guanidinium treatment of aorta depleted the tissue in elastin-associated microfibrils. At that time, little was known about the chemical composition of microfibrils. The extracted material was insoluble in the absence of dissociative salts and heterogenous as shown by SDSelectrophoresis. However, staining of the gels with a Schiff reagent revealed that it contained only one glycoprotein band with an apparent molecular mass of 128 kDa which was degraded by trypsin and chymotrypsin but not by collagenase. This glycoprotein was first named GP128, and biochemical

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studies have shown that GP128 was partially degraded by pepsin, indicating the presence of helicoidal structures. The amino acid analysis revealed a high number of glutamic acid residues and the absence of hydroxyproline and hydroxylysine,indicating that the extracted microfibrils did not contain a collagenous protein. GP128 is synthesized by endothelial cells in culture together with other glycoproteins,such as fibronectin and thrombospondin, and we showed that GP128 and thrombospondin presented similar affinities for heparin and hydroxyapatite (Fauvel et aL, 1984). In order to determine whether thrombospondin (TSP) and GP128 represent the same protein, we looked for the presence of common antigenic determinants. An antibody directed against human platelet TSP recognized only GP128 in the microfibrillar extract (Fauvel-Lafkve and Legrand, 1988). We obtained an antibody directed against GP128 by injecting GP128 isolated by electrophoresis according to a method suitable for insoluble and heterogeneous compounds (Knudsen, 1985). In immunoblotting studies, this antibody recognized GP128 in microfibrils and TSP in a platelet lysate (Fauvel-Lafkve et al., 1988). This antibody did not react with other matrix proteins such as fibronectin or different types of collagen, including type VI collagen. In early experiments, GP128 was extracted in the presence of EDTA in order to inhibit calcium-dependent proteases. Because TSP is a calciumbinding protein and calcium protects it against enzymatic degradation (Lawler and Simons, 1983),microfibrils extracted in the presence of calcium ions contained the TSP molecule with a molecular mass after reduction of 180-kDa reacting with the anti-thrombospondin antibody. GP128, which is a degradation product of TSP, is no longer detected in these extracts. Moreover, immunohistochemical studies in situ have confirmed the presence of TSP in the microfibrils associated with elastin or present beyond the basement membrane (Fig. 6). In an immunofluorescence study, TSP was first located in the vascular subendothelium (Wight et al., 1985). At the electron microscopy level, as shown in Fig. 6, anti-TSP antibodies recognized elastin-linked microfibrils as well as microfibrils adjacent to the basement membrane in the vascular subendothelium (Fauvel-Lafkveet al., 1996). In skin, TSP was associated with microfibrils of the dermoepidermal junction and was also present in a nonelastic tissue (placenta) in the microfibrils linked to the syncytiotrophoblast epithelial basement membrane (Arbeille et aL, 1991). In the subendothelium, anti-TSP antibodies decorated a meshwork of microfibrils formed by fine 8-nm-diameter microfibrils interconnected at the level of 40-nm zones (Fig. 6). We have shown that TSP is present in these interconnections, whereas an anti-fibrillin-1antibody recognized the 8-nm microfibrils indicating that TSP could play a role in the stabilization of the fibrillin microfibrils.

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17

FIG. 6 In the human umbilical cord arteries, the anti-TSP antibodies(arrows) label the typical microfibrils present in the vinicity of endothelial cells (EC). Scale bar = 200 nm.

Several forms of TSP have been described (TSP-1-5) (Adams and Lawler, 1993). TSP-1 was first identified in blood platelets by Baenziger et al. (1971) and has been extensively studied. TSP-1 is synthesized by endothelial cells and present in the subendothelium. TSP-1 mRNA are observed in capillaries of Days 16-18 mouse embryos, whereas TSP-2 transcripts were expressed earlier in these embryos (Iruela-Arispe et al., 1993). TSP1and TSP-2 have related trimeric structures but differential tissue distributions. TSP-3-5 are pentameric molecules having specific tissue distribution. The gene coding for TSP-1 is located on the human chromosome 15 at the 15q loci. This gene is close to the gene coding for fibrilin l(15q15-21) which indicates that these molecules could be linked in situ. TSP-1 is a 420-kDa homotrimer, and each chain is composed of several structural domains (Fig. 7): two globular N- and C-terminal regions separated by a procollagen sequence followed by type I, 11, and I11 repeats with homologies to the circumsporozoite protein from Plasmodium falciparum, EGF, and parvalbumin or calmodulin. TSP is a calcium-binding protein: TSP-1 could

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0

EGF-like motif

4I

calmodulin-likerepeat

FIG. 7 Domain structure of thrombospondin. TSP is formed by a N-terminal heparin-binding domain followed by a procollagen sequence, a series of type I, I1 (EGF-like),and 111repeats, and a globular C-terminal domain. R, RGD sequence.

bind 12 calcium ions per subunit and calcium deprivation induces a molecular conformational change, increases the susceptibility to proteolysis, and alters the adhesive properties (Lawler and Simons, 1983;Lawler et al., 1988). TSP-1 reacts with other tissue components such as collagen (Lahav et al., 1982; Mumby et aZ., 1984; Galvin et al., 1987), fibronectin (Lahav et al., 1984; Dardik and Lahav, 1989; Homandberg and Kramer-Bjerke, 1987), laminin (Lawler et aZ., 1986), SPARC (Sage et al., 1989), and TGF-P (Murphy-Ullrich et al., 1992). TSP in the matrix activates TGF-P (SchultzCherry and Murphy-Ullrich, 1993) and the presence of TSP and latent TGF-/3 on subendothelial microfibrils could lead the latent complex to rapid catabolism and activation (Taipale et aZ., 1996). TSP is involved in several physiopathological processes, such as hemostasis, thrombosis, inflammation, metastasis, and vasoocclusive disorders associated with malaria infection and sickle cell anemia (Legrand et al., 1997). Finally, TSP is a modulator of cell functions in wound healing and angiogenesis (Bornstein, 1995; Tuszynski et al., 1996; Roberts, 1996; DiPietro, 1997). 9. Fibronectin

Fibronectin has been shown by immunohistochemistry to localize to microfibrils in a number of tissues (Krauhs, 1983; Fleischmajer and Timpl, 1984, Schwartz et al., 1985; Inoue et al., 1989;Davis et al., 1994). Fibronectin is colocated with fibrillin in microfibrils anchoring endothelial cells to the elastin lamina in the developing mouse aortic intima (Davis et al., 1994). Fibronectin appears to coat fibrillin-containing microfibrils since no fibronectin was found in the guanidinium chloride extracts. However, the pres-

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19

ence of fibronectin on the surface of endothelial cell anchoring filaments in situ may provide an important structural link between the connecting filaments and the endothelial cell surface. All these studies show that microfibrils represent a family of filamentous structures presenting different structural entities with common properties, and in situ, microfibrils appear as core proteins, probably fibrilin, LTBPs, and MAGP coated by glycoproteins. The binding between these glycoproteins and the core protein could be due to the calcium-binding properties of the different microfibrillar proteins or to weaker associations such as those existing between fibrillin and fibronectin or plasma protein deposits.

111. Microfibrils and Elastogenesis The elastic fiber formation occurs via an ordered series of events (Cleary and Gibson, 1996). During the elastic tissue development, elastin-synthesizing cells first assemble fibrillin, MAGP, and other constitutive molecules into microfibrils, which are laid down as precisely aligned parallel aggregates close to the cell surface: Microfibrils are the first visible component of the elastic fibers. In aorta as well as nuchal ligament, the period of the highest elastin synthesis coincides with an increase in the expression of fibrillins-1 and -2 (Mariencheck et aL, 1994). The increase in fibrillin-1 expression is greater in ligament, in which the elastic fibers are linear, whereas the rise in fibrillin-2 expression is more marked in aorta, in which elastic fibers form sheets. This is due to the hydrophobic glycine-rich domain in fibrillin2 which is similar to numerous elastin fragments and able to form /3 sheets and /3 turns. MAGP is strongly express at relatively constant levels throughout fetal development in both tissues and, as previously indicated, MAGP2 is secreted with fibrillin-2. Microfibril aggregates form a scaffold for the binding of tropoelastin molecules which are then cross-linked by lysyl oxidase to form polymeric elastin. Elastin appears as amorphous material inside microfibrillar aggregates. Progressively, these amorphous areas assemble to form the central core of elastin. Microfibrils are then shifted around the fiber where they are visualized in adult tissues.

IV. The Thrombogenicityof the Subendothelium A. Subendothelial Thrombogenic Macromolecules The adhesion of blood platelets to the subendothelium is the first step of hemostasis and thrombosis and is implicated in the development of

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arteriosclerosis. Hugues (1962) and Hugues and Lapike (1964) observed platelet adhesion to aortic connective tissue fragments and more particularly to purified collagen fibers. Baumgartner and Haudenschild (1972) developed a technique to study the adhesion of platelets to the subendothelium. Rabbit aortas were deendothelialized by a balloon catheter, and aortic segmentswere everted and submitted to the blood flow in a plastic chamber. Platelet-subendothelium interactionshave been quantified and subdivided into contact, adhesion, spreading, and thrombus formation. This technique was used to elucidate the nature of the subendothelial molecules that react with platelets. For this purpose, Sakariassen et al. (1983) developed a parallel-plate flow chamber in which the aortic segments were replaced by a coverslide coated by endothelial cell matrix or purified proteins. In addition to the different types of collagen (Saelman et d.,1994), platelets under flow adhered to purified fibronectin (Houdijk et aZ., 1986), laminin (Ili et al., 1984), and thrombospondin (Agbanyo et aZ., 1993). Nevertheless, ultrastructure studies in situ on the interactions of platelets with the subendothelium showed that platelets reacted with the fibrillar components of the subendothelium (Cazenave et aZ., 1979). These fibrillar structures are composed of type I11 and type VI collagens and microfibrils. Only sparse type I11 collagen fibers are observed in the subendothelium (Gay et aL, 1975) and could not explain the high platelet adhesion observed. Baumgartner and Haudenschild (1972) noted a disappearance of platelet-subendothelium interaction after treatment of aortic segments with collagenase. Stemerman and Baumgartner (1971), however, observed that other elements, such as collagenase-resistantmicrofibrils,were also involved in platelet deposition. In these studies, crude preparations of collagenase, often contaminated by other nonspecificproteolytic activities, were used. We have incubated aortic segments with highly purSed achromobacter iophagus collagenase and showed that platelets reacted with the elastin-associatedmicrofibrils which were characterized by their specific staining by tannic acid, ruthenium red, peroxidase-labeled concanavalin A, or Ricinus cornrnunis lectins (Birembaut et aL, 1982). When the aortas were successively incubated with collagenase and chymotrypsin, platelets did not adhere to the amorphous elastin. These studies were performed before the discovery of type VI collagen and its resistance to collagenase. Thus, two structures could be possible candidates involved in the subendothelium thrombogenicity: microfibrils either associated or not to elastin and type VI collagen. Before examining the thrombogenic properties of these two structures I describe the physiological requirements of platelet adhesion to the matrix and more precisely the role of the von Willebrand factor and platelet membrane proteins involved in this interaction. All the studies on platelet adhesion to the subendothelium were performed under flow using the Baumgartner perfusion chamber or the parallel plate perfusion chamber developed by Sakariassen.

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B. von Willebrand Factor von Willebrand factor (vWF) is a platelet-adhesive protein found in plasma, subendothelium, and platelet a granules (Nachman and Jaffe, 1975; Rand et al., 1980). vWF is synthesized by endothelial cells (Jaffe et al., 1973) and megakaryocytes. vWF is evenly distributed throughout the matrix, forming fibers in close association with other matrix proteins such as fibronectin, collagen, and thrombospondin (Wagner et al., 1984). In severe von Willebrand diseases characterized by absent or reduced vWF, platelet adhesion to the subendothelium is decreased at high shear rate (Weiss et al., 1978; Sakariassen et al., 1984). Platelet adhesion to the subendothelium (Fig. 8) is dependent not only on circulating vWF but also to a significant extent on vWF present in the subendothelium (Stel et al., 1985; Turitto et al., 1985). The effect of vWF on platelet adhesion was only observed at high shear rates since adhesive forces provided by thrombogenic molecules are not sufficient and vWF is required for adhesion and thrombus growth. Although fibrillar type I and I11 collagens bind vWF with a high affinity in vitro, immunolocalization studies of vWF in the subendothelium failed to show any significant association with collagen fibers, but, as shown in Figure 9, vWF is located on microfibrillar structures (Fauvel-Lefeve et al., 1996; Wu et al., 1996). Moreover, treating endothelial cell-derived extracellular matrix with collagenase did not remove vWF and treatment of cells with an inhibitor of collagen synthesis did not block the expression of v W F in the extracellular matrix (Wagner et al., 1984), vWF binding to type VI collagen and to microfibrils will be discussed further.

BLOOD

vWF

ECM Type VI collagen

Fibrillar collagen (type Ill)

FIG. 8 Platelet adhesion to the subendothelium.vWF synthesized by endothelial cells (EC) to the plasma or the extracellular matrix (ECM) binds on intimal microfibrillar structures (type VI collagenand microfibrils)and reinforcesthe platelet (P) adhesionto these components at high shear rates.

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FIG. 9 Anti-vWFantibodiesdecorate the microfibrillar structures (arrows)from the subendothelium of umbilical cord arteries. (EC), endothelial cell. Scale bar = 200 nm.

C. Platelet Membrane Glycoprotein Reacting with the Subendothelium Some bleeding disorders are due to an abnormal adhesion or aggregation of platelets to the subendothelium. In these cases, platelets also show a defect in their surface receptors. The interaction of platelets with the subendothelium is abnormal when platelets are deficient in GPIb as in BernardSoulier syndrome (Nurden et al., 1983), in GPIIb-IIIa as in Glanzmann’s thrombathenia (Sakariassen et al., 1986), and in GPIa (Nieuwenhuis et al., 1986). The Bernard-Soulier syndrome is characterized by a thrombocytopenia with the presence of large platelets which lack glycoproteins Ib, V, and IX. In perfusion studies, such platelets do not adhere to the subendothelium (Sakariassen et al., 1986). Bernard-Soulier platelets do not bind vWF and are not aggregatedby ristocetin (Ruggeri et al., 1983).Human alloantibodies against the glycoprotein Ib inhibit platelet adhesion to the subendothelium (Tobelem et al., 1978). AN51, a monoclonal anti-GPIb antibody, inhibits platelet adhesion to normal and collagenase-treatedsubendothelium (Ruan

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23

et aZ., 1981), and there is a role for this glycoprotein in microfibril-induced platelet aggregation. Glanzmann’s thromboasthenia is a bleeding disorder in which the platelet membrane glycoprotein complex IIb-IIIa (integrin a2bp3) is abnormal or absent (Nurden and Caen, 1975). Thrombasthenic platelets show an abnormality of adhesion at high shear rates and an absence of thrombus formation at all shear rates. At high shear rates, Glanzmann’s platelets are less spread on the surface and the adhesion is somewhat diminished (Sakariassen et aZ., 1986; Weiss et al., 1986). Similar inhibition is observed with monoclonal antibodies to the GPIIb-IIIa glycoprotein complex and studies with RGDS peptides show that vWF is involved in glycoprotein complex IIb-IIIa-dependent adhesion, suggesting that GPIIb-IIIa participates in vWF-mediated platelet adhesion at high shear rate (Nievelstein and de Groot, 1988). Perfusion studies performed with patient platelet presenting a 80% deficiency in GPIa-IIa glycoprotein (integrin a2pl or VLA2) showed a marked decrease in adhesion. Platelets do not adhere to subendothelium or to isolated collagen (Nieuwenhuis et aZ., 1985, 1986). In summary, the collagenase-resistant fibrillar structures present in the subendothelium which react with blood platelets have to induce platelet adhesion and aggregation, bind vWF, and react with platelet glycoproteins GPIb, IIb-IIIa, and Ia-IIa. I discuss type VI collagen and microfibrils reactivity with vWF and platelets in the following section.

V. The Thrombogenicity of Type W Collagen A. von Willebrand Factor Binding to Type VI Collagen Rand and coworkers (1991) studying the vWF binding site in the subendothelium have shown that vWF reacts with a 150-kDaprotein obtained after the extraction of umbilical vein endothelium with 2% SDS-8 M urea. This protein was further identilied as type VI collagen. The association of type VI collagen and vWF in situ was then investigated by immunolocalization studies (Rand et al. 1993; Wu et aZ., 1996). Early observations were performed at the level of immunofluorescence microscopy and it was thus difficult to identify the nature of the microfibrillar structure(s). At the electron microscope level, it was shown that vWF binds to type VI collagen and not to fibrillin-containing microfibrils or to fibrillar collagen. These studies were performed with very high concentrations of anti-type VI monoclonal and polyclonal(1: 5) antibodies: Using the same polyclonal antibody at a 1:500 dilution, we did not observe a colocalization of vWF with type

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VI collagen in the subendothelium of human umbilical cord arteries (Fig. 10) (Fauvel-Lafi3ve et al., 1996). In vitro, type VI collagen binds vWF with a very low affinity compared to fibrillar (type I and type 111) collagens. Denis et al. (1993), calculating the binding of lZI-labeled v W F to purified fibrillar type VI collagen, found 10 times less binding than on type I11 collagen or the endothelial cell extracellular matrix (Baruch et al., 1991) indicating that type VI collagen is probably not the major vWF-binding protein in the vessel wall. Hoylaerts et al. (1997) showed that vWF binds with the same affinity to type VI collagen and the extracellular matrix of a lung fibroblast cell line MRC-5, but this matrix is enriched in type VI collagen (Hessle and Engval, 1984) and thus probably different from the subendothelium. For Denis et al. (1993) and Hoylaerts et al. (1997), the dimeric T116 fragment of v W F correspondingto the vWF amino acid (aa) residues 449-728 and comprising the v W F A1 domain is able to block vWF binding to the endothelial cell matrix and to type VI collagen. Furthermore, Denis et al. (1993) showed

FIG. 10 In the human umbilical cord artery, anti-type VI collagen (5-nm gold beads) and the anti-vWF antibodiesare located on different microfibrillar structures.The anti-vWFis present on the typical microfibrils (arrow), whereas the anti-type VI collagen labels smaller fibrils (arrowheads). Scale bar = 200 nm.

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25

that the SpI fragment of vWF (aa 911-1365) binds to the matrix and type VI collagen but found no interaction for the C-terminal vWF segment (aa 1366-2050). B. Blood Platelet Reactivity with Type VI Collagen

In view of the association of vWF with type VI collagen, it is important to know the reactivity of type VI collagen with platelets. It has been shown by Zangari et al. (1995) that purified type VI collagen is a weak platelet agonist. Moreover, a bovine aorta microfibrillar extract containing type VI collagen and fibrillin in their native state does not induce platelet aggregation (Fauvel-Lafhve et al., 1996). However, a weak but significant platelet adhesion was observed which was inhibited by an anti-type VI collagen antibody. In a static adhesion assay, type VI collagen supports the adhesion of human platelets very weakly and this adhesion was not mediated by vWF. In control inhibition studies with calin (a collagen inhibitor derived from the saliva of the leech Hirudo medicinalis), anti-type VI collagen, and anti-vWF antibodies, it was shown that the strong binding of washed platelets to the subendothelium does not involve collagens (including collagen VI) or matrix-associated vWF (Hoylaerts et al., 1997). In flow system, Ross et al. (1995) demonstrated that type VI collagen stimulated platelet reactivity at low shear rates, equivalent to that observed in venous circulation, but not at arterial shear conditions. The same authors showed that reactivity of platelets to type VI collagen is reduced in the absence of vWF and is inhibited in the presence of monoclonal antibodies which block the platelet GPIb or GPIIb-IIIa receptors.

W. The Thrombogenicity of Elastin-Associated Microfibrils A. Platelet Interaction with Microfibrils: The Role of Thrombospondin

Fauvel et al. (1983) showed that a guanidiniumchloride extract from bovine aorta was able to induce platelet adhesion and aggregation different from collagen by the requirement of plasma or vWF. Moreover, an antibody directed against the platelet glycoprotein GPIb (AN51) inhibits platelet adhesion and aggregation induced by microfibrils and not by collagen. By examining the reactivity of the enzyme-treated microfibrils toward blood platelets, we established that GP128 (thrombospondin) was essential for

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the thrombogenicity of microfibrils. By ELISA, we verified that the microfibrillar extract did not contain fibrillin or type VI collagen which normally are not present in guanidinium chloride tissue extractsand whose extraction needs the presence of a reducing agent. The interaction between platelets and microfibrils was not inhibited by anti-fibrillin or anti-type VI collagen antibodies (Fauvel-Laf2ve et aL, 1996). Anti-GP128, as well as anti-TSP antibodies, totally inhibited platelet aggregation induced by microfibrils but was without effect on the aggregation induced by ADP or collagen (Fauvel-Lafhve et d.,1988). These works gave an important role to tissue TSP in platelet interaction with the subendothelium. This role was confirmed by the work of Agbyano et al. (1993) showing a divalent cation-dependent adhesion of platelets to TSP under flow conditions.

6 . The Role of von Willebrand Factor in Platelet-Microfibril Interaction Contrary to collagen, washed platelets did not react with microfibrils indicating that a plasma factor was required (Legrand et aL, 1986). This factor was vWF because normal platelets resuspended in the plasma from a patient presenting a severe deficit in vWF could not be aggregated by microfibrils; the aggregationwas restored after injection into the patient of a cryoprecipitate enriched with vWF (Fauvel et aZ., 1983). The absence of platelet aggregation and platelet adhesion was observed for all the severe deficiencies in vWF studied. Moreover, the percentage yield of platelet adhesion to microfibrils is in direct ratio to the vWF concentration and depends on the presence of high-molecular-weight vWF polymers (Legrand and FauvelLafhe, 1992). In a solid phase assay, we have shown that 125iodinatedv W F binds to microfibrils with the same affinity as it does to type I11 collagen. This binding was not inhibited by anti-collagen (type I, 111, or VI), anti-TSP-1, or anti-GP128 antibodies. In fact, after the electrophoretic separation of microfibrillarproteins and blotting, labeled v W Fbinds to a 97-kDa nonidentified protein (Fauvel-Lafhve and Legrand, 1990). Immunoelectron microscopy in situ showed a colabeling of the subendothelial microfibrils with the anti-vWF and the anti-TSP-1 antibodies (Fig. 11). No binding was observed at the level of the basement membrane, collagen fibers, or microfibers (type VI) as shown in Fig. 10. Pig aortic fragments which do not contain v W F were not decorated by the anti-vWF, after an incubation with fresh plasma before fixation, we observed a binding of plasma vWF to the intimal TSP-labeled microfibrils

SUBENDOTHELIAL MICROFIBRILS

27

FIG. 11 In the subendothelium of human umbilical arteries, anti-TSP (5-nm gold beads) and anti-vWF (15-nm beads) antibodies decorate the same microfibrils (A) as shown at higher magnification (B). Scale bars = 100 nm.

but not to collagen fibers (Arbeille et al., 1991). Moreover, Barabino et al. (1997) showed a direct binding of vWF to purified TSP-1. Thus, in the vessel wall, vWF binds to thrombospondin-containing microfibrils and not to collagens and the binding affinity is the same for these microfibrils and the subendothelium.

C. Microfibril Interactions with Platelet Membrane Glycoproteins Among platelet membrane glycoproteins, GPIb is involved in platelet adhesion to the subendothelium since platelets from patients affected by Bernard-Soulier syndrome are devoid of GPIb and do not adhere to the subendothelium. A monoclonal anti-GPIb antibody (AN51) directed against GPIb-vWF binding site inhibits the platelet aggregation induced by microfibrils (Fauvel et aL, 1983) and is without effect on the aggregation induced by collagen. Two other antibodies directed against the GPIb-GP IX complex (FMC 25) and the central GPIb macroglycopeptide (WM23) do not inhibit the platelet interaction with microfibrils.

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In order to study the h t step of platelet interaction with microfibrils, namely, platelet adhesion, we developed an adhesion assay in which we measured platelet adhesion in the absence of platelet activation (FauvelLafkve et al., 1993a).In this assay it was shown that the adhesion of platelets from patients with Bernard-Soulier syndrome to microfibrils was diminished, whereas the adhesion to collagen was normal. These results indicated that the abnormal attachment of these platelets to the subendothelium was due to a defect in platelet adhesion to the noncollagenous microfibrils (Fauvel-Laf6ve et al., 1993b). In this adhesion assay, the binding of platelet to microfibrils was not inhibited by other anti-platelet glycoprotein antibodies, and platelets from patients with Glanzmann’s thromboasthenia, devoid of glycoprotein GPIIb-IIIa, adhered normally to microfibrils.On the other hand, antibodies directed against GPIIb-IIIa, GPIaIIa, or GPIV did not inhibit the adhesion but prevented platelet aggregation induced by microfibrils, indicating that these antibodies are involved in the later phases of platelet activation. In a recent study submitted for publication, we show that GPIaIIa is also not a primary binding site for collagen because it did not affect the recognition of collagen by platelet but GPIaIIa was involved in all other steps of platelet activation (spreading and aggregation).

D. The Model for Platelet-Microfibril Interaction These different studies on the function of thrombospondin-containing microfibrils allow me to propose a model for their thrombogenicity in the subendothelium(Fig. 12). vWF present in the subendothelium or adsorbed from the plasma after an endothelial cell lesion is fixed on the microfibrils. Fixed vWF is modified and able to react with platelet GPIb: This interaction is the first phase in the adhesion of platelet since the contact between platelets and microfibril interaction depends on the presence of vWF. The binding of the vWF to GPIb could induce an “outside-in’’ activation phenomenon which would activate other platelets glycoproteins: GPIaIIa, GP IV, GP IIbIIIa, or other TSP receptors such as the integrin-associated protein (CD 47) which reacts with the C-terminal TSP peptide RFYVVMK, which is also involved in platelet aggregation and in the potentialization of platelet aggregation induced by ADP (Fukimoto el aZ., 1995; Dorahy et al., 1997). These studies have demonstrated the presence of thrombospondin in microfibrillar structures of the arterial subendothelium and show the evident role of these microfibrils in vWF binding and in thrombogenicity of the subendothelium.

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AN 51 Anti-TSP1

97 kD

TSP

FIG. 12 Model for the interaction between platelet and thrombospondin-containing microfibrils. v W F bound to microfibrils is recognized by the platelet GPIb. This binding allows the fixation of the platelet TSP receptor on microfibrils.

VII. Conclusion and Perspectives Despite real progress in the characterization of microfibril proteins constituents, little is known about the extact structure of subendothelial microfibrils. The situation is particularly complex in the vessel wall if compared to ligament or other elastin-rich tissues. Some questions remain: Are fibronectin-containing microfibrils composed of only fibrillin and fibronectin? Do these microfibrils also contain thrombospondin? Do thrombospondin-containing microfibrils belong to a microfibrillar meshwork also composed of fibronectin-containing microfibrils and fibrillin microfibrils? The situation is also not clear for latent TGF-&binding proteins: Do they really serve as a substitute for fibrilin in some microfibrils? This point is probably very important for the localization of thrombospondin in the subendothelium because TSP is generally present at the level of 40-nm structures which bind fibrillin microfibrils together. Do TSPs colocalize with the latent TGFP-binding proteins? Due to the TGF-/3 activation by TSP the coexistence of both molecules on the same microfibrils should be studied. Such microfibrils could be involved in the activation of smooth muscle cells by TGF-fl and consequently in the migration of these cells and arteriosclerotic lesion formation. Moreover, TSP-containing microfibrils induce blood platelet

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activation and platelet granules contain large amounts of TGF-P which could be activated by TSP. Thus, endogenous vessel wall and exogenous platelet TGF-P could contribute, after an endothelial cell lesion, to the intimal thickning. Concerning platelet adhesion to the subendothelium, there is evidence of a primordial role of TSP-containingmicrofibrils in the thrombogenicity of the subendothelium,with microfibrilsbeing able to bind vWF and to react with platelet membrane glycoproteins directly involved in the interaction between platelets and the intima. Rand et al. (1997) and Hoylaerts et al. (1997) tend to minimize the role of type VI collagen because its affinity for von Willebrand factor is too low and it only weakly reacts with blood platelets at low shear rates, where vWF is not necessary to reinforce the adhesion to the subendothelium. In fact, Rand et al. (1997) found that platelets adhered at low shear rate to purified fibrillin-containing elastinassociated microfibrils, but purified fibrillin fibrils do not contain thrombospondin as demonstratedin ELISA studies. In their conclusions on a graded platelet response according to the degree of the vessel wall lesion, they attributed a role to type VI collagen in superficial endothelial trauma, whereas elastin-associatedmicrofibrilswere suggested to be more important for deeper lesions. However, they did not mention that, in the arterial wall, microfibrils are not only bound to the elastin lamina but also present just beneath the basement membrane together with type VI collagen. Hoylaerts et al. (1997) also evoked the role of a molecule other than type VI collagen for the reactivity of platelets with the vessel wall, and they proposed fibronectin in microfibrillar structures (Kawai et al., 1989). Thus, it will be important in the next few years to determine functional roles for the different microfibrils present in the subendothelium and in particular to study the relation between fibronectin,latent TGF-&binding proteins, and thrombospondin-containing microfibrils.

Acknowledgments The author is particularly grateN to Dr. B. Arbeille and her team from Unite de Microscopie Electronique, CHU de Tours, France, for hnunoelectron microscopy. This work was supported by INSERM.

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Lawler, J., Connoly, J. E., Ferro, P., and Derick, L. H. (1986). Thrombin and chymotrypsin interactions with thrombospondin. Ann. N. Y.Acad. Sci. 485,273-287. Lawler, J., Weinstein, R., and Hynes, R. 0. (1988). Cell attachment to thrombospondin: The role of-Arg-Gly-Asp, calcium and integrin receptors. J. Cell Biol. 107,2351-2361. LeBaron, R. G., Bezverkov, K. I., Zimber, M. P., Pavelec, R., Skonier, J., and Purchio, A. F. (1995). Beta IG-H3, a novel secretory protein inducible by transforming growth factor-beta, is present in normal skin and promotes the adhesion and spreading of dermal fibroblasts in vitro. J. Invest. DermatoL 104,844-849. Lee, B., Godfrey, M., Vitale, E., Hori, H., Mattei, M. G., Sarfarazi,M., Tsipouras,P., Ramirez, F., and Hollister, D. (1991). Linkage of Marfan syndrome and a phenotypically related disorder to two different fibrillin genes. Nature 352,330-334. Legrand, C., Dubernard, V., Rabhi-Sabile, S., and Morandi da Silva, V. (1997). Functional and clinical significance of thrombospondin. Platelets 8,211-223. Legrand, Y. J., and Fauvel-Weve, F. (1992). Molecular mechanism of the interaction of subendothelial microfibrils with blood platelets. Nouv. Rev. Fr. Hematol. 34,17-25. Legrand, Y . J., Fauvel, F., Arbeille, B., Mouhli, H., Gutman, N., and Muh, J. P. (1986). Activation of platelets by microfibrils and collagen: A comparative study. Lab. Invest. 54 566-573. Liu, W., Faraco, J., Qian, C., and Francke, U. (1997). The gene for microfibril-associated protein-1 (MFAP1) is located several megabases centromeric to FBNl and is not mutated in Marfan-syndrome. Hum. Genet. 99,578-584. Microfibrils, a small extracellular component of connective tissue. Anat. Low, F. N. (l%l). Rec. US, 250. Maddox, B. K., Sakai,L. Y., Keene, D. R., and Glanville, R. W. (1989). Connective tissue microfibrils. Isolation and characterizationof three large pepsin-resistant domains of fibrillin. J. Bwl. Chem. 264,21381-21385. Magenis, R. E., Maslen, C. L., Smith, L., Allen, L., and Sakai, L. Y. (1991). Localization of the fibrillin (FBN) gene to chromosome 15, band q21.1 Genomics 11,346-351. Malsen, C. L., Corson, G. M., Maddox, B. K., Glanville, R. W., and S a k i L. (1991). Partial sequence for a candidate gene for the Marfan Syndrome. Nature 352,334-337. Mariencheck, M. C., Davis, E. C., Zhang, H., Ramirez, F., Rosenbloom, J., Gibson, M. A., Parks, W. C., and Mecham, R. P. (1994). Fibrillin-1 and fibrillin-2show temporal and tissuespecific regulation of expressionin developingelastictissues. Connective Tissue Res. 31,l-11. Mayer, B. W., Hay, E. D., and Hynes, R. 0. (1981). Immunoqtochemical localization of fibronectin in embryonic chick trunck and area vasculosa. Dev. Biol. 82,267-286. Mayne, R. (1987). Vascular connective tissue. Normal biology and derangement in human diseases. In “Connective Tissue Disease. Molecular Pathology of the Extracellular Matrix” (J. Uitto and A. J. Perejda, eds.), pp. 163-183. Dekker, New York. Miyazono, K., Hellman, U., Wernstedt, C., and Heldin, C. H (1988). Latent high molecular weight complex of transforming growth factor-pl. Purification from human blood platelets and structural characterization. J. B i d Chem. 263,6407-6415. Miyazono, K., Olofsson, A., Colosetti, P., and Heldin, C. H. (1991). A role of the latent TGFpl-binding protein in the assembly and secretion of TGF-p1. EMBO J. 10,1091-1101. Moren, A., Olofsson, A., Stenman, G., Sahlin, P., Kanzaki, T., Claesson-Welsh,L., ten Dijke, P., Miyazono, K., and Heldin, C. H. (1994). Identification and characterization of LTBP-2, a novel latent transforming growth factor-binding protein. J. Biol. Chem. 269,32469-32478. Mumby, S. M., Raugi, G. J., and Bornstein, P. (1984). Interactions of thrombospondin with extracellular matrix proteins: Selective binding to type V collagen. J. Cell Biol. !J& 646-652. Murata, K., and Matoyama, T. (1990). Collagen species in various sized human arteries and their changes with intimal proliferation. Artery 17,%-106. Murphy-Ullrich, J. E., Schultz-Cherry, S., and Hook, M. (1992). Transforming growth factorp complexes with thrombospondin. Mol. Biol. Cell 3,181-188.

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Cultivation and Transplantation of Epidermal Keratinocytes V. V. Terskikh and A. V. Vasiliev Institute of Developmental Biology, Russian Academy of Sciences, 117334 Moscow, Russia

Transplantation of autologous cultured keratinocytes is the most advanced area of tissue engineering which has clinical application in restoration of skin lesions. In vitro, disaggregated keratinocytes undergo activation and after adhesion and histogenic aggregation form three-dimensional epithelial sheets suitable for grafting on prepared wounds that provide a reparative environment. Epidermal stem cells survive and proliferate in culture, retaining their potential to differentiate and to produce neoepidermis. Reconstructed skin is physiologically compatible to split-thicknessautografts. Autotransplantation of cultured keratinocytes is a promising technique for gene therapy. In many cases allografting of cultured keratinocytes promotes wound healing by stimulation of epithelialization. Banking of cryopreserved keratinocytes is a significant improvement in usage of cultured keratinocytes for wound healing. Skin substitutes reconstructed in vitro that have morphological, biochemical, and functional features of the native tissue are of interest as model systems that enable extrapolation to situations in vivo. KEY WORDS: Epidermal keratinocytes, Cell culture, Transplantation, Wound healing, Skin reconstruction. o 1999~cademiipress.

1. Introduction

In vitro culture of human epithelial cells has attracted a remarkable interest in the past two decades, not only because of the importance of its practical applications but also because of the wide range of basic investigations which can be performed on it. The most striking practical application is undoubtedly the successful use of in vitro cultured epithelial sheets as autografts or allografts on patients with extensive bums. Replacement of International Review of Cytology, VoL I 8 8 0074-7696l99 $30.00

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Copyright 0 1999 by Academic Press. All rights of reproduction in any form reserved.

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V. V. TERSKIKH AND A. V. VASlLlEV

missing skin to restore protective skin barrier has long been a problem for physicians and surgeons. In the United States, more than 1 million people a year suffer thermal injuries. Of these, approximately 100,OOO patients are hospitalized and 10,000die (Hansbrough, 1992).Autologous skin grafts were used for wound closure, but in severe burn victims there is not enough full-thickness skin available to cover the wounds. Because the ultimate outcome of heavy burn patients is determined by wound coverage, temporary coverage of wounds with fresh or cadaver skin allografts is used. However, such allografts are rejected and regrafting is necessary. Use of bioartificial tissues is the only choice in the absence of any remaining functional tissues to replace missing epidermal keratinocytes. Bioartificial tissues are of two general types: the temporary type serves to stimulate the regeneration of host tissue, whereas the artificial tissue itself is degrading, and the other type is permanent. Epidermis, which is probably the most complex stratified epithelium, is mainly composed of one cell type-the keratinocyte. Moreover, the epidermis is not directly vascularized but receives nutrients from the blood vessels of the dermis. These properties allowed the reconstruction of epidermis in vitro. In heavy full-thickness burns reparation does not occur because of the lack of progenitor keratinocytes, although the reparative environment contains mitogen and extracellular matrix molecules that support cell growth. Also, it was experimentally demonstrated that cultured keratinocyte sheets transplanted to the wound bed can survive and organize neoepidermis. Reconstruction of skin is the most remarkable example of a new strategy for the restoration of biological structure and function, called tissue engineering, which has emerged during the past few decades through interdisciplinary research in cell physiology, cell and molecular biology, and biochemistry of extracellular matrix. In 1948,Medawar demonstratedthat explanted fragments of rabbit epidermis produced epithelium upon transplantation back to the donor animal. Billingham and Reynolds (1952) described a method for preparation of sheets of pure epidermis and of epidermal cell suspensions which can be transplanted successfully. Later it was shown that proliferating keratinocytes in outgrowth cultures might be transplanted (Karasek, 1968). Igel et al. (1974) proved that skin epithelium cultured on dead pigskin can produce true epithelium upon transplantation to the donor rabbit. Early investigations provided convincing proof that cultured epidermal cells retain morphological features of epithelial cells and produce true epithelium upon transplantation. Large-scale grafting became possible when new culture systems were developed. In 1979, Green et al. brought to the attention of surgeons the possibility of using autologous culture-

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grown keratinocyte sheets to restore defects in epidermis. Later, human epithelial sheets grown from suspensions of disaggregated keratinocytes were transplanted onto a graft bed prepared in athymic mice. Such grafts formed epidermis complete with stratum corneum and remained healthy for as long as 108 days after grafting (Banks-Schlegel and Green, 1980).

II. Culture of Keratinocytes A. Methods of Culture 1. Explant Cultures

Historically, the first approach was the culture of explants, either as epidermal or as organ cultures. In explant cultures small split-thickness fragments of skin are cultured on different substrates. The explants can yield either organ culture if the substrate does not allow the epidermal cells to migrate and the original organization of skin is preserved or epidermal cell cultures through the expansion of epidermis and formation of a peripheral outgrowth. Epidermal cell outgrowths derived from explants are easy to obtain. They represent a simple and efficient way to culture adult epidermal cells and have been used for decades (Karasek, 1966). Large numbers of human keratinocytes can be obtained in primary cultures by multiplying the number of explants, allowing biochemical studies to be made without subculturing (Flaxman and Harper, 1975).

2. Fibroblast Contamination The phenomenon of fibroblast overgrowth is one of the major problems of explant cultures of keratinocytes. Fibroblast contamination is usually negligible during the first days but eventually fibroblasts overgrow the epidermal cells. A variety of methods of reducing the extent of fibroblast contamination have been reported. Fibroblasts were selectively removed by trypsin and EDTA treatment (Price et al., 1983),by incubation in Hanks’ salt solution (Pal and Grover, 1983),and by lowering the incubation temperature to 32 or 33°C (Jensen and Therkelsen, 1981). A technique was described for selective elimination of fibroblasts in human keratinocyte cultures using a monoclonal antibody raised against human fibroblasts (Gusterson et al., 1981). Linge et al. (1989) used mouse monoclonal antibody raised against human brain Thy-1, a cell surface glycoprotein, to attach the contaminating dermal fibroblasts to a goat antimouse immunoglobulin-

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coated plastic surface. Saalbach et al. (1997) used fibroblast-specific mAb AS02 as a tool for elimination of contaminating fibroblasts. 3. Dispersed Cultures

Another approach is to separate epidermis from dermis and use the former to initiate pure cultures of epidermal cells. Basically, this has been possible since the work of Medawar (1941), who showed that it was possible to completely separate epidermis from dermis and to maintain it in culture. Cruickshank et al. (1960) were the first to show that guinea pig epidermal cells could be cultured in v i m and exhibit mitotic activity in the absence of dermal constituents. Usually, interfollicular keratinocytes are used for cultivation, and Limat et al. (1986) serially cultivated keratinocytes isolated from the outer root sheath of anagen human hair follices. The first important procedure is the isolation of keratinocytes.Trypsinization, the widely used method of cell separation, is a critical factor in the process of keratinocyte culture. The trypsin-floating technique of Yuspa and Harris (1974) is widely used, with either short trypsinization at 37°C or prolonged incubation at 4°C. By further dissociatingthe epidermal tissue, cell suspensions could be prepared and set in culture. This method led to many attempts at cultivation of epidermal cells from various species. Highdensity dispersed cultures were usually started in modified Eagle’s medium supplemented with fetal calf serum by inoculation of no less than 105 cells/ cm2. By carefully controlling the various steps of preparation, epidermal keratinocyte suspensions were produced with negligible fibroblast contamination. Such cultures grew to confluency and formed multilayered sheets. Some cell multiplication took place, but generally the population of basal cells declined. It was not possible to cultivate keratinocyte serially, and separated single cells were not able to efficiently produce colonies (Fusenig and Worst, 1975; Liu and Karasek, 1978a; Marcel0 et al., 1978; Eisinger et al., 1979). Low-density dispersed cultures became feasible when Rheinwald and Green (1975) discovered that rapid growth of neonatal and adult human epidermal keratinocytes could be supported by cocultivation with a feeder layer of lethally irradiated 3T3 cells in Dulbecco’s modified Eagle’s medium (DMEM) with hydrocortisone and 20% fetal bovine serum. Later, the medium was improved by addition of EGF (Green, 1977) and cholera toxin or isoproterenol to stimulate production of cyclic AMP (Green, 1978). Because EGF was found to reduce colony-forming efficiency when added at the time of inoculation, it was routinely added to cultures at the time of the first medium change. EGF extended the culture life of keratinocytes by augmenting the ability of cells to survive subculture and initiate new colonies. Use of cholera toxin increased the number of small cells (9.5-

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14 pm) with high proliferative potential. Under these conditions keratinocytes were serially cultivated through many transfers and more than 100 cell generations. Peehl and Ham (1980a,b) developed an improved procedure for clonal growth of human keratinocytes without feeder layer in medium 199 with the addition of hydrocortisone and FBS or in medium F-12 or MCDB 151 supplemented with dialyzed fetal bovine serum protein rather than whole serum. Thompson et al. (1985) described a method which enabled optimized growth of human keratinocytes without the use of a feeder layer or collagen substrate. They found that high-calcium DMEM supplemented with 10% FCS, 10 ng/ml EGF, 10-l' M cholera toxin was superior to all other media in producing the most rapid linear expansion of keratinocytes. However, a high proportion of cells differentiated. At very low calcium concentrations (0.06 mM) cells were grown readily and formed a monolayer which was morphologically similar to the basal layer of the epidermis. 4. Three-Dimensional Cultures

In conventional submerged cultures on plastic, incomplete differentiation of keratinocytes was observed, which was likely the result of the microenvironment being drastically different from that existing in vivo. To improve differentiation of cultured keratinocytes in vitro and render the culture conditions more physiological, three-dimensional cultures have been developed. These cultures might be raised to the air-medium interface by use of rigid support. Collagen gels or rafts (Lillie et al., 1988) and dead deepidermized dermis (Regnier et aZ.,1981) were used as substrate to culture epidermal cells. In 1971 Karasek and Charlton demonstrated that keratinocytes attached, spread, and grew on collagen gel. Elsdale and Barde (1972) used collagen gel for cultivation of fibroblasts. Bell et al. (1981,1991) manufactured a living skin equivalent that resembled human skin. The skin equivalent consisted of dermal equivalent, reconstituted with collagen gel and dermal fibroblasts, and cultured keratinocytes plated on the surface of dermal equivalent.

5. Serum-Free Cultures Another approach is to produce defined serum-free medium. Serum is critical in the culture of keratinocytes. Some lots of serum were found to inhibit keratinocyte growth (Hawly-Nelson et al., 1980). Bertolero et al. (1986) demonstrated that at least three factors present in FBS (fetuin, highdensity lipoprotein, and platelet-derived growth factor) inhibited growth of epidermal keratinocytes. Ham and coworkers (Peehl and Ham, 1980,a,b; Tsao et aZ., 1982; Boyce and Ham, 1983) reported the development of

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defined media consisting of nutrients, hormones, growth factors, and trace elements optimized for the culture of epidermal keratinocytes in the absence of a feeder layer and contaminating fibroblasts. Good colony formation was obtained with an inoculum of 2000 cells per 5.0 ml of medium in a 60-mm petri dish (Tsao et aZ., 1982). It was possible to initiate cultures directly from disaggregated foreskin cells.

6. Microcarrier Cultures Katayama et aZ. (1987) used collagen-coated Cytodex 3 microcamers to culture human keratinocytes without stirring. The cells attached to the beads rather loosely. Rapid cessation of growth and differentiation of keratinocyteswas observed. We cultured primary human keratinocytes on collagen microcarriersin DMEM :F12 medium supplemented with EGF, insulin, and isoproterenol. Significant growth was observed within a week and cultures were suitable for transplantation (Fig. 1). 7. Low-Calcium Cultures

In primary murine keratinocytes optimal proliferation occurs at calcium concentrations below 0.1 mM. Reduced concentration of Cazc prevents

FIG. 1 Human epidermal keratinocytes grown on microcarriers. Scale bar = 50 pm.

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differentiation and stratification. Optimum Ca24concentration for growth of human keratinocytes at clonal seeding densities is about 0.3 mM (Boyce and Ham, 1983). In confluent cultures human keratinocyte proliferation and epithelial thickness were maximal at 1or 2 mM Ca2+.Confluent cultures may be models of intact epidermis, whereas subconfluent cultures may be models of wounded epidermis (Milstone, 1987). Raising the concentration of Ca2+in the medium rapidly induces a complex set of events which are collectively referred to as terminal differentiation (Hennings et aL, 1980) and assembly of adherence junctions (O’Keefe et af., 1987),which precedes desmosome formation (Inohara et aZ., 1990).

111. Behavior of Keratinocytes in Culture A. Proliferation of Keratinocytes Growth of keratinocytes in culture is regulated by a delicate balance of influences which promote either proliferation or differentiation. The average doubling time for cells in exponential growth is about 24 hr in both the presence and absence of EGF. The culture lifetime of human epidermal cells is increased from 50 to 150 generations by adding EGF to the medium. EGF extends the culture life by providing the cells with a greater ability to survive subculture and initiate new colonies but not necessarily by an increased growth rate (Rheinwald and Green, 1977). Transforming growth factor-p (TGF-P) is a potent reversible inhibitor of growth that abrogates the stimulatory action of TGF-a and EGF (Coffey et aZ., 1988b). TGF-P selectivelyreduces expression of certain genes associated with cell proliferation (c-mycand KC) that does not result in induction of terminal differentiation (Coffey et aZ., 1988a; Pietenpol et aL, 1990). Acidic and basic fibroblast growth factors were determined to be potent mitogens in serum-free keratinocyte cultures (Shipley et aZ., 1989).A growth factor with a high degree of specificity for epithelial cells was identified by Rubin et aZ. (1989). This factor, designated KGF, is a member of the FGF family and has the properties of a paracrine effector of keratinocytes (Finch et af., 1989). In response to the differentiation signal induced by a high Ca2+concentration, KGF-treated keratinocytes ceased to proliferate. Under these conditions human keratinocytes express both K1 and filaggrin, early and late markers of biochemical differentiation, respectively. In contrast to KGF, EGF significantly retarded the expression of these markers (Marchese et aZ., 1990). Rapidly growing normal human neonetal keratinocytes cultured in serum-free MCDB 153medium can be induced to undergo either reversible or irreversible growth arrest at distinct cell cycle states. Reversible GI arrest was induced by cultivation of low-density cells in

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human lymphocyte conditioned medium, by cultivation in high-density stationary phase conditioned medium, and by cultivation in isoleucinedeficient medium. Irreversible arrest of growth predominantly in G1 was induced by culture in growth factor-deficient medium (Pittelkow et al., 1986). To proliferate, keratinocytes must adhear to a substrate. Cells suspended in methylcellulose maintained a high rate of DNA synthesis for the first 10 h and by 14 h DNA synthesis had ceased. Suspended G1 phase cells were completely blocked from entering the S phase, whereas S-phase cells continued to traverse the S phase and enter the G2 and M phases in which they accumulated (Pittelkow et al., 1986). The population of keratinocytes in culture is highly heterogeneous. Measurements of cellular DNA and RNA revealed kinetically distinct subpopulations with different generation times and different cellular and nuclear RNA content (Staiano-CoicoetaZ., 1989). Jensen et al. (1985) demonstrated that cycling basal keratinocytes may be divided into at least two subpopulations that differ with respect to the rate of DNA replication and in the presence of EGF and cholera toxin dramatic changes were found between these subpopulations.

B. Stem Cells Stem cells are defined as cells that retain a high capacity for self-renewal throughout adult life and also have the ability to produce daughters that undergo terminal differentation (Lajtha, 1979). Epidermis is a wellcharacterized example of permanently renewing tissue in which terminally differentiated cells have a short life span (Halland Watt, 1989). In keratinocyte cultures, like in epidermis, proliferation is largely confined to the basal layer of cells, and keratinocytes undergo terminal differentiation as they migrate upward. Cultured autologous keratinocyte sheets persist on the recipient for many years, indicating that stem cells have been retained in culture. From analyzing keratinocyte colonies, Barrandon and Green (1987a) proposed that holoclones were probably founded by stem cells and shortlived paraclones by transit amplifying cells. it is more difficult to classify the founders of meroclones, but they may be stem cells that generate transit amplifymg daughters at higher frequency than the stem cells of holoclones. The proportion of putative stem cells in keratinocyte cultures (10-30%) varied with cell strain, passage number, and state of confluence (Jones and Watt, 1993). These authors found that in culture keratinocytes that were committed to differentiation divided one to five times, after which all their progeny underwent terminal differentiation. These results are in good

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agreement with the data of Potten (1981), who predicted that, in vivo, transit amplifying cells divide one to three times prior to differentiation. Jones et al. (1995) showed that in vivo stem cells express higher levels of the ad1and a& integrins than transit-amplifying cells. The distribution of stem cells and transit-amplifying cells is not random: Patches of integrinbright and integrin-dull cells have specific locations with respect to the epidermal-dermal junction. Stem cell pattering can be re-created in culture, in the absence of dermis, and appears to be subject to autoregulation.

C. Migration Cultivation in vitro results in “a~tivation~~ of keratinocytes, including increased cell attachment, spreading, and migration (Grinnell, 1990). In migrating keratinocytes traction forces were not detected at the rapidly extending front edge of the cell. Instead, the largest traction forces were exerted perpendicular to the left and right cell margins. Lamella extension proceeds until it is inhibited by increasing tension between the front and rear of the cell. Following a short lag, retraction of the cell rear occurs and tension is released, leading to another phase of lamellar extension (Lee et al., 1994). In low-calcium medium keratinocytes are more motile and had a higher rate of membrane activity than keratinocytes in normal medium; however, high motility does not result in significant translocation of cells across the substratum. In low-calcium medium about 20% of the cells at the margin of the wound migrated into it over 9 h, forming fan-shaped lamellae (Magee et al., 1987). It seems reasonable to suggest that for active lateral migration keratinocytes should be organized in a sheet. The migration behavior of cultured keratinocytes is influenced by extracellular matrix molecules. Fibronectin and collagen types I and IV significantly promoted migration (Woodley et al., 1988; O’Keefe et al., 1985). Human keratinocytes express vitronectin receptors and use a,pS receptor for cellular locomotion (Kim et al., 1994). Small colonies of keratinocytes are polarized and actively migrate on plastic (Fig. 2). Inhibition of cell proliferation with mitomycin C treatment or addition of excess thymidine resulted in cessation of migration of keratinocyte colonies (Vasiliev et aL, 1993). In A-431 cells it was shown that the locomotion of colonies correlated with proliferative potential of all cells of the colony but the direction of migration did not depend on local cell proliferation (Samarova et al., 1997). Some data suggest integrated control of growth and migration in normal human keratinocytes. Several growth factors efficiently stimulate keratino-

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FIG. 2 Toluidine blue-stained migrating colony in primary keratinocyte culture on plastic. Scale bar = 100 pm.

cyte migration. Barrandon and Green (1987b) found that EGF and TGFLY added to a culture induce centripetal migration of marginal keratinocytes in colonies within 15 min. TGF-LY stimulated collagenase and gelatinase production by keratinocytes, was cultured on floating lattice of collagen, and enhanced invasion of basal cells into the collagen matrix (Turksen et al., 1991). Urokinase plasminogen activator binding sites were detected on the plasma membrane of human keratinocytes at the leading edge of the migrating epithelial sheet (McNeill and Jensen, 1990).Interaction of epidermal keratinocytes with uPA A chain at a concentration able to induce migration in vitro stimulates an increase in PA release into the culture medium (Del Rosso et d.,1990;Fibbi et al., 1990), suggesting that keratinocyte migration may be regulated by autocriny. The results obtained in A431 cells suggest that processes of proliferation and migration are closely related but may be partially dissociated. EGF stimulates cell migration and its effect may be potentiated by insulin, which is also a mitogen for keratinocytes. Mitomycine C treatment abrogated the epidermal growth factor effect, whereas basal-level migration, independent of mitogens, persisted (Vorotelyak et aL, 1997).

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D. Differentiation In cell culture conditionsepidermal keratinocytes retain their basic differentiation phenotype but the route of differentiation is different from that of normal epidermis and depends on culture microenvironments. Suspended keratinocytes lose the proliferative potential with a half-life of 3 or 4 h and later acquire markers characteristic of terminal differentiation (Rheinwald and Green, 1977). After 3 h in suspension all cells were capable of initiating colonies; virtually no colonies were detected when cells were seeded 8 or 16 h after suspension (Pittelkow et al., 1986).Selective detachment of differentiated keratinocytes from basement membrane proteins depends on the loss of ligand-binding ability of the PI integrins (Adams and Watt, 1990; Hotchin et aZ., 1993).Hotchin et al. (1995) studied the mechanisms by which integrin levels are controlled. In suspension cultures intracellular,transport of integrinsto the cell surfacewas blocked and the level of P1integrin mRNA declines and the receptors on the surface were endocytosed. During suspension-induced terminal differentiation of human epidermal keratinocytes, the asp1integrin is downregulated in two stages: (i) The ability of the receptor to bind fibronectin is reduced and (ii) the receptor is lost from the cell surface. Loss of as& from the cell surface reflects both inhibition of transcription of the subunit genes and inhibition of maturation and intracellular transport of newly synthesized subunits (Hotchin and Watt, 1992). Fibronectin inhibits the terminal differentiation of human keratinocytes (Adams and Watt, 1989).EGF and cholera toxin inhibit terminal differentiation resulting in a reduced multilayering and reduced formation of the cornified envelope (Jensen et aZ., 1985). Primary murine keratinocytes proliferate in low-calcium medium (below 0.1 mM) with limited spontaneous differentiation. When extracellular Ca2+ concentration is increased, keratinocytes cease proliferation and commence terminal differentiation (Hennings et aZ., 1980;Boyce and Ham, 1983;Yuspa et aZ., 1989). Using the intracellular calcium chelator BAPTA it was found that inhibition of keratinocyte differentiation marker expression may result from depletion of the calcium stores (Li et aZ., 1995). Culture environment is not sufficient to allow the full expression of the keratinocyte differentiation program. However, the complete differentiation program is recovered after transplantation, and human keratinocytes seem to posess an intrinsic sitespecific differentiation program that is maintained independently of the grafted body area (Compton et aZ., 1989; De Luca et aZ., 1990). E. Formation of Culture

Only a small fraction of trypsinized keratinocytes derived from normal epidermis could be identified as basal cells that constitute the proliferative

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pool; the rest of the cells are keratinocytes at different stages of differentiation. Thus, the colony-forming efficiency for typsinized keratinocytes ranged from slightly less than 1%for older donors to as high as 4% for newborns. The colony-forming efficiency of a subpopulation of basal cells might be more than four-fold higher than the average values. The trypsinized cells of subconfluent primary and secondary cultures formed new colonies with an efficiencyof up to 30% (Green et aZ., 1979). These data are in good agreement with the proportion of putative stem cells in keratinocyte cultures (Jones and Watt, 1993). When mouse keratinocytes were fractionated using Percoll density gradient centrifugation, the fractions containing basal cells had a plating efficiency of 70-85% on collagen-coated plastic (Gross et aL, 1987). When cell suspension is placed in culture flask moderate clumping occurs. It was also noted that aggregation of single cells before attachment to the substratum noticeably increased the number of subsequent epithelial colonies (Milo et aZ., 1980). Malcovati and Tenchini (1991) suggested that populations of keratinocytes contain a definite and constant proportion of clusterogenic cells and “gregarious” cells that attach to the clusters. Keratinocytes secrete a diffusible “spreading” factor and the percentage of spread keratinocytes shows an increase at increasing cell density (Malcovati and Tenchini, 1991). Within 1day of culture in the absence of growth factors, a striking reorganization of colony morphology is observed, namely, all single cells disappear and are replaced by different-sized aggregates of cohesive colonies. Keratinocytes form multicell clusters in the absence of cell division. The epithelial sheet formed in culture is a self-assembled structure and does not require the participation of any other cell types resident in the skin. Several days after inoculation small colonies are seen; they migrate, expand, and eventually fuse to form a single multilayered keratinocyte sheet. In the confluent sheet division continues in the basal layer but at a rate much lower than that in the growing colonies. Electron micrographs show that keratinocyte cultures resembling those of epidermis even when the state of differentiation is incomplete. Though the cells are flattened, the basal layer cells correspond most closely to the germinative cells in normal epidermis (Fig. 3). The surface adhering to the plastic support appears flat. Keratinocyte intercellular adhesion might be mediated by interaction of integrins a& and (Symington et aZ., 1993). Proliferation is largely confined to the basal layer of keratinocytes and cells undergo terminal differentiation as they migrate upward and eventually desquamate in the medium (Franzi et aZ., 1992). Upward migration of keratinocytes can be triggered at all points in the cell cycle with equal probability and is not restricted to those cells that already contain involucrin

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FIG. 3 Basal keratinocytes in multilayered sheet prepared for transplantation. Scale bar 0.5 Wm.

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=

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(Heenen et aL, 1992). The surface of the epithelial sheets in culture shows several lines of flattened cells which often maintain the nuclei in a variable state of degeneration. Melanocytes and Langerhans cells are present in primary keratinocyte cultures. Melanocytes are located among the basal keratinocytes and are surrounded by circular space separating them from keratinocytes. The culture lifetime of primary human keratinocytes is finite and, unlike rodent keratinocytes, human keratinocytes do not undergo transformation and do not spontaneously become established as cell lines (Green, 1977, 1978). Long-lived lines of human keratinocytes are rare (Baden et aZ., 1987). However, primary or low-passage keratinocyte cultures are a unique model system for oncogene studies which are highly valid with regard to in vivo situations (Dlugosz et aZ., 1995). Rockwell et aZ. (1987) found that calcium concentration of growth medium significantly influenced the culture growth period in vitro. In low-calcium medium cells underwent 35-40 population doublings over 16 or 17 passages, whereas cells grown in high-calcium medium ceased to proliferate after 20 population doublings over 7 passages. When cells are transferred from low-calcium conditions to high-calcium conditions they immediately limit proliferation and die within a few passages. On the other hand, when a population of cells that was destined to cease proliferation in high-calcium medium was transferred to low-calcium medium it recovered and continued to grow and divide. By starting with 1 cm2of newborn skin and using an inoculation density of lo4 cells, the area of cultured epithelium can be expanded to 0.6 m2 in 14-21 days. The increase in area (extention factor) is about 6OOO in 14-21 days. Because the epithelium shrinks after its detachment to about onefourth its area while attached, the overall expansion of epithelium might be 1500-fold. Subcultivation of pdmary subconlluent cultures might yield 3 m2 epithelium (Green et aZ., 1979).

IV. Transplantation of Cultud Keratinocyms

As methods of keratinocyte cultivation were developing, several attempts were made to cover skin wounds with cultured keratinocytes. However, earlier methods of keratinocyte cultivation were insufficient to produce large epithelial sheets required for grafting. The situation changed in 1975 when Rheinwald and Green developed a method of serial cultivation of human keratinocytes.

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A. Transplantation Technique Transplantation of autologous keratinocyte sheets to treat burns was used, although the technique had some limitations. The skin biopsy used for cultivation of keratinocytes should be taken as soon as possible after the injury and 3 or 4 weeks are required to produce epithelial sheets suitable for grafting. An alternative way is the use of allogenic keratinocyte sheets. Allografts offer some advantages: immediate graft availability, almost unlimited supply, and the possibility to store cultured cells in skin banks. Serologic control of donors must be conducted to rule out potential contamination of recipients with HIV, hepatitis B viruses, and other infectious agents. To date, allograft skin is the most frequently used and the most effective biological dressing. It formes the “gold standard” by which all other dressings are evaluated. Following observations that cultured keratinocytes do not express class I1 antigens (Morhenn et al., 1982) and are not clinically rejected in bum patients (Hefton et al., 1983; Madden et al., 1986), it was suggested that cultured allografts might be accepted permanently in unmatched recipients. Later it was shown that grafting of allogenic cultured keratinocytes does not lead to permanent but rather to slightly prolonged graft survival (Aubock et al., 1988). DNA fingerprinting of the wound covering epidermal cells taken after successful grafting with allogenic keratinocyte cultures showed that DNA originated from the recipient and not the donor cells (van der Merwe et al., 1990). Zhao and coworkers (1992) employed two methods to identlfy the presence of the cultured allografts on the wounds: (i) an indirect enzymeconjugated Staphylococcus protein A to detect A or B blood groups between donor and recipient and (ii) a polymerase chain reaction to ampllfy the Y chromosome-specificDNA sequence of samples taken from allografts on female patients who were grafted with cultured male keratinocytes. It was concluded that the survival time of cultured allografts was prolonged up to 35 days and that recipient tissue and the graft tissue combined to provide graft coverage of mixed host and donor cells. The technology of transplantation is the same for autologous and allogenic keratinocyte sheets. When confluent epithelial sheets are ready for grafting they are usually detached from the plastic surface with Dispase. They are then washed with serum-free medium and placed in petri dishes basal side up onto sterile vaseline gauze cut into suitable pieces. Then the dishes are transported to the bedside. Vaseline gauze was removed 10 days after grafting when the grafts began to form a stratum comeurn (O’Connor et al., 1981; Gallic0 and O’Connor, 1985). The dressing, wound bed, infection, and the provision of dermal component are factors influencing graft take. Dressing was studied by Carver et al. (1993). After grafting, occlusion

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was necessary for keratinocyte survival and attachment to the wound bed, but after this a semiocclusive or dry environment was important for the formation of differentiated epidermis (Fig. 4). However, this method has some inconveniences: the Dispase treatment, which is time-consuming and requires further steps in the laboratory; the necessity of having confluent multilayered epithelium ready for grafting; and accurate application of cultured epithelium to large areas requires substantial time. Nevertheless, the great advantage of the method is the provision of large areas of permanent wound coverage in patients for whom there are few other therapeutic options. In addition to wound coverage cultured keratinocytes inhibit bacterial growth (Maier et ale, 1992), which is in accord with the bacterial defense mechanisms of the skin. Currently, many burn centers employ transplantationof cultured keratinocyte sheets, but do so in various working environments and different facilities. Green et al. (1979) indicated that detachment of keratinocyte sheets by Dispase I1 had virtually no effect on the viability of cells. However, Boucher et al. (1991) studied the effect of storage of Dispase-detached cultures on proliferative potential and adhesion of keratinocytes. It was shown that the storage period of detached cultures must be as short as

FIG. 4 Cultured grafts were placed onto the thigh. Ten days after transplantation Paranet dressing was removed from low right graft. The graft appears as a thin wafer.

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possible to preserve the keratinocytes’ growth potential. It was also shown that in the absence of contact with basement membrane extracellular matrix epithelial cells lose correct tissue architecture and undergo apoptosis (Boudreau et al., 1996). Data indicate that keratinocytes synthesize and secrete urokinase-type plasminogen activator, which is bound in an autocrine manner to a specific receptor, uPA-R, bound to their surface. Experimental detachment with Dispase provides signals for the concomitant upregulation of uPA-R and PA (Schaffer et al., 1996). Urokinase plasminogen activators are among the most widely distributed cell surface proteinases. GrondhalHansen et al. (1988) showed overproduction of u-AP in skin wounds. There is a definite correlation between the increase in plasminogen activator activity and decrease in EGF binding resulting from receptor inactivation in A-431 epidermoid carcinoma cells (Gross et aZ., 1983).These data indicate why cultured epithelial sheets detached by Dispase have a limited shelflife in the fresh form. To facilitate the transfer of epidermal sheets from the culture flasks onto the wound bed Ronfard et al. (1991) cultured keratinocytes on a fibrin glue. Burns were grafted by simply placing the sheet onto the prepared site so that the cell layer was directly on the wound bed and the fibrin glue was on the outside. Two forms of cultured epithelium were used: 2-day-old cultures, with clones of 10-20 cells, and 10-day-old cultures, with one to four layers of cells. Both graftings were successful. The newly formed epidermis was fully differentiated and histologically normal after 1 year. To decrease the time before the cultured cells were available for transplantation Kaiser et al. (1994) covered debrided wounds with keratinocytes suspended in fibrin glue and overgrafted them with glycerolized cadaver skin. Hafemann et al. (1989) proposed a combination of in vitro cultured autogenic keratinocytes with allogenic split-thickness skin in the form of an intermingled graft. Cultured keratinocytes were scraped from the culture dishes with a rubber policeman, washed in PBS buffer, and centrifuged, and the cell pellet was used to fill holes, with a diameter of 4 mm, of the punched allograft. When allograft was rejected, spreading of autoepidermis as a fragile membrane in the form of a ring up to 3 mm wide was seen. To reduce the time required for cell growth and facilitate transfer of autologous cells to the wound bed, Burt and Clarke (1997) cultivated keratinocytes on a polymer film. The film discs with actively proliferating subconfluent cultures were inverted onto the wound. The lack of dermis seems to be a major problem in deep, full skinthickness burns. It was shown that human dermal fibroblasts facilitate the long-term maintenance of the reorganized epidermis after xenotransplantation of cultured human keratinocytes in immunodeficient mice (Inokuchi et aZ., 1995). Attempts have been directed toward the production of cultured

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or artificial derma substitute for combination with cultured keratinocytes (Cuono et al., 1987). Matouskova et al. (1994) cultured human keratinocytes on cell-free pig dermis and applied the graft with the keratinocyte layer facing the wound. Vasiliev et al. (1997) used allogenic keratinocytes cultured on microcarrierswhich were spread on the prepared wound bed. To improve grafting microcarriers were imbedded in collagen gel. Keratinocytes migrated from microcarriers and produced islands of epithelialization within 3 days. The search continues for the perfect technology of keratinocyte transplantation which might be immediately available and may give excellent cosmetic results with no wound contraction and scarring.

6.Clinical Application The first successful application of this technique was reported by O’Connor et al. (1981). Cultured autografts, prepared from small skin biopsy from burn patients, were placed on full-thickness wounds on the arms of two adult bum patients. Gallico et al. (1984) reported permanent restoration of skin in two children with burns covering 97 and 98%of the body surface (83 and 89% full skin-thickness burns). The reconstructed epithelium was stable for the first 3 years after grafting (Gallico and O’Connor, 1985). This technique was successfully used for much larger burn wounds and proved to be lifesaving. Transplantation of allogenic dermis covered with autologous keratinocytes and immunosuppression with cyclosporin A permitted replacement of large areas of burned skin at the earliest time possible (Krupp et al., 1994). Carter et al. (1987) used autologous cultured keratinocytes grown on collagen sponges for successful treatment of chronic facial erosions in three boys with junctional epidermolysis bullosa. By 1989 more than 200 burn patients at various centers throughout the world were successfully grafted with cultured autologous epidermis (Green, 1989). Allogeneic cultured keratinocytes were used for treatment of recessive dystrophic epidermolysis bullosa (McGuire et al., 1987) and nonhealing area on an adult patient with refractory ulcers (Beele et al., 1991). Two cases of toxic epidermal necrolysis (Lyell’s syndrome) were treated with cryopreserved cultured homologous keratinocytes which promoted extremely rapid reepithelialization of the lesions (48 h) and led to complete healing (Napoli et al., 1996). Sonlers et al. (1997) used cultured allogeneic keratinocyte sheets for treatment of chronic otorrhea. Cultured skin equivalent was used in 233 patients. It is responsive to individual wound conditions and thus acts as a “smart material” in the chronic wound (Sabolinski et al., 19%).

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C. Reconstruction of Skin

1. Reconstruction in V i m Several methods of three-dimensional cultivation of keratinocytes were used to modulate epidermal reconstruction that simulates the in vivo situation. PruniCras et al. (1983) cultured keratinocytes on dead deepidennized dermis (DDED) raised to the air-medium interface. After seeding of cell suspension on the DDED a new skin had formed with epidermis reminiscent of a normal in vivo epidermis. When 4-mm punch biopsy is deposited on DDED a well-differentiated epidermis develops which resembles normal skin. Differentiation markers such as involucrin and filaggrin were detected. The keratin profile is comparable to that observed in other skin culture models: 58-56-kDa and 48-kDa keratins were identified. After serum delipidization an increase in the amount of 67-kDa keratin was observed. Reconstitution of normal basement membrane zone-like structure could be seen, both hemidesmosomes and anchoring filaments were observed, and the presence of bullous pemphigoid antigen was revealed in the dermoepiderma1 junction (Basset-SCguin et al., 1990). Involucrin, transglutaminase, and filaggrin were expressed in spinous layer. Keratin 6, which is absent in normal epidermis, was expressed in reconstructed epidermis. Keratins 1 and 10were found to be expressed in reconstructed epidermis in the correct location. Air-exposed reconstructed epidermis reproduces to a high degree the lipids of the native epidermis, with the exception of higher triglyceride and lower glycospingolipid content and a very low content of linoleic acid. The differences in the expression of differentiation-specificprotein markers, as well as in the lipid composition, can be most probably attributed to the culture conditions used since on culturing freshly excised skin under the same conditions similar deviations from the noncultured tissue were observed (Ponec, 1991). In skin equivalent reconstituted epithelium is thicker than epidermis in vivo and basal keratinocytes lack typical cuboid form. Some changes in the topology of the expression of differentiation markers were described. Alterations in the distribution of actin, laminin, and bullous pemphigoid antigen suggest altered attachment of basal keratinocytes to the collagen lattice. Also, precocious expression of involucrin and delayed expression of 67-kDa keratin were observed (Asselineau et al., 1986).These findings suggest that air-exposed reconstructed human epidermis shares some common features with hyperproliferating epidermis. 2. Reconstruction in Vivo

Compton et al. (1989) and Compton (1992) studied reconstruction of skin in patients treated with cultured autologous keratinocytes transplanted to

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full-thicknessburn wounds excised to muscle fascia. Six days after grafting, a mildly hypertrophic flat epidermis with all strata was formed and the process of de novo formation of the dermal-epidermal junction had begun. Within 3 or 4 weeks formation of mature hemidesmosomes and the dermalepidermal junction was complete. Rete ridges regenerated from 6 weeks to 1 year after grafting. Anchoring fibrils appeared immature until 6-12 months after grafting. Hyperproliferationmarkers were strongly expressed up to a year postgrafting. Figure 5 presents reconstituted epidermis 4 weeks after transplantation of cultured keratinocytes. Normalization of keratinocyte differentiation occurred rapidly as judged by expression of involucrin and flaggrin. The subjacent connective tissue initially healed to form normal scar, but it remodeled dramatically,regenerated elastin, and resembled a true dermis within 4 or 5 years. Stabilization of the dermoepidermaljunction by transamidation of basal membrane zone was completed only 4 or 5 months after grafting. Collagen VII was identified as a substrate for tissue transglutaminase. Distinct regions on the central portion of anchoring fibrils were positive for monodansylcadaverine in normal skin which were negative during the initial phase of de novo forma-

FIG. 5 Reconstructed epidermis at l month posttransplantation of cultured keratinocyte sheets. Epidermis lacks rete ridges. Lamellar arrays of collagen bundles are oriented parallel to the overlying flat epidermis. Scale bar = 100 pm.

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tion of anchoring fibrils in regenerating skin (Raghunath et al., 1996). The reconstructed skin was physiologically comparable to standard meshed split-thickness autografts and more closely resembled normal skin than meshed split-thickness autograft interstice controls. Cultured keratinocytes possess a site-specificdifferentiation program which is expressed after grafting independently of the underlying connective tissue (Compton et al., 1989; De Luca et d., 1990). Normal histologic features of the reconstructed epidermis were maintained for years after grafting (Fig. 6). Transitory pathologic changes, including parakeratosis, dyskeratosis, and intraepithelial friction blister formation, were infrequently observed. No dysplastic or premalignant changes were found (Compton et al., 1989).

D. Cryopreservation of Keratinocytes To increase the ready supply of cultured allogenic keratinocyte sheets attempts were made to cryopreserve cultured epithelial sheets. The development of reliable methods of cryopreservation would allow banking epithelial sheets for long-term storage, increase the shelf life of grafts, and

FIG. 6 Electron micrograph of a basal keratinocyte in reconstructed epidermis 1 year after transplantation of autologous keratinocyte sheet. Dispersed chromatin, keratin bundles, and desmosomes are seen. Scale bar = 0.5 pm.

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provide ready access for burn victims when needed. May et al. (1988) developed optimal conditions for freezing and thawing of cultured human keratinocytes. The cryopreservation agent consisted of 10% DMSO in MCDB 153 medium with 0.15 M calcium, 10 ng/rnl hydrocortizone, and 0.4% v/v bovine pituitary extract. The optimum cooling-warming rate combination was -1"C/min cooling and +173"C/min warming with 73-91% maximum viability. Teepe et al. (1990) preserved sheets detached with Dispase in cryoprotective medium containing 7.5% DMSO, EGF, and 10% FCS. The initial cooling rate to subzero was -1"C/min. Then the cultures were cooled to -70°C and stored up to 6 weeks before grafting. After thawing, recovery rate of keratinocytes tested with Trypan blue exclusion and colony-forming efficiency were approximately 50% of the index values of the cells before cryopreservation. It was shown that the rate of healing of chronic skin ulcers with cryopreserved grafts was not significantly different from control. Epithelial sheets cryopreserved for 7 months significantly accelerated the rate of re-epithelialization of donor sites. The sheets were placed in MEM supplemented with 10% DMSO and 50% fetal bovine serum and were cooled at -1"C/min to -7°C before rapid cooling and storage in liquid nitrogen (Madden et al., 1996). It was also demonstrated that cultured human epithelium might be stored up to 2 years in MEM containing 10% FCS, 5% glycerol, and 5% DMSO (Roseeuw et al., 1991). Madden et al. (1996) cryopreserved cultured allografts by cooling the cell sheets at - 1"C/ min to -7°C before more rapid cooling and then storage in liquid nitrogen. Retention of approximately 92% of the original viability and no loss in basal keratinocytes were found. We cryopreserved keratinocytes cultured on microcarriers in DMEM:F12 medium containing 7% DMSO for 8 months with the retention of 80% viability. A simple model for the assessment of in vitro attachment efficiency of stored cultured grafts was described (Ghosh et al., 1995).

E. Gene Therapy The development of the gene transfer technique made it possible to introduce normal functional genes stably into mammalian cells and provided tools for a new approach to genetic disease treatment. Keratinocytes may be advantageous for gene therapy because they are easily accessible and grafting procedures are well developed and produce reconstructed epidermis. The grafts are easily monitored and removed if necessary. Proteins as large as apolipoprotein E (299 amino acids) secreted by keratinocyte grafts can traverse the epidermal-dermal barrier (Fenjves et al., 1990).In dogs it was demonstratedthat keratinocytes could be efficiently

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infected with a neo gene-containing retroviral vector and persisted for at least 130 days after transplantation into the skin donor (Flowers et al., 1990). Retrovirus-mediated gene transfer was used to introduce a recombinant growth hormone gene into cultured human keratinocytes. The transduced keratinocytes secreted biologically active growth hormone into the culture medium. When grafted as an epithelial sheet onto athymic mice, these keratinocytes reconstituted an epidermis that was similar in appearance to that resulting from normal cells. Human growth hormone could be detected in the blood of the mice at concentrations in the physiological range for more than 4 weeks (Morgan et al., 1987; Teumer et al., 1990). Jensen et al. (1994) found that differentiation of transfected keratinocytes was the main reason why human growth hormone was produced for a short time after transplantation of keratinocytes onto mice. There are indications that hemophilia B may be treated by gene therapy. Gerrard et al. (1993) introduced recombinant human factor IX cDNA into primary human keratinocytes by means of a retroviral vector. After transplantation of transduced keratinocytes onto nude mice human factor IX was detected in the blood stream in small quantities for 1 week. Page and Brownlee (1997) described preclinical model for gene therapy of hemophilia B. Factor IX was secreted by cultured keratinocytes transduced by factor IX-expressing retroviral vectors, and after grafting into nude mice human factor IX was detected in plasma for 4 or 5 weeks.

V. Concluding Remarks The development of human keratinocyte cultures had a great impact on biochemical as well as molecular-biological studies and provided techniques for covering skin defects such as burn wounds. Cultured keratinocyte grafting-the most advanced area of tissue engineering-is based on some fundamental events. Disaggregated keratinocytes can adhere to the culture substrates, polarize, and, after histogenic aggregation,form epithelial sheets suitable for grafting. Human keratinocytes in culture undergo “activation” but are not transformed, and after transplantation no neoplastic growth was observed. Epidermal stem cells survive and proliferate in culture, and they fully retain their potential to differentiate and to produce neoepidermis. Transplanted keratinocyte sheets adhere to the underlying wound bed and make tight contact that provides feeding of grafted cells. The wound provides the reparative environment necessary for proliferation and migration of grafted keratinocytes. Now the problem of keratinocyte cultivation is actually solved, but the “take” of cultured keratinocytes remains to be improved. The role of keratinocyte allografts in promotion of wound heal-

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ing must be investigated. In the near future xenotransplantation might be included in medical practice. Pigs could be especially good donors and breeds of pigs now exist that are free from certain known pathogens. The induction of immunological tolerance is an area of vigorous research, and advances are sure to be made. Coculture of keratinocytes with other cellular components of skin may contribute to the study of cutaneous immunological defense mechanisms. Skin substitutes reconstructed in vitro that exhibit morphological, biochemical, and functional features of the native tissue are of interst as in vitro model systems that enable extrapolation to the in vivo situation. Experiments on live-skin equivalents may contribute to our understanding of interactions of skin cells with extracellular matrix. Use of epidermal keratinocyte cultures is of great importance in the development of modern pharmacology at the molecular level. Cultured keratinocytes offer a unique and valid model of neoplasia research with regard to in vivo biology.

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O’Connor, N. E.,Mulliken, J. B., Banks-Schlegel, S., Kehinde, O., and Green, H. (1981). Grafting of bums with cultured epithelium prepared from autologousepidermal cells. Lancet 1, 75-78. O’Keefe, E. J., Payne, R. E., Russel, N., and Woodley, D. T. (1985). Spreading and enhanced motility of human keratinocytes on fibronectin. J. Invest. Dennatol. 85,125-130. O’Keefe, E. J., Briggaman, R. A., and Herman, B. (1987). Calcium-induced assembly of adherence junctions in keratinocytes. J. Cell Biol. 105,807-817. Page, S . M., and Brownlee, G. G. (1997). An ex vivo keratinocyte model for gene therapy of hemophilia B. J. Invest. Dermatol. 107,139-145. Pal, K., and Grover, P. L. (1983). A simple method for the removal of contaminatingfibroblasts from cultures of rat mammary epithelial cells. Cell BioL Int. Rep. 7,779-783. Peehl, D. M., and Ham, R. G. (1980a). Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium. In Vitro 16,516-525. Peehl, D. M., and Ham, R. G. (1980b). Clonal growth of human kertinocytes with small amounts of dialysed serum. In Vitro 16,526-538. Pietenpol, J. A., Holt, J. T., Stein, R. W., and Moses, H. L. (1990). Transforming growth factor pl suppressionof c-myc gene transcription:Role in inhibition of keratinocyte proliferation. Proc. NatL Acad. Sci USA 87,3758-3762. Pittelkow, M. R., Wille, J. J., and Scott, R. E. (1986). Two functionally distinct classes of growth arrest states in human prokeratinocytes that regulate clonogenic potential. J. Invest. Dermatol. 86,410-417. ponec, M. (1991). Reconstruction of human epidermis on deepidermized dermis: Expression of differentiation specificprotein markers and lipid composition. Toxicol. in Vitro 35,597-606. Potten, C. S. (1981). Cell replacement in epidermis (keratopoiesis) via discrete units of proliferation. Int. Rev. Cytol. 69,271-318. Price, F. M., Taylor, W. G., Camalier, R. F., and Sanford, K. K. (1983). Approaches to I Natl. . Cancer enhance proliferation of human epidermal keratinocytes in mass culture. . Inst. 70,853-862. PruniBras,M., Delescluse, C., and RBgnier, M. (1976). The culture of skin. A review of theories and experimental methods. J. Invest. Dermatol. 67,58-65. Pruni6ras, M., Regnier, M., and Woodley, D. (1983). Methods for cultivation of keratinocytes with an air-liquid interface. J. Invest. Dermatol. 8%28s-33s. Raghunath, M., Hoefner, B., Aeschlimann, D., Luethi, U., Meuli, M., Altermalt, S., Gobet, R., Bruckner-Tuderman, L., and Steinmann, B. (1996). Cross-linkingof the dermo-epidermal junction of skin regenerating from keratinocyte autografts: Anchoring fibrils are a target for tissue transglutaminase. J. Clin. Invest. 98, 1174-1184. Regnier, M., Pnmieras, M., and Woodley, D. (1981). Growth and differentiation of adult human epidermal cells on dermal substrates. Front. Matrix. Biol. 9,4-35. Rheinwald, J. G., and Green, H. (1975). Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells. Cell 6, 331-344. Rheinwald, J. G., and Green H. (1977). Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature (London) 265,421-424. Rockwell, G. A., Johnson, G., and Sibatani, A. (1987). In vitro senescence of human keratinocyte cultures. Cell Struct. Fund 12,539-548. Ronfard, V., Broly, H., Mitchell, V., Galizia, J. P., Hochart, D., Chambon, E., Pellerin, P., and Huart, J. J. (1991). Use of human keratinocytes cultured on fibrin glue in the treatment of burn W O U ~ S . B u m 17,181-184. Roseeuw, D., De Coninck, A., Neven, A. M., Vandenberghe, Y., Kets, E., Verleye, G., and Rogiers, V. (1991). Fresh and cryopreserved cultured keratinocyte allografts for wound healing. Toxicol. in Vitro 5,579-583. Rubin, J. S., Osada, H., Finch, P. W., Taylor, W. G., Rudikoff, S., and Aaronson, S. A. (1989). Purification and characterization of a newly identified growth factor specific for epithelial cells. Proc. Natl. Acad. Sci USA 86, 802-806.

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Saalbach,A., Aust, G.,Haustein, U. F., Hemnann, K., and Anderegg, U. (1997). The fibroblastspecific Mab A S 0 2 A novel tool for detection and elimination of human fibroblasts. Cell Tissue Res. 290, 593-599. Sabolinski,M. L., Alvarez, O., Auletta, M., Mulder, G., and Parenteau, N. L. (1996). Cultured skin as a “smart material” for healing wounds: Experience in venous ulcers. Biomaterials 17,311-320. Samarova,A. V., Vorotelyak, E. A., Vasiliev, A. V., and Terskikh, V. V. (1997). Cell proliferation in migrating colonies of cultured epidermoid carcinoma A-431 cells. Ontogenez (Russia) 28,288-292. Schaffer, B. M., Reinartz, J., Bechel, M. J., Inndorf, S., Lang, E., and Kramer, M. D. (1996). Dispase-mediated basal detachment of cultured keratinocytes induces urokinase-type plasminogen activator (uPA) and its receptor (uPA-R, CD87). Exp. Cell Res. 228,246-253. Shipley, G. D., Keeble, W. W., Hendrickson, J. E., Coffey, R. J., and Pittelkow, M. R. (1989). Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor. J. Cell. Physiol. WS, 511-518. Somers, T., Verbeken, G., Delaey, B., Duinslaeger, L., Govaerts, P., and Offeciers, E. (1997). Treatment of chronic postoperative otorrhea with cultured keratinocyte sheets. Ann. Otol. Rhinol. Laryngol. 106, 15-20. Staiano-Coico,L., Darzynkiewicz, Z., and McMahon, C. K. (1989). Cultured human keratinocytes: Discrimination of different cell cycle compartments based upon measurements of nuclear RNA or total cellular RNA content. Cell Tissue Kinet. 22, 235-243. Sun, T.-T., and Green, H. (1976). Differentiation of epidermal keratinocyte in culture: Formation of the cornified envelope. Cell 39, 511-521. Symington, B. E., Takada, Y., and Carter, W. G. (1993). Interaction of integrins ab and a b Potential role in keratinocyte intercellular adhesion. J. Cell Biol. 120, 523-535. Teepe, R. G. C., Koebrugge, E. J., Ponec, M., and Vermeer, B. J. (1990). Fresh versus cryopreserved cultured allografts for the treatment of chronic skin ulcers. Br. J. Dermatol. 122,81439. Teumer, J., Lindahl, A., and Green, H. (1990). Human growth hormone in the blood of athymic mice grafted with cultures of hormone-secreting human keratinocytes. FASEB J. 4, 3245-3250. Thompson, C. H., Rose, B. R., and Cossart, Y. E. (1985). Optimised growth of human epidermal cells in vitro without the use of a feeder layer or collagen substrate. Austr. J. Exp. B i d . Med. Sci. 63(2), 147-156. Tsao, M. C., Walthall, B. J., and Ham, R. G. (1982). Clonal growth of normal human epidermal keratinocytes in a defined medium. J. Cell. Physiol. 110, 219-229. Turksen, K., Choi, Y., and Fuchs, E. (1991). Transforming growth factor alpha induces collagen degradation and cell migration in differentiating human epidermal raft cultures. J. Cell Regul. 2,613-625. van der Merwe, A. E., Mattheyse, F. J., Bedford, M., van Helden, P. D., and Rossouw, D. J. (1990). Allografted keratinocytes used to accelerate the treatment of bum wounds are replaced by recipient cells. Burns 16, 193-197. Vasiliev, A. V., Smirnov, S. V., Ermolinsky, I. I., Zaikonnikova A. P., Samarova, A. V., Kiceliov, I. V., and Terskikh, V. V. (1997). Restoration of epithelial lesionsby transplanation of human allogenic keratinocytes. International-European A.I.R.R. Conference 1997 at Cologne, Germany. Vasiliev, A. V., Voloshin, A. V., Vorotelyak, E. A., and Terskikh, V. V. (1993). Migration of colonies of human epidermal keratinocytes in culture. Dokl. Akad. Nauk. (Russia) 329,232-235. Vorotelyak, E. A,, Samarova, A. V., Vasiliev, A. V., and Terskikh, V. V. (1997). The effect of epidermal growth factor on migration of A-431 cells. Zzvestia A N Ser. Biol. (Russia) 6,724-727,

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Watt, F. M. (1988). Epidermal stem cells in culture. J. Cell Sci Suppl. 10, 85-94. Woodley, D. T., Bachmann, P. M.,and OKeefe, E. J. (1988).Laminin inhibits human keratinocyte migration. J. Cell Physiol. 136,140-146. Yuspa, S. H., and Harris,C. C. (1974). Altered differentiation of mouse epidermal cells treated with retinyl acetate in vitro. Exp. Cell Res. 86, 95-105. Yuspa, S. H., Kilhenny, A. E., Steinert, P. M., and Roop, D. R. (1989). Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J. Cell B i d 109,1207-1217. Zhao, Y. B., Zhao, X. F., Li, A., Lu, S. Z., Wang, X., Huang, S. Z., and Zhuo, X. T. (1992). Clinical observations and methodsfor identifymg the existence of cultured epidermal allografts. B u m 1% 4-8.

Retinoids and Mammalian Development G. M. Morris-Kay* and S. J. Wardt *Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom; and ?Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom

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All vertebrate embryos require retinoic acid (RA) for fulfilment of the developmental program encoded in the genome. In mammals, maternal homeostatic mechanisms minimize variation of retinoid levels reaching the embryo. Retinol is transported as a complex with retinol-binding protein (RBP): transplacental transfer of retinol and its uptake by the embryonic tissues involves binding to an RBP receptor at the cell surface. Embryonic tissues in which this receptor is present also contain the retinol-binding protein CRBP I and the enzymes involved in RA synthesis; the same tissues are particularly vulnerable to vitamin A deficiency. In the nucleus, the RA signal is transduced by binding to a heterodimeric pair of retinoid receptors (RAWRXR). In general, the receptors show functional plasticity, disruption of one RAR or RXR gene having minor or no effects on embtyogenesis. However, genetic studies indicate that RXRa is essential for normal development of the heart and eye. Excess RA causes abnormalities of many systems; altered susceptibility to RA excess in mice lacking RARy or RXRa suggests that the teratogenic signal is transduced through different receptors compared with physiological RA function in the same tissue. KEY WORDS: Vitamin A, Retinoic acid, Mammalian embryo, Retinol-binding protein, Craniofacial, Limb, Placenta, Heart, Eye, Urogenital, Lung, Skeleton. o 1999 Academic Press.

1. Introduction

A. Vitamin A and Pregnancy 1. The Mammalian Embryo in the Context of Pregnancy The retinoids are a large family of natural and synthetic compounds related to vitamin A (all-trans-retinol). Although the vitamin A derivative retinoic International Review of Cytology, VoL 188 0074-76%/99 $30.00

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acid (RA) is the most significant retinoid for vertebrate embryogenesis, mammalian embryos also show a dependence on retinol for the maintenance of pregnancy. Therefore, when considering vitamin A and RA in embryogenesis, the embryo must be considered as part of a maternalplacental-embryonic unit. This is the approach taken in this review; our aim is to determine the roles of vitamin A and RA in the context of normal pregnancy and how retinoid-related nutritional and genetic defects cause congenital abnormality. Mammalian embryogenesis is initiated as a program of development controlled by information laid down in the cytoplasm and nucleus of the fertilized oocyte. During implantation in the wall of the uterus, an intimate relationship between the maternal and embryonic tissues is established; from then on, the maternal environment and the placenta actively contribute toward the events unfolding within the embryo itself. Vitamin A plays a unique role in mammalian development: It is essential for the well-being of the mother, for maintenance of the placenta, and as a source of RA within the embryo. F U , mainly synthesized within embryonic tissues, interacts directly with the embryo’s genetic control mechanisms and is essential for fulfillment of the genetically determined developmental program (Fig. 1). Clearly, this interaction between maternal factors and the embryonic genome requires the integrated control of supply mechanisms located in

FIG. 1 Diagrammatic representation of the interaction between retinoids of maternal origin and embryonic genotype. Both vitamin A deficiency (left) and retinoid excess (right) are associatedwith developmentalabnormality,through altering the normal relationship between cellular retinoid levels and the embryo’s genetic program for development.

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the maternal body, the placenta, and the embryo, Congenital abnormalities may result from failure at any level of this system. 2. Vitamin A Deficiency

Inadequate dietary intake of vitamin A among children in developing countries remains the leading cause of preventable illness and disability, including severe visual impairment and blindness, infection and death from diarrhea, and measles (Underwood and Arthur, 1996). Vitamin A deficiency (VAD) increases the likelihood of other complications in women and children, such as iron-deficiency anemia and poor maternal performance during pregnancy and lactation. Animal models have established that severe vitamin A deficiency results in early embryonic death, possibly due to placental failure. Less severe VAD, however, results in developmental malformations known collectively as the vitamin A deficiency syndrome which has been described in rat (Thompson et al., 1964; Warkany and Schraffenberger, 1946; Wilson and Warkany, 1947,1948,1949), pig (Hale, 1933,1935), quail (Dersch and Zile, 1993), and mouse (Morriss-Kay and Sokolova, 1996). The features of the VAD syndrome include cleft face, palate, and lip; small eyes; abnormalities of the urogenital system, especially of the kidneys; abnormalities of the heart and great vessels; and malformation of the forelimbs. All these defects have been described as human congenital malformations, commonly with two or more in association. Severe VAD is unlikely to be a significant cause of human malformations, but a moderate level of deficiency, combined with genetic defects, may be an underestimated cause of human abnormality.

B. Historical Perspective

1. Early Studies Early nutritional studies established that vitamin A, defined as a dietary substance soluble in fat and unstable in the presence of oxygen, was essential for normal growth, vision, and reproduction (McCollum and Davies, 1913; McCollum et al., 1922). Diets lacking vitamin A cause changes in the differentiated state of many tissues, especially epithelia (Wolbach and Howe, 1925). Experimental studies on the effects of vitamin A deficiency on embryogenesis (Hale, 1933,1935,1937) were initiated following observations of the effects of the depression on pig breeding in the United States. Pigs fed vitamin A-deficient diet before and during pregnancy gave birth to piglets with congenital anomalies of the eyes, face (cleft lip and palate), unascended kidneys, and undescended gonads. The most consistent effect

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was microphthalmia (small eyes), seen in all of the deficient young. This important observation is again topical, and the roles of vitamin A in eye development are now understood in some detail at the molecular level (see Section VI1,C). Vitamin A acetate was isolated in crystalline form in 1940 and was available in quantity from 1947 onwards (Moore, 1957), enabling experimental studies on the effects of vitamin A excess to be camed out as well as specific vitamin A supplementation of deficient animals. Prior to this, vitamin A excess and supplementation studies had used cod-liver oil. Oral doses of a cod-liver oil extract 10,OOO times the daily minimum administered to young rats, mice, and dogs caused alopecia, paraplegia, emaciation, fatty degeneration of the liver, kidneys, and heart, hemorrhage into the gut and lungs, and death within 2 weeks (Takahashi et al., 1925).This study, together with Wolbach and Howe’s observations on vitamin A deficiency published the same year, established that the differentiated state of tissues can be altered by both a deficiency and an excess of vitamin A. 2. Cell Proliferation, Differentiation, and Congenital Malformations Vitamin A excess was also found to inhibit growth of the long bones of young animals, and studies on the mechanism of this effect led to the discovery of its teratogenicity. The primary site of pathogenesis was found to be the epiphyseal cartilage, in which the matrix surrounding the differentiated chondrocytes lost its structure and staining properties. The effects of vitamin A on cartilage differentiation were studied both in vitro (Fell and Mellanby, 1952) and in vivo (Thomas et al., 1960). The loss of matrix was attributed to the labilization by retinol of the lysosomal membranes, which then released their hydrolytic enzymes into the extracellular material (Fell and Rinaldini, 1965). Although the involvement of gene expression in the vitamin A-induced release of proteoglycans from cartilage was not established until much later (Kistler, 1978), it was recognized at an early stage of the investigations that the effect was most pronounced when the chondrocytes were healthy, and that it was not associated with necrosis. The discovery that excess vitamin A causes congenital malformations was a chance observation during a study of its effects on bone growth in young rats (Cohlan, 1953). Giroud and Martinet (1955) showed that the effect on morphogenesiswas dependent on the stage of pregnancy (i.e., the stage of embryonicdevelopment) at the time of dosing. Stage dependency is one of the classic features of teratogenesis and has also been shown in the reverse context: If the diet of vitamin A-deficient rats is supplemented during the organogenesis period of pregnancy, embryos that would other-

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wise die will survive to be born malformed, but they are free of late organogenesis-associated abnormalities (Wilson et al., 1953). Retinoids, especially RA, can induce differentiation in a number of other systems. In teratocarcinoma cells, RA-induced endodermal differentiation has been a widely used model for monitoring RA-inducible gene expression (e.g., Hosler et al., 1989). In promyelocytic leukemia, in which the RA receptora (RARa) gene is rearranged, the therapeutic value of all-transRA is due to its induction of differentiation of promyelocytes to myelocytes (De The et al., 1995). Skin cancers, especially melanoma and basal cell carcinomas, also respond to retinoid therapy by undergoing differentiation (Pastorino et al., 1995). The use of synthetic retinoids in other aspects of dermatology has had a major impact on skin conditions such as severe nodulocystic acne and psoriasis (Peck, 1986). All these effects suggest that RA plays a fundamental role in controlling events during the cell cycle, affecting decisions to continue proliferation or to differentiate along a specific pathway. These decisions are a key feature of normal embryogenesis: In every developing organ, the exchange of information by molecular signaling between interacting tissues controls gene expression, which in turn either maintains the proliferative state or contributes toward a specific differentiation pathway. The teratogenic effects of retinoid excess and the congenital malformations caused by VAD demonstrate the potential of retinoids to influence developmental processes and the importance of keeping the supply of retinoids to the embryos within the normal physiological range. It was very disappointing that the teratogenic effects of RA had to be discovered anew in human pregnancy 30 years after Cohlan’s initial report, when 13-cis-RA (isotretinoin) was used as a treatment for cystic acne (Lammer et al., 1985).

II. The Retinoic Acid Signaling Pathway in Embryogenesis The requirement for RA within embryos is tissue-specific and stage-specific. One of the most interesting current lines of investigation involves the elucidation of tissue-specific mechanisms of RA synthesis. This is the end point of a sequence of events that begins with the maternal diet. In Sections 11-IV we will follow the retinoid pathway from maternal nutritional uptake through the stages of storage, metabolism, transport, and homeostasis of retinoids, transport across the placenta, uptake by the embryonic tissues, synthesis and turnover of the RA ligands, their activation of the nuclear receptors, and their transcriptional effects. The pathway is summarized in the scheme shown in Table I.

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TABLE I The Retinoic Acid Signaling Pathway in Mammalian Development

Nutritional sources of RA precursors Intestinal uptake and transport to storage sites

Retinyl esters (animal fats) and p-carotene (plant sources) Conversion to retinol in the intestinal lumen; uptake as retinol; conversion to retinyl esters in enterocytes; transport within chylomicrons

Storage of retinyl esters

Lipid droplets in stellate cells of liver and extrahepatic sites

Transport in maternal bloodstream

Retinyl esters are hydrolyzed to retinol and bound to retinol-binding protein (RBP) and transthyretin for transport A balance is maintained between blood retinol and stored esters to maintain blood retinol levels within the physiological range

Homeostasis

Uptake by the placenta

The RBP component of the complex binds to cell surface receptor, RBPr,retinol traverses the cell membrane into the cytoplasm

Transport within the embryo

Retinol is released from the placenta into the embryonic blood bound to embryonic RBP RBP binds to RBPr on certain embryonic cells; retinol enters cells AU-tram-retinoic acid is synthesized from retinol via retinaldehyde and catalyzed by specific dehydrogenases. Cytoplasmic RBPs may facilitate these conversions All-tram-retinoic acid is reversibly converted to 9-15sretinoic acid and other isomers

Uptake by specific embryonic tissues Synthesis of RA in embryonic cells

Isomerization of RA Activation of nuclear receptors

All-trans-retinoic acid and 9-cis-retinoic acid enter the nucleus, where they bind to and activate two families of nuclear retinoic acid receptors, RARs and RXRs

Transcription of target genes

Activated RARRXR heterodimers bind to RA responsive elements present within or near the promoters of certain genes, e.g., the 3’ €€ox genes Tissue-specificdistribution of machinery for retinol uptake and RA synthesis; tissue-specific distribution of nuclear receptors and controlled access of RA to the nucleus are probably all important in spatial regulation of signal transduction; localized RA catabolism may also be significant Direct and downstream effects of RA signaling affect the expression of genes involved in tissue interactions and hence morphogenesis,organogenesis,and pattern formation

Localization of the interaction of RA with the embryonic genome

Morphogenesis, organogenesis, and pattern formation

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A. Maternal Factors 1. Nutritional Sources of Vitamin A In birds and other egg-laying vertebrates, retinoids are supplied to the egg yolk during oogenesis and are responsible for its characteristic yellow color. In placental mammals, retinol is supplied continuously during pregnancy, requiring transport and uptake mechanisms. After birth, mammalian young continue to rely on the mother for vitamin A, which is an important constituent of milk. In monotreme and marsupial mammals, milk is the sole source of retinoids and other nutrients from much earlier stages of development. Most of the available information on nutritional sources of vitamin A is based on the human diet, and the following details are from McCance and Widdowson (1995). Vitamin A activity is derived from a variety of retinoids and carotenoids (provitamin A) found in animal and plant foods. Vitamin A potency, or retinol equivalent (RE) activity, is expressed in relation to the activity of 1pg all-trans-retinol.13-cis-retinol (commonly found with all-trans-retinol) and retinaldehyde (present in avian eggs and fish roe) have approximately 75 and 90% respectively, RE activity (Sivell et aL, 1984). Retinyl esters, predominantly as retinyl palmitate, have 87% RE activity. @-Carotene,the most common and most potent carotenoid, has approximately 16.7% (onesixth) RE activity; the other useful carotenoids are a-carotene and a- and P-cryptoxanthins, which have half the activity of p-carotene. The human recommended daily allowance for vitamin A (expressed as RE activity) is 1000 pg (United Kingdom) or 1500 pg (United States). Liver is a particularly rich dietary source of vitamin A: Calf liver contains around 30,000 pg RE activity per 100 grams, many times greater than other rich sources, e.g., egg yolk (535 pg) and butter (815 pg per 100 grams). Vegetable sources of carotenoids range from carrots (old ones can contain as much as 11,000 pg p-carotene equivalents, i.e., 1833 RE) to potatoes, which contain only a trace. Sweet potato and spinach are rich sources of carotenoids, with both possessing almost 4000 pg &carotene equivalents. In general, the amount of carotene in leaf vegetables is directly related to the amount of chlorophyll so that although cabbage contains on average 385 pg, the outer green leaves may contain 50 times as much as the inner pale ones. Although retinoids are specifically confined to animal food sources, plant-derived carotenoids are present in some animal-derived foods, especially milk and other dairy products. &carotene accounts for about 40% of human RE (Underwood, 1984). 2. Absorption and Storage of Retinoids

Physiological and biochemical aspects of vitamin A uptake and storage have mainly been studied in rodents. Retinyl esters are enzymatically con-

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verted to retinol in the lumen of the small intestinebefore being absorbed by the enterocytic lining. The hydrolase activity is derived from both pancreatic secretion and the enterocytic brush border (see Ong, 1994, for details). Retinol is absorbed much more efficiently than /3-carotene (37 and 3-6% respectively in a series of experiments by Huang and Goodman, 1965). Absorption of free retinol is carrier mediated; however, there is good evidence that a species-specificprotein component of milk forms a complex with retinol, making its absorption much more efficient (Said et al., 1988). This mechanism is particularly important in suckling animals, whose vitamin A requirement is higher than that of older animals. After absorption by the enterocytes, cartenoids are mainly converted to retinol via retinal, although there is also evidence for some RA formation (Napoli and Race, 1988).Enterocytes contain high levels of the cytoplasmic retinol-binding protein CRBP I1 (Ong, 1994). Unlike CRF'B I, which is expressed in the developing but not the postnatal intestine, CRBP I1 binds with equal affinity to retinol and retinal. Its affinity for retinoids correlates closely to their vitamin A activity, suggestingthat CRBP I1 may be functionally linked to the bioavailability of dietary retinoids. It is expressed specifically in mature, functional enterocytes of the absorptive region of the gut, with its level increasing during pregnancy and lactation. In rodents CRBP I1 expression begins a few days before birth, providing high levels of CRBP I1 protein before absorption of the first milk meal. Retinol is esterified within the enterocytes; the esters, together with any remaining carotenoids, are packaged into chylomicrons for transport to the liver via the lymphatic system. Carotenoids are stored in the body fat of many species, including human and rat, giving it a yellow color, whereas other species, including rabbit, do not store carotenoids in this way and so have white body fat. Retinyl esters are stored in fat droplets within specialized lipocytes (stellate or Ito cells) in the liver, accounting for 40% of their lipid content. The lungs, kidneys, gonads, and eyes are also significant sites of storage, but the liver is the major site (Wake, 1980). The major events of dietary uptake, absorption, and storage are shown in Figure 2.

FIG. 2 Absorption, transport, and storage of vitamin A. (A) Dietary intake. Dietary retinyl esters (RE)are hydrolyzed to retinol before absorption. Carotenoids are converted to retinol in enterocytes, where retinol is esterified and incorporated into chylomicrons together with triacylglycerol. Chylomicrons are taken up by the intestinal lymph vessels and transported to the systemic circulation; they are cleared mainly by the liver parenchymal cells. (B) Storage. In the liver parenchymal cells, retinyl esters are hydrolized to retinol which binds to retinolbinding protein (RBP).Most of the retinol in parenchymal cells is transferred to perisinusoidal stellate cells (lipocytes), which store retinyl esters in lipid droplets. RBP-retinol is secreted from the liver and most complexes with transthyretins in the plasma.

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3. Homeostasis and Transport In practice, human and nonlaboratory animal diets show wide daily and seasonal fluctuations in vitamin A content, and mechanisms for longterm storage and continuous homeostasis are vital components of retinoid physiology. The most important observations relating to these mechanisms are those showing that retinoid metabolism is regulated by the level of available retinoids. Stored retinyl esters are converted to retinol for circulation in the blood, in which retinol forms a complex with two proteins, retinol-binding protein (RBP) and transthyretin (Soprano et al., 1986). Metabolic studies have shown that blood retinol levels are maintained within a narrow range despite variations in dietary intake. When blood retinol levels fall, hydrolysis of stored retinyl esters restores the levels to normal (Napoli, 1993). This effect is mediated by control of the activity of lecithin acyltransferase (LRAT), which mediates conversion of retinol to retinyl ester: It is decreased to undetectable levels following vitamin A depletion but returns to normal when oral retinyl ester is given (Randolf and Ross, 1991). The binding protein CRBP I plays an important role in this process (Fig. 2; see also Section III,B,2). Administration of RA to the depleted animals leads to a more rapid return to normal LRAT synthesis and is more potent than an equimolar dose of retinol (Matsuura and Ross, 1993). These results suggest that although the goal of retinoid homeostasis is to maintain blood levels of retinol as a source for all tissues, the mechanism is mediated by RAresponsive gene expression. Although the precise nature of the pathway involved is not understood, it is remarkable that many of the genes encoding retinoid receptors (RAW, RARal, RARy, and RXRa) and many of those encoding RPBs have RA response elements in their promoters and hence depend on the presence of RA for their expression (Mangelsdorf et al., 1990; Soprano et aZ., 1993). During pregnancy, these mechanisms for retinoid homeostasis ensure that adequate levels of retinol are maintained in the maternal blood and are hence available to the embryo/fetus. Blood retinol levels are unaffected by dietary deficiency until the liver stores fall to very low levels (Ross and Gardner, 1994). Evidence from vitamin A deficiency studies in mice suggests that even when maternal blood levels fall, the embryo is privileged Female mice in our laboratory do not become sufficiently vitamin A deficient to produce congenitally abnormal young until after they have shown deficiency symptoms for weeks; furthermore, they only reach this state after their stores have been raided by previous pregnancy and lactation (Sokolova, 1996). These observations suggest that mechanisms of retinol uptake by the placenta are more efficient than those of other tissues.

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B. Transfer of Retinol from Maternal Blood to Embryonic Tissues 1. Transplacental Transfer Transplacental transfer of retinoids has been studied in humans, mice, rats, and monkeys (Creech Kraft et al., 1987,1988, 1989a,b;Kochhar et al., 1988; Satre and Kochhar, 1989; Ward and Momss-Kay, 1995; Tzimas et al., 1996). Accumulation of RA and its major metabolites within the embryo is rapid, with peak concentrations appearing 2-4 h after maternal administration. The rate of transplacental transfer is retinoid specific (measurements for alltrunsretinol, 13-cis-,9-cis-, and all-trans-RA and their glycuronoconjugates; Nau et al., 1996). However, vitamin A palmitate is not transferred (Geelen, 1972). These studies suggest that a specific uptake pathway exists for the transfer of vitamin A from the mother to the embryo. The placenta can metabolize some retinoids and is able to produce retinoids from maternally delivered precursors (Dimenstein et al., 1996). At early stages of rodent development the yolk sac (Fig. 3) is the primary placenta and is responsible for nutrient uptake (Freeman et al., 1981;Jollie, 1986). Maternal RBP-bound retinol is actively accumulated at the outer (maternal) face of the yolk sac placenta; this surface is composed of visceral endoderm, a highly endocytotic tissue responsible for nutrient uptake into the yolk sac placenta. The mechanism underlying retinol-RBP accumulation has been demonstrated to involve a cell surface receptor for RBP (RBPr) (BAvik et al., 1997; Ward et al., 1997). RBPr-mediated retinol uptake was first identified in the adult eye (BAvik et al., 1991,1992). The interaction between RBP and RBPr in the eye has been characterized both biochemically and by means of a monoclonal antibody that binds the RBP-RBPr complex (BAvik et al., 1991,1992). The same antibody detects an epitope in the rodent visceral yolk sac endoderm, whose credentials as a receptor have been confirmed by measurement of the kinetics of uptake by the visceral yolk sac of radiolabeled retinol from holo-RBP (Ward et aZ., 1997). Developingrodent embryos therefore acquire retinol from the neighboring maternal blood sinuses via interaction of maternal RBP-retinol with an RBPr in the visceral yolk sac endoderm. Expression of RBP receptors in the placental brush border of the human embryo has also been reported (Sivaprasadarao and Findlay, 1988). Once within the endodermal cells, retinol must be transferred to the yolk sac vasculature for transport to the embryo. This process depends on RBP synthesized in the endodermal cells (BAvik et al., 1996). The visceral yolk sac endoderm is a major site of BFW synthesis (Soprano et al., 1986; Sapin et al., 1997a). If synthesis of yolk sac RBP is inhibited by injecting antisense oligodeoxynucleotides into the exocoelomic cavities of cultured

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ctoplacental cone trophoblastic giant cells) rioallantoic membrane uture labyrinthinezone of placenta) ocoelomic cavity

h visceral yolk sac (outer endoderm, inner vascularised mesoderm) tal yolk sac (Reichert's membrane) FIG. 3 The maternal-embryonic relationship in early postimplantation stage rodent embryos (Day 9 mouse and Day 10 rat), showing the structures referred to in the text.

mouse embryos, embryonic uptake of retinol and production of RA is impaired (B5vik et aL, 1996; Ward et aL, 1997). Induction of RA deficiency by this method resulted in developmental abnormalities, some of which are characteristic of the VAD syndrome and have also been observed in mutant mice lacking two RA receptors, and some of which had not been observed previously. The onset of each of the malformations could be prevented by addition of RA to the culture medium of the embryos at the time of injection of antisense FU3P oligonucleotides. This technique allows RA deficiency to be induced suddenly at selected developmental stages, pinpointing the specific stages of morphogenesis during which different tissues rely on adequate RA levels for normal development: For example, cranial neural tube closure was prevented by inhibiting retinol uptake before, but not after, the five-somite stage. RBP synthesized by the endodermal cells is detected at the endoplasmic reticulum, where it can bind retinol ready for exocytosis; retinol is then released to the yolk sac circulation bound to visceral yolk sac-derived RBP (Ward et aL, 1997). Thus, embryonic accumulation of retinol depends on

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a relay of retinol from maternal RBP to RI3P synthesized in the yolk sac via interaction with a cell surface RBP-receptor (Fig. 4). 2. Vitamin A and Placental Function

There is good evidence that several aspects of placental function are dependent on retinoid activity, including control of estradiol production (Piao et al., 1997) and regulation of production of the peptide hormones chorionic gonadotrophin and placental lactogen (Chou, 1992; Stephanou and Hanwerger, 1995). During placental development, RA may help regulate trophoblastic invasion of the uterus through moderating the production of metalloproteinase inhibitors or the spatiotemporal pattern of expression of the connexins implicated in this process (Winterhager et al., 1996). It acts synergistically with epidermal growth factor on hCG secretion and transcriptionally regulates the EGF receptor (Roulier et al., 1994, 1996). Expression of the cytoplasmic RBPs (see Section 111) and of some nuclear retinoid receptors in human trophoblast cells also supports an involvement of the retinoids in chorioallantoic placental function (Roulier et al., 1994; Stephanou et al., 1994). Furthermore, the level of expression of some RA receptors varies during the differentiation of cultured cytotrophoblasts into syncytiotrophoblasts (Stephanou et al., 1994). Pregnancy can be maintained in vitamin A-deficient rats by maternal supplementation with RA alone up to Day 10, but after this stage some retinol is required (Thompson et al., 1964; Wellik and DeLuca, 1995). The onset of this physiological requirement for retinol coincides with the stage at which the chorioallantoic placenta replaces the yolk sac placenta as the functional site of maternal-embryonic exchange, suggesting that the placental site which specifically requires retinol is the syncytiotrophoblast. In contrast, the main site of retinol transfer to the embryo continues to be the yolk sac placenta even after formation of the chorioallantoic placenta (see Section III,B,2). The role of retinoids in placental structure and function is further discussed in Section VI,E.

111. Synthesis and Catabolism of Retinoic Acid in Embryonic Tissues A. A Mechanism for TissueSpecific Provision of Retinoic Acid As discussed previously, embryonic accumulation of retinol depends on synthesis of RBP in the visceral yolk sac endoderm. Distribution of retinol

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to developing tissues is also facilitated by RBP. However, with the exception of the visual process and spermatogenesis (Wellick et al., 1997), and possibly the chorioallantoic placenta, retinol has a low biological activity when compared with its metabolites all-trans- and 9-cis-RA. These active derivatives of retinol are the ligands for two closely related families of transcription factors, the retinoic acid receptors (RARs) and the retinoid “X” receptors (RXRs) (see Section IV,A). Distribution of the RAR and RXR subtypes is widespread throughout the developing embryo, suggestingthat an important aspect of their cell-specificregulation must lie in control of the availability of activating ligands. In principle, control at the ligand level could act through (i) site-restricted synthesis of RA from retinol, (ii) restriction of access of RA to the nucleus, and (iii) RA catabolism. There is experimental evidence for the existence of all three mechanisms. The hypothesis that production of RA is localized to sites in which it is required implies localized induction of the molecular machinery for the sequestration of retinol from the circulation and for its conversion to RA. Candidate proteins forming this machinery have been found to be colocalized in appropriate sites. RBPr has been detected in embryonic tissues by means of the same antibody by which it was localized to the visceral yolk sac endoderm (BAvik et al., 1997). As described previously, this cell surface receptor has been functionally demonstrated to mediate the cellular uptake of retinol from maternal RBP (Ward et al., 1997), and it seems reasonable to assume that it performs an equivalent function in the embryo with respect to embryonic RBP. Accumulation of retinol within embryonic cells has been demonstrated to occur selectively in cells that express CRBP I (Gustafson et al., 1993). CRBP I is a highaffinity intracellular RBP (Ong et al., 1994). Several retinol dehydrogenase (RoDH) enzymes belonging to the short-chain alcohol dehydrogenase family have been identified that are able to catalyze the first rate-limiting step in the production of RA (Chai et al., 1995a,b, 1996). An important feature of these enzymes is their ability to use CRBP I-bound retinol as a substrate (Napoli, 1996). The final step in generating RA involves

FIG. 4 Cellular uptake and metabolic channeling of retinol. Cell surface receptors for RBP facilitate retinol uptake into target cells by an unknown mechanism; retinol then binds to cellular retinol-binding protein (CRBP I) in the cytosol. It is then converted to retinyl esters for storage (lower left), transferred to the endoplasmic reticulum and bound to newly synthesized RBP ready for exocytosis (lower middle), or transferred to hydrogenases and converted to RAs (upper right). The RAs (mainly all-trans- and 9-&-) are transferred to the nucleus where they act as ligands for the nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs).

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the retinal dehydrogenases (RalDH), a family of enzymes that are able to convert retinal to RA (Bhat et al., 1995; Penzes et al., 1997). These events are summarized in Fig. 4. Immunohistochemical studies reveal that these proteins are in fact restricted to tissues that depend on vitamin A for normal development, including tissues previously identified as RA signaling centers (BBvik et al., 1997; Fig. 5 ) . One of these is the developing eye, which is one of the most sensitive organs to maternal vitamin A deficiency (Hale, 1933). VAD very early in development results in failure to form lens placodes (BBvik et al., 1996);similarly, agenesis of the lens (aphakia) has been reported in animals lacking some of the nuclear receptors necessary for mediating the retinoid signal (Lohnes et al., 1994). Induction of the lens requires contact between the optic vesicle, an outgrowth from the telencephalic region of the forebrain, and the surface ecoderm (see Gilbert, 1997, for a full account of the experimental basis of this statement). Immunohistochemical staining demonstrates the colocalization of RBPr, CRBP I, RoDH, and RalDH proteins in all cells of the presumptive lens placodes of early rat embryos (BBvik et al., 1997). RA activity has been detected in the developing lens by two transgenic mouse strains constructed to report RA activity during embryogenesis using a P-galactosidase gene linked to a RA response ele-

FIG. 5 Immunohistochemical localizationof CRBP I in the developing rat embryo. CRBP I staining is colocalized with staining for RBPr, RoDH, and RalDH proteins in each site (see text and Bfivik et al., 1997, for further details). (A) Dorsal ectoderm (de) and lateral mesenchyme (lm)of the limb bud (25-somite stage embryo); (B) lens placode (lp) (25-somitestage); (C) cardiogenic plate (cp) (3-somite stage). C, coelom; fb, forebrain, nf, cranial neural folds; op, optic vesicle. Scale bar = 50 pm.

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ment (RARE) (Rossant et al., 1991;Balkan et al., 1992). RA is also involved in differentiation stages of lens development: The yF-crystallin promoter contains an RARE (Tini et al., 1993). In summary, during the initial phase of eye development, molecular signals (as yet unidentified) from the epithelium of the optic vesicle induce synthesis of the molecular machinery for RA synthesis within the adjacent ectoderm cells. The newly synthesized RA initiates lens-specific differentiation by binding to and activating nuclear retinoid receptors which themselves activate the RARE of the yF-crystallin gene. RA continues to play essential roles in formation of the eye, including development of the retina and cornea (see Section VI1,C). The previous account of the role of RA in lens induction is one of many examples in which all the proteins of the RA synthetic machinery are localized in embryonic tissues known to be affected by VAD and by the loss of two or more alleles of the genes encoding RARs. Three examples are illustrated in Fig. 5. Comparison of the results of all three types of study enables us to develop a picture of the sites in which RA, derived from a maternal dietary precursor, is essential for the embryo to fulfill its genetically determined developmental program. A summary correlating the results of these separate approaches is presented in Table 11. Some of the correlations between VAD and retinoid receptor null mutant phenotypes will be considered in more detail later.

B. Cytoplasmic Retinol and Retinoic Acid-Binding Proteins

1. Nature and Functions of Cellular Retinol-Binding Proteins and Cellular Retinoic Acid-Binding Proteins Theoretically, once retinol is within a cell it could be stored, transported out of the cell (exocytosis), or produce RA to drive transcriptional regulation of target genes. Similarly, cells in which RA is synthesized, or imported from an adjacent tissue, could move it into the nucleus for transcriptional purposes, store it, export it, or catabolize it. The molecular machinery for controlling such events is thought to involve cytoplasmic-binding proteins, the cellular retinol-binding proteins (CRBPs) and the cellular retinoic acidbinding proteins (CRABPs). Current understanding of the contribution of the RBPs to site-specific RA activity comes mainly from in vitro biochemical studies; investigations of the effects of the absence of binding proteins have not been informative. The major physiological form of all the binding proteins is the liganded (holo-) form; the balance between holo- and apoforms of binding proteins within cells plays an important role in regulation of retinoid metabolism (Napoli, 1996).

TABLE II Correspondence between Embryonic Tissue Localization of the Machinery for RA Synthesis, Susceptibility to Vitamin A Deficiency, and the Effects of Null Mutation of Retinoid Receptors _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

Colocalization of CRBP, REtPr, RoDH, and RalDH proteins Extraembryonic/placental tissues Ectoplacental cone Extraembryonic ectoderm Extraembryonic endoderm

8

Dorsal (paraxial) mesoderm and vertebrae Somites Myotome Sensory placodes Eye Optic vesicle Lens placode Ear: Otic pit or vesicle Gut derivatives Gut endoderm Hepatic diverticulum Rathke’s pouch Trachea and lungs

~

~

~

When colocalization occurs [presomitic30 somite pairs (sp)] Presomitic-30 Presomitic Presomitic-30

8-12 25-30

~

____

~

VAD susceptible

____~

RAR/RXR mutants

Not reported Placental dysfunction evident, resulting in early termination of pregnancy

Not reported

Homeotic transformations: R A R q , RARaly, RAWZY

8-12 25-30 8-30 8-30 8-12 8-12 8-12

Not reported Not reported Not reported

Liver defects: RXRa Respiratory tract: RARa&, RARaPz+’-, RARa&

Diaphragm Septum transversum Pericardioperitoneal canal Pharyngeal arches 1st pharyngeal cleft 1st pharyngeal pouch Pharyngeal arch ectoderm Pharyngeal arch mesenchyme

8-30 8-12

J J

8-12 8-12 8-30 8-30

Lungs and hard palate affected; thyroid, parathyroid, and thymus unaffected

Heart Cardiogenic platelheart

1-30

J

Neurulation Notochordal platelnotochord Primitive streakhode

8-30 8-12

Limb bud Dorsal ectoderm Other tissues Surface ectoderm of body Mesenchyme of body wall Coelomic epithelium of urogenital ridge

25-30 25-30 8-30 25-30

Postulated centers of RA signaling

J Not reported Not reported

J

Diaphragmatic hernia: RARa&+’-, RARaP2

Skeletal malformations: RARa, RARy, RARalyl, RARc~lyc~2+’-, R A R q , RARPzy, RARaPz; cartilage malformations: RARa& RARalY, R A R q , RARalyaZ+’-;thymus malformations: RARalPZ, RAR& R A R q , RARaPz, RAR~IYCY~+’-. RAR&y, RARalP, RARalP2; thyroid malformations: RARa&, RARcYP~, RARalyaz+‘-, R A R q , RAR&y

Not reported

Abnormal limb shape: R A R q Urogenital malformations: RARa1P2, RARa&+‘-, RARaPz, RARalyaz+‘-, R A R q , RARPzy, RAR@, RXRP

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The RBPs were originally identified from cytosolic preparations of adult rat tissues incubated with radiolabeled retinol or RA (Bashor et aL, 1973; Ong and Chytil, 1975). It is now known that there are two different forms of each protein. The genes of all four binding proteins have been cloned from bovine (Levin et al., 1987), mouse (Perez-Castro et aL, 1989; Stoner and Gudas, 1989;Vaessen et al., 1989,1990;Wei et al., 1987),and rat sources (Shubeita et aL, 1987). They are all located on chromosome 9 in the mouse (Vaessen et aL, 1989; Wei et aL, 1987). This, and their structural similarity, suggests a relatively recent common evolutionary origin. CRBP I is a 15.7-kDaprotein (Sundelinet aL, 1985)which can be detected in virtually all adult rat tissues with the notable exceptions of the adrenal glands, ileal mucosa, and skin (Ong et aZ., 1982). It binds retinol in vivo with a high degree of specificity (Saari et al., 1982). During morphogenesis and organogenesis stages of mouse development, it is frequently coexpressed with the FL4-inducible RAW, suggesting an association with relatively high RA levels (Doll6 et aZ., 1990; Ruberte et aZ., 1991). CRBP I1 is a 16-kDa protein (Ong, 1984). The distribution of CRBP I1 in adult rat tissues is limited to the mucosal absorptive cells of the small intestine (Crow and Ong, 1985). Its function here may be to facilitate initial retinol uptake from the gut. It is not expressed during organogenesis, appearing for the first time in the fetal liver and gut on Day 15 of prenatal development in the mouse (H. Nakshatri and P. Chambon, personal communcation).CRBP I is therefore the main CRBP of interest in this review and will henceforth be written as CRBP. CRABP I also has a widespread distribution in adult rat tissues, but it is absent from adrenal glands, liver, and ileal and jejunal mucosa (Ong et al., 1982). CRABP I has both a similar molecular weight (15.5 kDa) and related amino acid sequence to CRBP (Sundelin et aL, 1985), yet has a very different ligand specificity, binding one molecule of RA. CRABP I specifically binds all-trans-RA in vivo (Saari et al., 1982). An additional CRABP protein (CRABP 11) was discovered in chick embryos (Kitamoto et al., 1988) and rat pups (Bailey and Siu, 1988), with a slightly greater molecular weight (16.2 kDa). CRABP I1 has a lower affinity than CRABP I for RA, with a 15 times higher dissociation constant (KD, 4.2 nM for CRABP I and 65 nM for CRABP 11) (Ong and Chytil, 1975; Bailey and Siu, 1988). It also differs from CRABP I in being RA inducible through a RARE (GiguBre et aL, 1990). CRABP I and CRABP I1 show overlapping and dynamic paterns of expression during mouse embryogenesis, suggesting that their roles may be both cooperative and complementary (Ruberte et al, 1992). 2. Cellular Retinoid Metabolism: A Role for CRBP Retinol is released to the cell either from storage via membrane-bound retinyl ester hydrolysis or from RJ3P-retinol-RBPr interaction. Release

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of retinol from storage is stimulated by apo-CRBP, which inhibits LRAT but stimulates bile salt-independent retinyl ester hydrolase activity (Napoli et aZ., 1991). Retinol is then able to bind CRBP, which protects it from oxidation and dehydrogenation. Holo-CRBP acts as a substrate for NADPdependent microsomal dehydrogenases which convert retinol to retinal (Napoli, 1993). Retinal is then transferred to cytosolic NAD-dependent retinal dehydrogenase to produce RA (Bhat et al., 1995;Penzes et al., 1997). Hence, in vitamin A deficiency, an increase in the apo-CRBP :holo-CRBP ratio would favor both retinol synthesis from stored esters and conversion of retinol to RA. In fact, vitamin A deficiency can induce CRBP synthesis: Culture of embryonic lungs in retinol-free medium results in higher CRBP levels compared with lungs cultured in medium containing physiological levels of retinol (Sokolova, 1996). CRBP may also be responsible for delivering retinol to newly synthesized RBP for exocytosis. An example of this process can be found within the endodermal cells of the visceral yolk sac, in which CRBP is expressed from the earliest stages analyzed (Sapin et al., 1997a). These cells have also been shown to accumulate radioactivity upon maternal administration of radiolabeled retinyl esters, suggesting that storage may occur in these cells in a similar fashion to that in the stellate cells of the liver (Johansson et al., 1997). The yolk sac endoderm continues to be the main site mediating transfer of maternal retinol to the mouse embryo and fetus throughout pregnancy: Accumulation of radioactivity in the yolk sac persisted until Day 18 postcoitum, with very little detectable in the chorioallantoic placenta. The expression patterns of the binding proteins have been described following both in situ hybridization and immunohistochemical techniques in developing amphibian (axolotl and Xenopus), chicken, and mouse embryos. In keeping with the mammalian theme of this review, we will refer only to the patterns observed in mouse embryos in the following sections. 3. Localized Generation of Retinoic Acid from Retinol in Embryonic Tissues

In situ hybridization revealed high levels of CRBP transcripts in the primitive streak region and allantois of the late presomite stage mouse embryo [Day 7.5 post coitum (pc)] (Ruberte et al., 1991). By Day 8 pc, CRBP can be detected in the neural epithelium, and by Day 8.5 or 9 pc, CRBP transcript levels are higher ventrally than dorsally within the neural tube. By Day 11.5, there is a clear association between CRBP expression and motor neurone cell bodies (Ruberte et al., 1993). During organogenesis, CRBP transcripts show a wide distribution, including the frontonasal mesenchyme, tongue, thyroid, thymus, tracheal and lung mesenchyme, gut

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epithelium and mesenchyme including the liver, endocardial cushions, walls of large blood vessels, epicardium, mesonephros, retina and lens, inner ear, olfactory pits, and the pituitary (DollC et al., 1990). Immunohistochemistry shows a more specific distribution of CRBP protein, localized within areas that are known to depend on vitamin A for normal development; furthermore, CRBP is colocalized with three other proteins involved in retinol uptake and RA synthesis: RBPr, RoDH, and RalDH (Bivik et al., 1997;Fig. 5). This study, carried out in morphogenesisstage rat embryos (presomite to 30-somite stages) provides convincing evidence that the synthesis of RA in embryos takes place at or adjacent to the sites in which it is used for transcriptional activation.

4. CRABPs in the Mouse Embryo: Distribution and Possible Roles

Northern analysis and immunoblotting have shown that CRABP I (Dencker et al., 1990, Vaessen et aZ., 1989) and I1 (Gigukre et al., 1990) are expressed at high levels in the mid- to late gestation stage embryo, but expression falls toward term. In the presomite stage mouse embryo (Day 7.5 or 8 pc), transcripts of CRABP I are detectable throughout the early mesoderm and allantois, persisting later in undifferentiated mesenchyme lateral to the primitive streak (Ruberte et al., 1991). By Day 8 pc, expression in the cranial mesenchyme becomes limited to the neural crest and crest-derived structures, e.g., in the first pharyngeal arch, frontonasal mesenchyme, and cranial ganglia; expression is also found in the neural epithelium, showing a dynamic pattern of expression in the developing hindbrain and midbrain and more restricted expression in the forebrain (Ruberte et al., 1992). At later stages of development (Days 10.5-14.5) CRABP I transcripts show a complex spatial distribution in the developing face (including tooth mesenchyme), dermis, areas delimiting the cartilaginous condensations, the mediastinal mesenchyme, dorsal mesentery of the stomach, endocardial cushions of the heart, walls of the large arterial trunks, nephrogenic mesenchyme, neural retina, and olfactory pit epithelium (DollC et al., 1990). In many of these sites it is complementarity to CFU3P transcript distribution. This complementarity is even clearer when the protein distributions are compared (Gustafson et al., 1993): During limb and craniofacial development, CRBP is synthesized mainly in epithelia and CRABP I in adjacent mesenchyme. These patterns suggest coordinated physiological roles for CRBP and CRABP I in tissue interactions occurring during development of these structures, which show sensitivity to both RA excess and VAD.

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5. The Function of CRABP Conflicting Evidence and Unanswered Questions The roles of CRABPs are less well understood than those of CRBP. Clarification of conflicting data is made more difficult by species differences in RA metabolism and utilization. For instance, 3,4-didehydro-RA is a potent retinoid in controlling skeletal patterning in chick wing (Thaller and Eichele, 1990), and 4-0x0-RA is important in Xenopus embryos, in which it has been proposed to modulate positional specification (Pijnappel et al., 1993). In constrast, mouse embryos and mammalian cell lines use RA as their major retinoid, and the different metabolic effects of CRABP binding of the different retinoids are less informative for understanding CRABP function in mouse than in nonmammalian species. It has been suggested that CRABP may play a role in delivering RA to the nucleus (Donovan et aL, 1995), but experimental evidence is more strongly supportive of the idea that CRABP I limits the transcriptional potential of RA. It is clear that when CRABP I is present in cells, RA binds to it with high affinity, and that degradation of RA by catabolic enzymes is enhanced when RA is bound to CRABP I (Napoli, 1993). CRABP I overexpression in F9 cells decreases access of RA to the nucleus (Boylan and Gudas, 1991). Ectopic expression of CRABP I in the lens, under the control of the aA-crystallin promoter, interferes with the development of the lens and lens fiber differentiation (Perez-Castro et al., 1993). Setting this information in the context of the observation that adjacent epithelial and mesencymal cells respectively synthesize CRBP and CRABP I (Gustafson et al., 1993), one can envisage a mechanism whereby CRBPmediated RA synthesis in an epithelium and CRABP-mediated sequestrationkatabolism in the mesenchyme enables quite different levels of RAmediated transcriptional activation in these adjacent tissues. Differences may also be generated in this way between different populations of cells within a differentiating epithelium (Fig. 6). Surprisingly,it appears that neither CRABP I nor CRABP I1 is essential for normal embryonic development or postnatal life under laboratory conditions. Double null-mutant mice (CRABP I and I1 null mutant homozygotes) have an apparently normal phenotype (Gorry et al., 1994; Lampron et al., 1995), apart from a partial supernumerary postaxial digit on one or both forelimbs that is also present in CRABP I1 null mice (Fawcett et al., 1995). Even more surprisingly, they show a normal, not an exaggerated, teratogenic response to exogenous RA. On the other hand, ectopic expression of CRABP I in transgenic mice results in poor postnatal development (Wei et al., 1992). To date, experimental studies have been unable to detect any differences in response to changes in retinoid levels through classical vitamin A deficiency or RA excess studies using the CRABP null mutants.

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G. M. MORRISS-KAY AND S. J. WARD apical cells (supporting cell line)

olfactory epithelium

middle cells (receptor cell line)

basal cells (stem cell line)

mesenchyme

FIG. 6 Semidiagrammatic representation of RA-related gene expression in the olfactory epithelium and subjacent mesenchyme in the 14.5-day mouse fetus. Differential expression in the three cell populations present within the epithelium and in the mesenchyme is consistent with the interpretation that RA is synthesized in the mesenchyme (the presence of CRBP I transcripts and the RA-inducible FtAR/3) and that lower levels of RA are present in the receptor cells (CRABP I transcripts) than in the supporting cells and basal cells (based on data from Doll6 et al., 1990, and Cushchieri and Bannister, 1975).

However,it may be that these studies do not reflect the normal physiological fluctuations that these proteins are usually controlling, and that outside of the relative protection of laboratory conditions mice lacking these binding proteins are more susceptible to fluctuation in vitamin A status. It is hard to believe that they are useless, given the clear correlation between the pattern of expression of CRABP I and tissues that are susceptible to RA excess. The expression pattern of CRABP 11, however, does not coincide precisely with RA-susceptibletissues (Ruberte et al., 1993,Lyn and Gigubre, 1994), and its developmental role is even less clear than that of CRABP I (Morriss-Kay, 1993). Despite the discouraging evidence from the CRABP double-knockout mouse phenotype, the hypothesis that CRABPs play physiologically significant roles continuesto be relevant to interpretation of new descriptive data. In the decidual tissue surrounding the early conceptus, the CRABP I and I1 genes are transcribed in the decidua before they appear in the embryo (Sapin et al., 1997a). The yolk-sac membranes express CRBP transcripts but not CRABP. This exclusivity may reflect the need to protect the early embryo from any free RA in the maternal blood passing through the sinuses close to the embryo. Clearly, regarding the developmentalfunction of the CRABPs, the co&cting information cannot be resolved on the basis of current understanding, but it is too early to dismiss them as redundant.

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IV. Nuclear Aspects of Retinoic Acid Signal Transduction A. Retinoic Acid as a Hormone-like Signaling Molecule Discovery of the nuclear RARs revolutionized the way in which the biological activity of vitamin A was viewed (Gigukre et al., 1987;Petkovitch et al., 1987). Their molecular structure and mode of action revealed that the nuclear component of the RA signaling pathway is equivalent to that of the steroidthyroid hormones, including vitamin D3, the RARs being members of the steroidkhyroid hormone receptor superfamily. Soon afterwards, a second structurally-related family of RA-responsive transcription factors, the RXRs, was identified (Mangelsdorf et al., 1990). The RXRs act as heterodimeric partners for RARs and for a number of other nuclear receptors, such as thyroid hormone receptors, vitamin D3 receptor, and peroxisome proliferator-activated receptors (Mangelsdorf et al., 1994; Mangelsdorf and Evans, 1995; Chambon, 1996). Retinoic acid receptors (RARa, RARp, and RARy) are activated by both all-trans-RA and 9-ck-RA, whereas retinoid X receptors (RXRa, RXRP, and RXRy) are activated only by 9-cis-RA (Mangelsdorf et al., 1994; Chambon, 1996). RARs bind cooperatively with RXRs on RAREs in the promoter region of target genes as heterodimers. RXRs have been shown in vitro to bind to certain DNA elements as homodimers, whereas RAR homodimers either do not bind to RAREs or have a weak affinity for them (Gudas et al., 1994). Each RAR and RXR gene produces at least two mRNA isoforms by alternative promoter usage and/or alternative splicing, leading to N-terminal variants of each isotype. These show considerable overlaps in embryonicexpression patterns and in functions (Subbarayan et al., 1997). Recent studies in F9 and P19 embryonal carcinoma cells have attempted to elucidate the degree of specificity of RAR and RXR types in controlling the expression of specific RA target genes. Synergistic activation of RAresponsive genes and induction of cell differentiation by an RAR selective agonist in combination with an RXR selective agonist in P19 and F9 embryonal carcinoma cells has revealed that although there is a significant degree of functional redundancy between the three RAR types, there is also some RA target gene-related specificity (Roy et al., 1995). A similar conclusion was arrived at by using F9 cells whose predominant RAR type, RARy, was absent (Taneja et al., 1995): In RARy-null F9 cells, most RA-inducible genes were not expressed; expression of most of them was restored by overexpression of RARa or RAW, but some were still not expressed, indicating that they specifically require RARy for RARE binding and

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transcriptional activation. The genes shown to have this requirement for RARy were Hoxal, Hoxa3, Tcj2, and Strud Furthermore, the transcription of these and other RA target genes, including CRABP ZI, Hoxbl, and RARp2, requires specific RARLRXR heterodimeric pairings (Chiba et al., 1997). In terms of understanding how these effects in cell lines may be applied to understanding RAR/RXR-related transcription in embryos, it is interesting to note that the two cell lines studied differ in an important respect: Only RARy can mediate the RA-induced differentiation of wild-type F9 cells, but both RARa and RARy can mediate differentiation of P19 cells (Taneja et al., 1996). These relatively simple systems are good models for the much more complex differences to be found within embryos. Genetic dissection of RAR function in mouse embryos has revealed a complex web of tissue-specificityand functional cooperation during embryogenesis.

B. Retinoid Receptors in Embryos The expression patterns of RARs and RXRs during mouse development have been described in detail (Ruberte et al., 1990,1991,1993;Doll6 et al., 1989,1990,1994;Mangelsdorf et uL, 1992). RARy shows a more restricted pattern of expression than RARa and RARp, in cartilage and precartilaginous cells from the sclerotome onwards, and in keratinizing epithelia (Ruberte et al., 1990). RARa is almost ubiquitous, but RARp is expressed in a diverse range of tissues. RXR expressionpatterns show less tissue specificity than RARs, particularly with respect to RXRa and RXRP; RXRy is present in the myogenic cell lineage from myotome stages onwards. Some tissues show clear correlations between expression of an RAR and an RXR, e.g., RXRyIRARP in the developing central nervous system, especially the caudate putamen of the brain and the ventral horn of the spinal cord (Doll6 et al., 1994). The assumption that these patterns reflect function has not been supported by analysis of null mutant mice lacking one receptor, but the phenotypes of these single knockout mice show some interesting features. For instance, the expression of RAR? in preossification-stage skeletal elements (Ruberte et al., 1990) is reflected in mice lacking RARy in the form of minor abnormalities of the upper cervical vertebrae and tracheal cartilages (Lohnes et al., 1993). The skeleton of these mice was otherwise reported as normal, suggesting that most of the functions of RARy in skeletogenesis can be taken over by another receptor in its absence. This receptor is likely to be RARa since loss of both RARy and RARa results in significant skeletal abnormalitiesinvolving the vertebrae, ribs, limbs, and skull (Lohnes et al., 1994).An intriguing finding in the RARy null mutants is that exposure

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to RA excess, which normally causes both vertebral and craniofacial abnormalities, has no effect on the vertebrae but induces the usual pattern of craniofacial defects. This observation indicates that RARy is essential for transduction of the teratogenic response to excess RA during development of the vertebral column, even though it is barely required for the normal development of this structure. It seems very odd that there is a genetic mechanism for transducing a damaging signal. There is a similar relationship between RXRa and RA-induced limb defects (Sucov et af.,1995; see Section VI). Double-RAR null mutant mice and RAR/RXR double mutants have been much more informative regarding the developmental roles of retinoid receptors (Lohnes et af., 1994; Mendelsohn et af., 1994; Kastner et af.,1995, 1997a; Ghyselinck et af., 1997). Analysis of RAFURXR double-null mutant phenotypes suggests that RXRdRAR heterodimers are the most common functional unit transducing the RA signal during embryogenesis (Kastner et af., 1997a). Some of the findings from these genetic studies will be described in the context of specific developmental systems in the following sections. Some spontaneous mouse mutations also have the potential to provide information on the development roles of RA signaling, but few of them are fully characterized genetically, limiting their usefulness. One example is the curly tail mouse, in which a neural tube defect phenotype is associated with a deficiency of RARy in the posterior neuropore; the defect can be partially rescued by maternal RA administration just prior to the onset of embryonic dysmorphogenesis (Chen et af., 1994, 1995).

V. Retinoic Acid in Craniofacial Development Recent studies indicate that RA is involved in two distinct aspects of craniofacial development: (i) patterning the hindbrain, including the neural crest cells that migrate from it to form the cranial ganglia and facial processes, and (ii) later development of the facial processes.

A. The Early Hindbrain and Neural Crest

1. Effects of Excess Retinoic Acid Evidence that RA plays an important role in hindbrain and neural crest cell development is well established from studies on retinoid excess (Morriss-Kay, 1993; Marshall et aZ., 1996) and has recently been confirmed in conditions of vitamin A deficiency in quail embryos (Maden et al., 1996).

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Neural crest cell migration is impaired in RA-treated embryos, and the migration pathways are abnormal. In the VAD quail, neither neural crest cell migration nor neuronal extension occur at all; these effects on neural crest emigration and neuroepithelial differentiation are almost certainly secondary to the effects on the hindbrain neuroepithelium. Except where stated, all the information in this section is derived from studies in mouse embryos. Before the molecular mechanism of action of RA was understood, the exposure of late gastrulation-stage rat embryos to retinoid excess was reported to cause shortening of the preotic hindbrain (Morriss, 1972). Understanding of this effect of RA, and of the role of RA in hindbrain development, has come through elucidation of the genetic control of hindbrain segmentation. The hindbrain consists of a series of seven segments (rhombomeres) and an unsegmented occipital region referred to as rhombomere (r) 8; the otic vesicle lies closely adjacent to r5. The zinc finger gene Krox20, which is expressed in r3 and r5, is crucially involved in the mechanism by which segmental boundaries are established between r2 and r3, r3 and r4, r4 and 1-5,and r5 and r6: Disruption of this gene results in the development of a hindbrain that is completely smooth from r2 to r6 (Schneider-Maunoury er aZ., 1993). Mouse homeobox (Hox)genes equivalent to the HOM genes of DrosophiZa (Akam,1989) show a colinear pattern of expression in the hindbrain, i.e., the rostral boundary of expression of each gene shows a sequential rostrocaudal position reflecting its 5’ to 3’ chromosomal position (Wilkinson et aZ., 1989). Within the hindbrain, the rostral boundary of each HoxB gene, from Hoxb2 to HoxbS, is two rhombomeres caudal to the gene 3‘ to it. Hoxbl (formerly Hox 2.9) does not follow this rule, being expressed only in r4 (Murphy et al., 1989;Wilkinson et al., 1989),whereas logically it should extend to the midbraidhindbrain boundary. It was therefore fascinating to observe that exposure of gastrulation-stage mouse embryos to excess RA resulted in the up-regulation of this gene in an expression domain extending from the midbrain-hindbrain junction to the rostral border of the otocyst (Morriss-Kay et aZ., 1991; Conlon and Rossant, 1992) (Figs. 7A and 7C). The r3 expression domain of Krox20 was abolished, and the r5 domain lost its clear segmental definition, reflecting the complete loss of morphological segmental boundaries from the preotic region (rl-r4). At early stages of neurulation, Hoxal is expressed in the same domain as Hoxbl, with a rostral limit corresponding to the future r3/4 boundary (Murphy and Hill, 1991); exposure to RA excess causes up-regulation throughout the entire embryonic ectoderm and mesoderm (Armstrong et aL, 1992). Although subsequent studies have shown that differences in concentration and timing of RA administration can induce different effects of RA on the hindbrain (Marshall et al., 1992, Wood er al., 1994; Figs. 7B and 7C),

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normal morphdogy. altered gene expression

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HoxW

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presomite stage RAtreated altered m q h o b g y , altered gene expression

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FIG. 7 Gene expressionin the hindbrain of mouse embryos on Day 9. (A) Normal hindbrain, indicating domains of RAR and RXR expression (left) and the rhombomere-specific expression of the HoxB genes and Krox20 (on the diagram); (B, C) effect of RA excess applied after (B) or before (C) the onset of somitogenesis on rhombomere morphology and gene expression (data from Ruberte et al., 1991; Wood et al., 1994; Doll6 et al., 1994).

the main point of these observations is that they indicate an involvement of RA both in the pattern of 3' Hox gene expression in the hindbrain and in the genetic mechanisms leading to morphological segmentation. They also reveal that RA excess affects only the most 3' HoxB genes, an effect that reflects the graded 3' to 5' effects of RA on upregulation of human HOX genes in embryonal carcinoma cell cultures (Simeone et al., 1990; Boncinelli et aZ., 1992). Some of these RA effects are mediated by RAREs. Hoxal was the first Hox gene in which the response to RA was shown to be functionally associated with an RARE (Langston and Gudas, 1992). This RARE is important for Hoxal regulation and is essential for the establishment of its rostra1 expression boundary in the hindbrain; however, it does not by itself mediate the Hoxal response to RA (DupC et aZ., 1997). Hoxbl is associated with two RAREs, a 3' one (homologous with that of Hoxal), which is essential for the response to RA excess, and a 5' one, which plays a role in sharpening the boundaries of the r4 domain in Hoxbl

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gene expression (Marshall et aL, 1996). A 3’ RARE has also been detected in Hoxd4, which is expressed in r7 and r8 (Popper1and Featherstone, 1993). 2. Vitamin A Deficiency and Hindbrain Development Recently, vitamin A deficiency has been shown to have complementary effects to those of retinoid excess: In the vitamin A-deficient quail embryo, shortening of the hindbrain is effected by compression of the otic and postotic regions, with concomitant loss of their morphological rhombomeric boundaries (Maden et aL, 1996). The most rostral parts of the Hoxbl and Hoxa2 domains of expression are lost in these embryos, and Krox20 expression is undetectable. These studies represent important additions to the evidence that RA is involved in regionalization of the hindbrain. Taken together, the studies on RA excess and VAD indicate that the rostral hindbrain (rl-r3) is unable to develop normally when RA levels are raised, and the caudal hindbrain (r4-r8) is similarly affected by low RA.

3. Retinoic Acid and Normal Hindbrain Patterning The observations discussed previously raise the question of how a physiological gradient of RA might be organized during normal hindbrain development. One possibility is that RA influences cell fate and gene expression during gastrulation, as the neurectoderm is determined. This hypothesis is consistent with the observation that exposure of the embryo to RA during gastrulation alters hindbrain patterning. Gastrulation is charaterized by the ingress of epiblast cells through the primitive streak and Hensen’s node to form mesoderm and notochord, respectively, and by the regression (caudal movement) of the streak and node as the embryonic axis grows in length. The neuroectoderm, which forms the neural tube and neural crest, does not pass through the node or streak but is induced continuously from mesodermal and notochordal tissue positioned just rostravanterior to the node as the node regresses. Hogan et al. (1992) have shown in mouse embryos that Hensen’s node is a site of RA synthesis and suggest that cells that leave the node early (i.e., those that form more rostral structures) have been exposed to RA for a shorter period of time than those that leave later (i.e., those that form more caudal structures). A second possible mechanism is that levels of available RA are differentially controlled within the hindbrain by the activity of the cytoplasmic REiPs. This hypothesis is not incompatible with the node-derived RA hypothesis and would act to maintain differential RA levels after the epiblast cells have lost contact with the node and differentiated as hindbrain neuroectoderm. In situ hybridizationstudies show that CRBP transcripts, which may indicate sites of RA synthesis (see Section III), are present throughout

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the presumptive hindbrain during prorhombomeric stages of development but are most abundant in the caudal hindbrain, CRABP Z levels are highest in the middle region of the hindbrain (future and actual r3-r6), possibly indicating lower availability of RA for transcriptional activation in this region (Ruberte et al., 1991). CRABP ZZ levels show a dynamic pattern of expression, with a specificallyhigh level in r4 and in the neural crest migrating from it (Ruberte et al., 1992). Although our ignorance of the developmental function of CRABP I1 protein precludes meaningful interpretation of these patterns, the coincidence between high levels of expression of CRABP Z, CRABP ZZ, and Hoxbl in r4 and its associated neural crest suggests some functional significance.

4. Retinoid Receptors in the Hindbrain The evidence that RA plays an active role in regional and segmentationrelated aspects of embryonic hindbrain development and in the induction of specific patterns of gene expression implies that retinoid receptors must be present to initiate signal transduction. We would therefore expect to find both RXR and RAR gene expression in the embryonic hindbrain. This is indeed the case (summarized in Fig. 7A). RXRa and RXRP are both present throughout the hindbrain (Doll6 et aZ., 1994). In Day 9 embryos, RARa is expressed in a domain extending caudally from r4, and RARP has a poorly defined rostral boundary within r7 (Ruberte et al., 1991), a pattern that is also shown by the RAR/32/P4 promoter (Mendelsohn et al., 1991). There is no detectable RAR expression within rl-r3, although low levels of RARa transcripts are detectable in the rostral hindbrain on Day 8.5. Hence, in normal Day 9 embryos, the rostral boundary of detectable RARa expression coincides with the rostral boundary of Hoxbl; the requirement for higher levels of RA in the caudal hindbrain is indicated both by its VAD sensitivity and by the expression of the RA-inducible RARP gene in r7 and r8. Failure to detect any RAR in rl-r3 suggests either that RA does not play an important role in this region or that signal transduction uses an RXR/RXR homodimer. The first possibility is supported by three observations: (i) RARaly double-null mutant embryos show an open rhombencephalon in which the open region does not include the most rostral part (Lohnes et al, 1994); (ii) rl-r3 are normal in VAD quail embryos, in which r4-r7 are lost (Maden et al., 1996); and (iii) neither Hoxal nor Hoxbl, both of which have RARE%,are expressed rostral to the future r3/r4 boundary at the stage when their expression domains are maximal (Murphy and Hill, 1991). This interpretation implies that the effects of RA on Hoxbl, Hoxb2, and Krox20 expression (Figs. 7B and 7C) are due to the duplication (Fig. 7B) or expansion (Fig. 7C) of the presumptive r4 tissue, in which RAR/

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RXR heterodimers are available for transducing the RA signal, at the expense of the presumptive rl-r3 tissue, in which RAR/RXR heterodimers cannot form.

B. Later Craniofacial Development 1. The Facial Processes The neural crest cells emigrating from r4 to r8 carry the same Hox gene code as their segment of origin (Hunt et al., 1991). Hindbrain-derived neural crest cells migrate into the face to form, together with the overlying ectoderm, the facial processes. The maxillary process of each side forms the maxillary region of the upper jaw and palate; the mandibular process forms the lower jaw; and the medial and lateral nasal dwellings, which are derived from the frontonasal mesenchyme and surround the olfactory placode, form the nose and premaxillary part of the upper jaw and palate. Failure of proper growth and morphogenesis of the facial processes results in clefts of the face and palate. Genetic control of development of the face is different from that of the hindbrain but also involves RA. Exposure of embryos to RA excess at later stages than those affecting hindbrain development causes cleft palate and shortening of the facial processes in rat embryos (Morriss, 1973) and shortening of the frontonasal and maxillary processes in the chick (Wedden and Tickle, 1988). Severe VAD causes midline facial clefts (Momss-Kay and Sokolova, 1996); these are also seen in RARdy double-null mutants (Lohnes et aZ., 1994). Mutant mice lacking the transcription factor AP-2 also show midline facial clefts (Schorle et al., 1996); AP-2 is expressed in cranial neural crest and may be involved in transcriptional regulation of RA-related morphogenetic events (Mitchell et aL, 1991). Midliie cleft face is also characteristic of homozygous embryos of the polydactylous mouse mutant Doublefoot ( D b f ) (Lyon et al., 1996): a RA signaling defect has not been shown in the face of this mutant, but an interaction between RA and the Dbfgene has been detected in the limb (Hayes, 1998; see Section VI,2). Many of the genes involved in facial development are common with those governing limb development (Winter, 1994), reflecting the observation that there are common features in the mechanisms of outgrowth of the facial processes and limbs, involving epithelial-mesenchymal interactions (Richman and Tickle, 1992). Recent studies aimed at identifying organizing centers in the facial processes equivalent to the zone of polarizing activity (ZPA) of the limb have shown that RA excess inhibits the expression of Sonic hedgehog (Shh) and its downstream target Patched (Ptc), but not Fg@, in the facial primordia of chick embryos (Helms et al., 1997) suggesting

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that the mechanism of the late effects of RA excess on the face may act through loss of Shh-inducedcell proliferation. Equivalent experiments have not been carried out on mouse embryos. 2. Osteogenesis and Chondrogenesis in the Skull Skeletogenesis of the skull is a complex combination of endochondral and intramembraneous bone formation; the two modes of ossification do not correspond to the mesectodermal and mesodermal origins of the skull bones. The neural crest origin of the facial region of the skull is well established for all vertebrae classes. Recent studies in chick embryos have suggested that the rostra1 skull base and most of the skull vault (frontal, parietal, and squamous temporal bones) is also neural crest-deprived, the mesodermal components being confined to the posterior part of the skull base and the occipital region (Couly et aZ., 1993). Alterations in skull morphogenesis in RARdRARy null mutants are not restricted to the neural crest-derived components, showing major alteration of the face, ear, skull vault, skull base, and occipital regions (Lohnes et aZ., 1994), These alterations are much less severe in mutants with a single allele of the RARa2 isoform (ie., RARa-’-/a2+’-/y-’-), suggesting that RARcu2 plays major roles in skull development. When assessing the reported abnormalities of skull development it is difficult to distinguish between patterning defects and defects of skeletogenesis, but it is clear that at least some are due to alterations in the balance between osteogenesis and chondrogenesis, e.g., the early loss of cartilaginous separation of the exoccipital and basioccipital bones in fetuses in which RARa and one or all RARP isoforms are disrupted (Lohnes et aZ., 1994; Ghyselinck et aZ., 1997). Altered skeletal patterns of osteogenesis and chondrogenesis in the vertebrae and ribs could similarly be due to differentiation rather than patterning defects. Abnormalities of the upper jaw, jaw articulation, and ear region have been particularly vulnerable to overinterpretation. In both Hoxa-2 null mutation and in various combinations of RAR double-null mutants, an ectopic cartilage links the incus with the alisphenoid; this has been interpreted as the atavistic formation of a reptilian pterygoquadrate cartilage, suggesting that RA-dependent mechanisms have been recruited during the reptilian-mammalian transition to modify some features of the reptilian skull (Rijli et al., 1993; Lohnes et aZ., 1994). However, null mutations are most unlikely to mimic earlier evolutionary stages and are certainly not associated with the presence of a pterygoquadrate cartilage in present-day reptiles. It is more likely that these ectopic structures result from RArelated alterations in the processes of tissue interaction, mesenchymal cell proliferation, and condensation that precede chondrogenesis (Smith and

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Schneider, 1998). Increased chondrogenesis in the skull with minimal changes in skeletal pattern can be induced in mouse embryos exposed to excess RA early on Day 10 of pregnancy (G. Morriss-Kay and S. Ward, unpublished observations), confirming that RA signaling plays important roles in determining skeletal differentiation as well as skeletal pattern in the skull, just as it does in the limbs. These two aspects of development are not always easy to distinguish when analyzing an abnormal phenotype.

VI. Retinob Acid and Limb Development A. Patterning the Limb Skeleton

1. Morphological Effects of Altered Retinoic Acid Signaling Only one observation has been reported of V A D effects on mouse limb development (Momss-Kay and Sokolova, 1996); the abnormalities were specifically of the forelimbs and included loss of digits and reduction in length of the limb in the most severe cases, as well as interdigital webbing and a supernumarary postaxial element in less severely affected fetuses. All these effects except the last have been observed in RARdy null mutants, including their forelimb specificity (Lohnes et al., 1994); the extra postaxial element appears to be identical to that of CRABP I1 null mutants (Fawcett et al., 1995). These observations provide strong evidence that RA signaling is essential for normal limb growth, differentiation, and patterning. Studies on RA excess are much simpler to carry out but do not necessarily provide information on the normal RA signaling pathway and its functions in limb development. Systemic exposure of the developing mammalian embryo to excess RA during the early stages of limb development results in limb skeletal malformations characterized by loss, reduction, or fusion of the digits and shortening of the long bones (Kochhar, 1973; Kistler, 1981).These effects represent alteration of both the anteroposterior (thumb to little finger) and proximodistal (shoulder to fingertips) limb axes. Limb bud outgrowth is initiated and maintained by fibroblast growth factors (Fgfs), mitogenic signals that are synthesized in the lateral plate mesoderm and apical ectodermal ridge (AER) (Niswander et al., 1993; Ohuchi et al., 1997). AER-derived Fgfs maintain the underlying mesenchyme (the progress zone) in a proliferating, undifferentiated state, thus maintaining limb bud outgrowth. Cell proliferation is significantly higher in the posterior than in the anterior limb bud mesenchyme, corresponding to the presence of Fgf4 in the posterior half of the AER, RA-induced reduction of digital number is preceded by rapid (within 5 h) down-regula-

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tion of Fgf4 and loss of the posteriorly biased cell proliferation rate (Hayes, 1998). The RA-inducible RARO214 promoter is up-regulated in the AER within 2 h of RA administration (Wood et al., 1996), suggesting that this tissue is a primary target of RA in these experiments. In the chick, local application of RA by means of an RA-soaked bead placed directly under the anterior margin of the developing wing bud has an apparently opposite effect on cell proliferation and skeletal pattern to that of mammals, resulting in the formation of supernumerary digits in a mirror-image pattern (Tickle et aZ., 1982). In these experiments, the RA bead mimics the effect of the ZPA, a discrete region of the posterior mesenchyme involved in anteroposterior patterning of the digits. This finding led to RA being hailed as a morphogen that emanated from the ZPA and diffused anteriorly across the limb mesenchyme. However, subsequent studies showed that the effect of RA is entirely local to an anteriorly placed bead and involves respecification of the RA-affected anterior cells so that they become “posteriorized” to ZPA-like cells (Wanek et al., 1991; Hayamizu and Bryant, 1992). RA-induced expansion of the limb bud prior to the formation of extra digits is associated with upregulation of the 5’ HoxD genes, which have been implicated in skeletal pattern formation (IzpistiaBelmonte et aL, 1992). Again, this is in contrast to the decreased domains of 5’ HoxD genes observed in the RA-exposed mouse limb (Wood et al., 1996); effects on the HoxD genes are likely to be secondary to the effects of Fgf expression. 2. Retinoic Acid and the Sonic hedgehog Signaling Pathway Understanding of the nature of the ZPA was revolutionizedby the discovery that this functionally defined mesenchymal domain coincided with the expression domain of Shh (Echelard et al., 1993; Riddle et al., 1993). Shh expression maintains, and is in turn maintained by, Fgf4 in the AER. Polarizing activity, as demonstrated by the ability to induce mirror-image duplication in the chick wing bud, was shown to reside at least partially in Shh; placing an RA-soaked bead in the anterior limb bud mesenchyme results in induction of Shh gene expression in the surrounding cells, confirming the local nature of the RA effect and showing that its action was to induce formation of a second ZPA. Although attempts to place RAsoaked beads in mouse limb buds have failed to induce digital duplication (Bryant et al., 1991), mouse mutants characterized by mirror-image-like polydactyly show that ectopic Shh expression is associated with anterior Fgf4 expression prior to the development of the extra digit-forming tissue (Masuya et al., 1997). Transplacental administration of RA to mouse embryos results in down-regulation of Fgf4 without any effect on Shh expression (Hayes, 1998).

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The Shh gene product remains local to its site of synthesis: Its active amino peptide, even when cleaved from the C-terminal domain, is anchored to the cell surface by cholesterol (Porter et aL, 1995,1996). RA and Shh are therefore both ruled out as candidates for a diffusible morphogen-like molecule, though both are clearly potent players in the mechanism of limb patterning. The new polydactylous mouse mutant, Db$ is beginning to clarify some of the apparently conflictingdata concerningRA, Shh, and limb patterning. Its gene product is a new component of the Shh signaling pathway which acts downstream of Shh and normally requires Shh for its activation; the mutant form of the Dbf gene product is constitutively active, converting the whole of the mesenchyme underlying the AER, from the ZPA to the anterior extremity of the AER, into one continuous ZPA-like structure without altering the expression of Shh (Hayes et aZ., 1998). RA excess does not alter the polydactylous phenotype of Dbf embryos but does cause shortening of the long bones, indicating that in wild-type RA-treated embryos the Dbf gene product mediates the effects of RA on the autopod but not its more proximal effects (Hayes, 1998). This may be because the proximal effects are due to effects on the control of skeletogenicdifferentiation rather than pattern, as discussed for the skull. Experiments using RA excess do not provide any information about the physiological role of RA in limb development. However, the recent discovery of RARE in the promoter of the zebrafish Shh gene (Chang et al., 1997) suggests that possible RA-Shh interactions should be investigated during normal anteroposterior limb patterning. It is also likely that the teratological effects of RA excess on limb development are transduced by a different pathway from the physiological effects. RXRa is not expressed at detectable levels in mouse limbs on Day 11.5 (the optimal time for RAinduced defects) (Mangelsdorf et uL, 1992; Doll6 et al., 1994, Sucov et al., 1995). Mouse embryos lacking RXRa have normal limbs but are resistant to RA-induced limb defects, indicating that although this receptor is not essential for normal development, it is required for transducing the teratogenic signal (Sucov et aZ., 1995). Control of dorsoventral (back of hand to palm) patterning in the limb bud depends on the early expression of Wnt-74 a mammalian homolog of the Drosophila wingless (wg)gene. Wnt7u is responsible for the correct expression of Shh (Parr and McMahon, 1995). The cellular machinery (binding proteins and enzymes) necessary for the production of RA has been detected specifically in the ectoderm overlyingthe prelimb bud region; at later stages it is restricted to the dorsal, proximal ectoderm, coinciding with the domain of Wnt-7u (Bzlvik et UL, 1997; Fig. 5A). It is an intriguing possibility that endogenously generated RA may influence the pattern of

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expression of Wnt-7a; the RA-responsiveness of this gene is not known, but the related gene mWnt-8 is RA-responsive (Bouillet et al., 1996).

3. Avian-Mammalian Differences The contrasting skeletal pattern effects of RA on chick and mouse limbs are consistent with the contrasting effects of the genes governing limb pattern: In the mouse, transplacental RA excess results in decreased digital number and down-regulation of genes implicated in cell proliferation and skeletal patterning; in the chick, local application of RA to the anterior aspect of the limb bud results in the formation of extra digits and upregulation of the same genes. It is not clear whether these species differences reflect physiological differences or are due to the different experimental approaches. Chick embryos are much larger than mouse embryos of equivalent stages and can be manipulated in ovo much more easily than mouse embryos in or ex utero; limb patterning occurs too late in relation to placenta formation for mouse embryo culture to be an option. A significant aspect of these species differences of RA responsiveness may be the different retinoids that are involved in signal transduction. 3,CDidehydroretinoic acid is a major retinoid in the chick limb (Thaller and Eichele, 1990) but is not synthesized by mouse embryos (Hogan et aL, 1992). There may be chick-mouse molecular differences in the RA-Shh relationship that do not enable RA to up-regulate Shh in the mouse limb as it does in the chick.

B. Limb Bud Outgrowth RA may play a role in the early initiation of limb bud outgrowth from the flank of the embryo (Cohn et aL, 1995; Crossley and Martin, 1995). Expression of the RA-inducible RARP2 gene is localized to the mouse embryonic flank before limb bud outgrowth and at higher levels in the limb bud regions than elsewhere during outgrowth (Mendelsohn et al., 1991). Administration of disulfiram, which inhibits RA synthesis, at pre- and early limb bud stages (stages 12-18) abolishes limb outgrowth in chick embryos (Stratford et al., 1997). Treatment with disulfiram also prevents expression of Shh and FgF 4 but has no effect on Fgf-8; local application of RA by bead implants into the limb region rescues normal limb development. Intriguingly, systemic (maternal) administration of RA to pregastrulation-stage mouse embryos results in supernumerary limbs (Rutledge et al., 1994; Niederreither et al., 1996). Most often, these arise as a pair of extra buds located caudally and ventrally to the normal hindlimb without duplication of the lower body axis. In accordance with the reversed polarity of these limbs, Shh and Fgf4 are expressed in the anterior margin of the ectopic buds.

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MI. Retinoic Acid in Other Developing Systems A. The Heart and Great Vessels

Aortic arch and cardiac abnormalities are common in congenital VAD (Wilson and Warkany, 1949).In rats, aortic arch anomalies can be prevented by supplementation of the dams prior to Day 10 of pregnancy (equivalent to mouse Day 9); however, defects of the heart itself, particularly interventricular septal defects, are not prevented by supplementation as early as Day 9 (equivalent to mouse Day 8) (Wilson et al., 1953). At this stage, the two heart tubes are just beginning to form around the rostral border of the neural plate and have yet to undergo fusion and descent to a ventral position. This remarkable observation indicates that RA is essential for the initial stages of heart development, abnormalityof which has irreparable effects on later development. CRBP, RElPr, and RoDH are all synthesized in the early cardiogenic tissue, indicating that it is a site of RA synthesis (Fig. 5C, B h i k et al., 1997). An abnormality seen only in the most severely affected VAD embryos was failure of the endocardial cushions to unite, leaving the atrioventricular canal undivided (Wilson et al., 1953). The presence of CRBP transcripts in the endocardial cushions (Doll6 et al., 1990) suggests that they are a site of RA synthesis. CRBP expression is also seen in the outer layer of the ventricular wall (Doll6 et al., 1990), reflecting both its pattern of growth and the fact that spongy myocardium is a common feature of VAD fetuses (Wilson and Warkany, 1949;Mahmood, 1992). Spongy myocardium and interventricular septal defects are also observed in some RARaly null mutants (Mendelsohn et al., 1994) and in RXRa null mutants (Kastner et al., 1994; Sucov et al., 1994). The histology of ventricular development in RXRa-’- fetuses shows an atrium-like structure (Dyson et al., 1995), although the persistent ventricular expression of the MLC2a marker described by Dyson et al. could not be detected by Kastner et al. (1997b). Study of the ontogeny of hypoplastic ventricle in embryos lacking functional RXRa and/or RXRP, RARa, or RAW, and in VAD embryos, indicates that all show premature ventricular myocytic differentiation as early as Day 8.5 (Kastner et aL, 1997b). The function of vitamin A early in heart development is clearly related to the inhibition of precocious differentiation, the maintenance of a high rate of proliferation, and the control of cell shape and cohesiveness in ventricular myocytes. A variety of abnormalities (loss or stenosis)of the aortic arches, especially affecting arches 4 and 6, have been observed in embryos and fetuses lacking RARa in combination with the lack of one or both alleles of RARp2 or with the lack of both alleles of RARy (Mendelsohn et al., 1994). Aortic

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arch anomalies were not observed in RXRa null mutants but were present in all fetuses lacking both RXRa and RARa, and in many of those lacking both RXRe alleles but heterozygous for RARa (Kastner et al., 1997a). Heart and aortic arch development is very complex. The observations on double-null mutants summarized previously suggest that different RXRd RAR heterodimers transduce the RA signal in different regions of the developing heart, with RXRe/RARa being the most important pairing. B. The Respiratory System

1. Lung Morphogenesis Evidence from VAD animals indicates that RA is involved in respiratory system development at all stages. In VAD rats, 6% of the offspring had a rudimentary lung or lung agenesis, mainly affecting the left lung; these abnormalities were prevented only when maternal supplementation was given at a developmental stage prior to outgrowth of the lung buds from the tracheoesophageal groove (Wilson et al., 1953). Recently, lung hypogenesis, again biased toward an effect on the left lung, has been reported in VAD mouse fetuses (Sokolova, 1996); the hypogenesis was correlated with a deficiency of airway branching. Branching morphogenesis can be enhanced by the addition of RA to lungs in organ culture (Schuger et aL, 1993). Throughout the period of lung morphogenesis, CRBP, which may indicate areas of RA synthesis, and RARP, which is induced by RA, are expressed in the mesenchyme and in the branching epithelium respectively (Doll6 et aZ., 1990). This pattern of expression suggests that RA may be involved in epithelial-mesenchymal interactions in lung morphogenesis. Mice lacking all isoforms of RAM have normal lungs (Luo et al., 1995; Ghyselinck et aZ., 1997),but mouse fetuses lacking both RARa and RARp2 show agenesis or hypoplasia of the left lung, together with hypoplasia of the right lung (Mendelsohn et al., 1994). RARa appears to be the most important RAR for lung morphogenesis since it is the only RAR disruption to be associated with lung hypoplasia in combination with RXRa disruption (Kastner et al., 1997a). The bias toward RA-related defects of the left lung in both VAD and double knockout studies suggests that RA signaling, in addition to playing an essential role in branching morphogenesis of the lungs, may be involved in the asymmetrical development of the normal respiratory system. 2. Lung Maturation

RA also plays an essential role in preparation of the lungs for their postnatal function. Both retinol and retinyl palmitate are detectable in the prenatal

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lung (Zachman et al., 1984), and vitamin A storage cells have been detected adjacent to pulmonary capillaries in near-term mouse fetuses (Sokolova, 1996). In rat fetuses, the concentration of retinyl palmitate in the lungs increases rapidly to a peak on Day 17 of pregnancy, then declines until just after birth, when the pups begin feeding (Masuyama et al., 1995). Retinoic acid administration stimulates lung surfactant phospholipid synthesis but inhibits synthesis of surfactant protein A (Chytil, 1996). Several studies on preterm infants have shown a correlation between vitamin A deficiency and severe bronchopulmonary dysplasia (Verma et al., 1996), but the claim is disputed by others (Chabra et al., 1994) and it is not clear whether retinoids will be able to provide a useful therapy for lung immaturity. 3. Metaplasia of Tracheobronchial Epithelium and Skin in V A D and Retinoid Excess

Retinoids continue to be essential for the maintenance of the differentiated phenotype of the epithelial lining of the respiratory system postnatally. Weanling rats fed a VAD diet quickly show metaplastic changes in the tracheal epithelium, which loses its mucous character and becomes keratinized (Wolbach and Howe, 1925). Similar effects have been reported in human infants with a low vitamin A intake (Bloch, 1921). The keratinizing effects of VAD in the tracheobronchial epithelium are mirrored by the effects of RA excess on developing skin. In mouse embryos, skin from 12day embryos placed in organ culture undergoes full mucous metaplasia when the culture medium is supplemented with 6 pg retinol per milliliter; there is a gradual loss of this plasticity during the next 4 days, and after the age of 16 days fetal skin is unaffected by retinoids (Hardy, 1992). C. Eye Development

Microphthalmia (small eyes) was one of the first reported characteristics of the congenital VAD syndrome (Hale, 1933). Other defects of eye development in V A D rat embryos and fetuses include postlenticular fibroplasia, coloboma (incomplete closure of the choroid fissure), folding of the neural retina, and defects of the eyelids and lens (Wilson et aL, 1953). These abnormalities were completely preventable by supplementation of the mothers beginning as late as Days 11 or 12, the early eye cupnens stage, suggesting that low levels of RA are sufficient for early morphogenesis of the eye cup and for lens induction and invagination but higher levels are required for subsequent growth and differentiation (the Day 11 or 12 rat embryo is equivalent to the Day 10 mouse).

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Cultured mouse embryos (Day 7.5 for 48 h), in which retinol uptake is inhibited by means of antisense oligodeoxynucleotides to RBP, show failure of the optic cup to induce a lens from the surface ectoderm (BAvik et aL, 1996); this effect can be prevented by adding RA to the culture medium, confirming that RA is required for early morphogenesis stages of eye development. As described in Section III,A, the presumptive lens ectoderm synthesizes the machinery for retinol uptake and RA synthesis (BAvik et aL, 1997). Complete anophthalmia has not been reported in congenital VAD, suggesting that the required level of deficiency is incompatible with pregnancy, or possibly even with life. The RA synthetic pathway uses different aldehyde dehydrogenases in the dorsal and ventral parts of the developing retina. The aldehyde dehydrogenase class 1isoform AHD-2 is present at high levels in the dorsal retina from lens induction stages onwards (McCaffery et uL, 1992). In the ventral retina, RA synthesis uses an acidic dehydrogenase, which is first detectable at the optic pit stage, a day before the first appearance of AHD-2 (McCaffery et al., 1993). Citral, a competitive inhibitor of aldehyde dehydrogenases, specificallyaffects the acidic dehydrogenase;treatment of zebrafish embryos with citral during the period of formation of the eye primordia decreases the production of RA in the ventral retina, leading to formation of eyes lacking a ventral retina (Marsh-Armstrong et aL, 1994). Reduction of the ventral retina is also seen in RXRa null mutant mice, the effect first becoming apparent at Day 11.5 (Kastner et ul., 1994). More severe reduction is present in RXRa+’-/RAR-’- double mutants, the ventral retina of RXRarlRARy mutants being virtually absent; the effect was at first thought to be specific to RXRa, but it also occurs in RARpI?I mutants (Kastner et al., 1997a; Ghyselinck et aL, 1997). Nevertheless, the greater severity in double mutants that include RXRa disruption suggests that this receptor may be particularly important for RA signal transduction in this site, and that the function of the unusual acidic aldehyde dehydrogenase detected in this region by McCaffery et al. (1993) may be to synthesize 9cis-RA. Lack of RXRa, RAR@/y, or RARPIy is also associated with other ocular anomalies including failure of the lens to separate from the (thickened) cornea to form the anterior chamber, failure of development of the sclera, the presence of a retrolenticular membrane, coloboma, and deficient eyelid development (Lohnes et aL, 1994; Kastner et aZ., 1997a; Ghyselinck et aL, 1997). Many of these abnormalities of the anterior chamber resemble VAD effects; they may be secondary to effects on the neural crest cells that contribute to this part of the eye. At neonatal and postnatal stages, effects on differentiation of the neural retinal are observed in RARp2/y2 mutants that are secondary to defects in the pigmented epithelium and/or periocular mesenchyme cells (Grondona et al., 1996).

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The investigations discussed in this section and in Section II1,A span eye development from lens induction to postnatal stages; the involvement of vitamin A in the visual pathway is well known. Although the involvement of retinoids in development, growth, differentiation, morphogenesis, and physiological function of other organs may be just as complex as those of the eye, this organ is clearly exceptionally sensitive to alterations of retinoid signaling throughout life.

D. The Urogenital System Development of the metanephros, the definitive mammalian kidney, is initiated when the ureteric bud grows out from the caudal part of the mesonephric duct into the adjacent metanephricmesenchyme. Tissue interactionsresult in branching of the ureteric bud epithelium to form the calyces and collecting ducts of the kidney, whereas ampullae at the tips of the later branches induce the mesenchyme to form nephrons, a process intimately involving blood vessels that invade the mesenchyme. These processes can fail completely, resulting in kidney agenesis, if the ureteric bud fails to make contact with the metanephric mesenchyme, or partially, if the epithelial-mesenchymal interactions and/or vascular involvement are impaired. RARs and RBPs show clear differential patterns of expression in the developing urogenital system, apart from RARa, the expression of which is apparently ubiquitous (DollC et al, 1990). In the developing metanephros, RAM transcripts are abundant in the cortical mesenchyme, where induction of new nephrons is taking place, and in the pelvis and ureter. The kidneys of R A R d P null mutant mice initiate apparently normal branching morphogenesisbut on Day 18.5 show a hypoplastic nephrogenic zone, often associated with hydronephrosis and ureteric abnormalities (Mendelsohn et al., 1994). These kidneys are also ectopic, failing to ascend from the pelvic region to their normal final position in the lumbar region; this is the only kidney abnormality seen in the RAR mutants which has previously been observed in VAD syndrome (Wilson et al., 1953). RARaly mutant mice show complete kidney agenesis, apparently due to failure of development of the caudal part of the mesonephric duct and hence the absence of the tissue from which the ureteric bud originates. This effect probably reflects the importance of RARa since RXRcr/RARa double mutants are the only RXWRAR combination to show kidney agenesis (Kastner et al., 1997a). RARy expression is not significant in the developing urogenital system except for the distal phallus (DollC et al, 1990);it is absent from the urogenital sinus epithelium but has not been investigatedin the caudal mesonephric duct or early ureteric bud.

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The genital ducts also require specific RA signaling pathways for their normal development. Male genital duct development is affected by failure of formation of the caudal part of the mesonephric (Wolffian) ducts in RARaly mutants; the seminal vesicles and caudal vas deferens, which derive from this part of the duct, are absent; the cranial part of the duct does form, but its derivatives, the epididymis and rostral vas deferens, are either absent or abnormal (Mendelsohn et aZ., 1994). In RXRdRARa mutants, the Wolffian ducts fail to become incorporated into the dorsal wall of the urogenital sinus (Kastner et al., 1997a). Complete agenesis of the female (Miillerian) duct system was observed in RARalP2 null mutants; the absence of the uterus and cranial vagina was observed in RARalIP2 and aly mutant females. These abnormalities have also been observed in VAD rats (Wilson and Warkany, 1948). Detailed analysis of the urogenital system abnormalities in RAWRAR and RXR/RAR double-null mutants indicates that RA signaling is involved at multiple developmental stages of these structures; there is much promiscuity of RAR function, but in some sites there is a clear requirement for a specific receptor, e.g., RARa in the cranial part of the Miillerian duct. Surprisingly, retention of both male and female duct systems (pseudohermaphroditism) has been observed in VAD rats but not RAR/RAR or RXWRAR mutants, possibly indicating involvement of an RXR/RXR homodimer.

E. The Chorioallantoic Placenta The yolk sac placenta, which is the functional organ for maternalembryonic exchange during early rodent development, is equivalent to the yolk sac of avian and reptilian embryos. It is the trophoblast, a specifically mammalian tissue, that has made intimate maternal-fetal exchange possible over longer periods of gestation though formation of the chorioallantoic placenta. The trophoblast also stores carbohydrates and lipids and synthesizes hormones and growth factors that are involved in the maintenance of pregnancy and fetal growth. In the mouse, the chorioallantoic placental circulation, which lies within the labyrinthine zone of the placenta, is functional at 12.5 days (Muntener and Hsu, 1977). As discussed in Section 11, C, 2, yolk sac placental function can be maintained in V A D animals by supplementation with RA, but normal chorioallantoic placental function specifically requires retinol (Thompson et al., 1964; Wellick and DeLuca, 1995). There is no evidence that the trophoblast uses retinol directly; it is more likely that the lack of a transport mechanism for RA limits its ability to enter the syncytiotrophoblast and traverse it to reach the fetal blood vessels.

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RXRa is essential for normal structure and function of the murine chorioallantoic placenta. This receptor is expressed within the trophoblastic giant cells and in the labyrinthine trophoblastic zone (Sapin ef af., 1997a). Mice with a targeted disruption of the RXRa gene show abnormality of placental tissue structure from Day 12.5 onwards, and by Day 16.5 almost all fetuses are dead (Sapin ef af, 1997b). Pooling of blood in the RXRa-’- placenta may be partly due to the myocardial defects referred to previously (Kastner et aL, 1994, see Section VII,A), but since the problem is mainly on the maternal side of the placenta, this seems unlikely. The progressive structural defects seen in these placentas are similar to those of VAD placentas (Howell et af., 1964; Noback and Takahashi, 1978). RXRa may heterodimerize with RARa and/or RARy, which are also present; the prenatal death of RXRa null mutants and RARdRARy double-null mutant fetuses (Lohnes ef af., 1994; Mendelsohn et af., 1994; Kastner et al., 1994) confirms the essential nature of these genes for placental function and limits the extent to which the development of the mutant phenotypes can be analyzed.

VIII. Conclusions A. Retinoic Acid Signaling and Embryonic Phenotype One of the most important aspects of recent studies on retinoid functions during embryogenesis is how detailed analysis of the phenotypes of knockout mutant mice, especially those lacking specific RAR or RXR receptors, have shed light on observations on vitamin A deficiency made many decades ago. An increasingly clear understanding of how and where vitamin A is used by embryos is emerging from these investigations. In particular, we know that although there are three RAR and three RXR nuclear receptors, most of the functions of RAM can be taken over by the other two RARs in its absence, and that RXRa is the only RXR that is indispensible for development (Kastner et aL, 1997a). In contrast to this detailed information on the roles of the receptors, there is still a significant gap in our knowledge linking the molecular events of RA signal transduction to the cell behavioral events of proliferation, morphogenesis, and differentiation that produce the phenotype. Although a large number of genes have been identified that are RA inducible, little is known of the normal chain of events within an intact embryonic tissue that lead from RA to, e.g., lens induction. In the hindbrain and the limb, the effects of RA excess have been described in detail in terms of altered gene expression and altered morphogenesis, but the physiological roles of RA in these embryonic structures can only be inferred.

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Observations on some of the systems described previously suggest that differential physiological levels of RA, controlled within specific tissues, are essential to maintain cell proliferation and to specify the timing of cell differentiation (e.g., of ventricular myoblasts to myocytes) and to define the choice of differentiation pathway (e.g., of epithelial cells to mucussecreting goblet cells or keratinocytes). These are fundamental developmental decisions that occur repeatedly during ontogeny. Although studies on VAD embryos and fetuses have shown RA to be essential for normal cell proliferation and differentiation, it is likely that in most or all of the VAD-sensitive tissues, normal development involves interactions between RA signaling and other signaling pathways. FGF signaling is particularly interesting in this respect. Its roles in controlling cell proliferation and differentiation in limb and craniofacial development (e.g., Niswander et al., 1993; Iseki et al., 1997) and in branching morphogenesis in the lung (Peters et al., 1994) suggest functional interactions with RA in these sites, such as that demonstrated in conditions of RA excess in the limb (Hayes, 1998). Demonstration that a MAP kinase phosphatase gene, XI7C, is RA inducible (Old et al., 1995)suggests a possible role for RA in modulating transduction of the FGF signal from cytoplasm to nucleus. Although significant progress has been made in understanding the mechanism of tissue-specific vitamin A uptake and RA synthesis in embryos (see Section 111), the potential for this aspect of retinoid signaling to be the cause of congenital abnormalities is much less well recognized than is the receptor component of the pathway. Mutations affecting retinoid metabolism, storage, or transport might have little effect on laboratory animals held in ideal and constant conditions or on healthy, well-fed pregnant women, but they could have major effects in conditions of temporary deficiency brought about by seasonal dietary fluctuationsor illness. Competition between ethanol and retinol can inhibit ADH-catalyzed RA synthesis and has been suggested as the mechanism underlying fetal alcohol syndrome (Duester, 1994); the effects of excessive alcohol ingestion on development would therefore be exacerbated by vitamin A deficiency. Investigation of the effects of long- and short-term VAD on embryos with mutations of genes affecting delivery of vitamin A to the embryo, and its metabolism within the cytoplasm, need to be carried out. Our understanding of the prenuclear aspects of RA signal transduction would then be able to match the current wealth of information on the roles of the retinoid receptors.

B. Retinoic Acid Signaling in Mammalian Development: A Uniquely Complex System Most of the conserved molecular signaling pathways that mediate tissue interactions in embryogenesis are paracrine systems, involving the synthesis

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and secretion by one tissue of a peptide (e.g., a “growth factor”) which is the ligand for a transmembrane receptor on an adjacent tissue; signal transduction from the activated receptor to the nucleus usually involves a chain of phosphorylation events in the cytoplasm, culminating in the activation of one or more transcription factors. The resulting transcriptional events affect cell behavior, especially proliferative activity or the initiation of a specifc pathway of differentiation. Retinoid signaling is similar in its effects but shows an amazing plasticity in its detail. Because RARs and RXRs are transcription factors as well as receptors, and entirely nuclear in location, regulation of their ligands can take place at many sites and at many levels before amving at the nuclear sites of action. Regulation of the synthesis, transport, uptake, storage, and catabolism of retinoids takes place from the maternal appetite at one extreme to the cytoplasm of a single embryonic cell at the other. Signaling may be paracrine, like other tissue interaction signaling, as suggested by the reciprocal synthesis of CRBP and CRAEiP I in adjacent epithelial and mesenchymal tissues (Gustafson et aZ., 1993); however, it may also be autocrine, when RA is synthesized for binding to nuclear receptors in the same cell, or long-distance, taking transport of the retinol precursor into account. The only other developmental signaling system that shows any resemblance to this is the action of androgens in sexual differentiation: Signaling can be short or (relatively) long distance, and there may be alteration prior to receptor binding, e.g., conversion of testosterone to the more potent androgen Sa-dihydrotestosteronefor masculinization of the external genitalia. However, there is no other system that shows anything like the dynamic range or functional variability shown by retinoid signaling in embryogenesis. It is this inherent pathway variability,together with the receptor diversity, that has enabled retinoids to take on such a fundamental position with respect to vertebrate developmental mechanisms. The evolutionary conservation of the vertebrate retinoid signaling pathway, and its apparently unique nature, suggests that the evolutionary origin of retinoid signaling may have played an essential role in the evolutionary origin of vertebrates. The effects of gene disruption, VAD, and retinoid excess studies indicate that concomitant with evolution of this important pathway, vertebrate embryos have evolved protective mechanisms against system failure. According to currently published literature, RA can induce or repress the expression of hundreds of genes, but loss of function of one RAR receptor type has surprisingly small effects; this phenomenon has been called functional redundancy, but it might be more appropriate to think of it as functional protection. Similarly,it takes a great deal of time and effort to induce VAD in laboratory mammals because of the maternal retinoid storage capacity and homeostatic mechanisms; these, too, are functional safeguards. Finally,

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RA levels many times greater than physiological levels are required to induce developmental abnormalities; whether or not CRABP I-mediated sequestration and catabolism are involved, it is clear that protective mechanisms against vitamin A excess exist in both mother and embryo. The existence of these belt-and-braces mechanisms is a particular problem for designing and interpreting experiments aimed at elucidating the cellular functions and molecular pathways of retinoids in mammalian development. Nevertheless, the research effort that has been invested in this field, particularly in the past decade, has been enormously fruitful. A picture has emerged of a signaling system that plays a role in all the fundamental embryonic processes:-cell proliferation, differentiation, and morphogenesis/pattem formation-from early embryogenesis to the organogenesis and maturation stages of (possibly) all organs and systems.

Acknowledgments We thank PhiLippe Kastner and Claes Bivik for their thorough and helpful comments on the manuscript and the many other colleagues, former colleagues, and funding agencies who have contributed to our research in this exciting field.

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Gene Expression in the Epididymis C.Kirchhoff IHF Institute for Hormone and Fertility Research, D-22529 Hamburg, Germany

The epididymis is a tubular organ exhibiting vectorial functions of sperm concentration, maturation, transport, and storage. The molecular basis for these functions is poorly understood. However, it has become increasingly clear that regional differences along the length of the duct play a role in epididymal physiology and that region-specific gene expression is involved in the formation of these differences. Although not an overtly segmented organ, the epididymis consists of a series of highly coiled “zones,” separated by connective tissue septulae and distinct by cell morphology and their pattern of gene expression. Thus, it constitutes an interesting mammalian model to study how pattern formation is achieved by differential gene activity. A large number of epididymis-expressed genes have been cloned and analyzed at the molecular level, most of them have been characterized by a distinct temporal and spatial expression pattern within the organ. Only recently have theories been developed about how and when during ontogenesis this pattern formation takes place and what its significance might be. This review summarizes the current knowledge on regionalized gene expression in the epididymis and presents hypotheses concerning its ontogenetic origin and regulation in the adult. KEY WORDS: Embryogenesis, Segmentation genes, Steroids, Transcription factors, Puberty, Aging, Epididymis. o 1% Academic PW.

1. Introduction A. Role of the Epididymis in Mammalian R e p r o d u c t i o n Most mammalian spermatozoado not have the ability to move progressively or to fertilize an egg by the time they leave the testis. In contrast to most invertebrates and vertebrates that practice external fertilization, they need a posttesticular maturation process which takes place in the epididymis International Review of Cytology, Vol. 188

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(Bedford, 1975; Blaquier et aZ., 1989; Brooks, 1987a; Orgebin-Crist, 1967, 1987). The adoption of internal fertilization may have been the major influence in this development (Bedford, 1992). The epididymal phase of sperm maturation is then followed by further changes occurring during ejaculation and Snally in the female genital tract. The latter is collectively termed “capacitation.” It seems that epididymal maturation confers upon spermatozoa the ability to capacitate, i.e., to respond to changes in intracellular calcium and pH by undergoing the acrosome reaction (Aitken et aZ., 1996). In addition, a system of regulated storage imposed on spermatozoa in the terminal region of the epididymis (Bedford, 1991) has developed in mammals, ensuring that sperm cells in storage are quiescent and unreactive. In order to fertilize, they need to escape from this stable state. In vivo, the environment of the female tract releases the safety mechanisms that have formed during epididymal transit (Yanagimachi, 1994). The evidence that sperm maturation and storage requires exposure to the specific microenvironment of the epididymis has been reviewed elsewhere (Cooper, 1986; Robaire and Hermo, 1988; Jones, 1989; Amann et aZ., 1993; Turner, 1995; Hinton et aZ., 1996; Nolan and Hammerstedt, 1997). Still, the functions of specific components of this microenvironment are poorly understood, and it has not been determined which maturational changes are intrinsic to sperm, requiring only time, and which depend on the interaction with the epididymis and specific products provided by various regions of this organ. To understand better on a molecular level the role of the epididymis in the metamorphosis of an immature, nonfunctional sperm into a mature, fertile but quiescent sperm, a careful analysis of its pattern of gene expression is helpful. A single convoluted duct, the epididymis can be divided grossly into three major regions: caput, corpus, and cauda. These regions can be distinguished by their epithelial cell morphology (Holstein, 1969;Hamilton, 1990, Robaire and Hermo, 1988) as well as by their specific pattern of gene expression, possibly reflecting the vectorial and progressive functions of sperm maturation and storage. Septulae of connective tissue within which blood vessels and nerves enter the organ (Setchell et aZ., 1994) further subdivide the organ into highly coiled “zones” or “segments.” These zones, although somewhat variable among individuals of the same species, may represent functional and regulatory subunits of the epididymis.

6. Regionalized Gene Expression in the Epididymal Duct Epithelium

The field of epididymal research has advanced to the molecular level. During the past 10 years a large number of genes have been cloned and

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analyzed which are involved in the establishment and maintenance of a unique and highly regionalized expression pattern along the duct. Epididyma1 mRNAs and proteins have been described in various species exhibiting region- and segment-specific expression patterns (Douglas et aZ., 1991; Garrett e t d , 1991;Krull et aZ., 1993;Pera et aZ., 1994;Winer and Wolgemuth, 1995; Orgebin-Crist, 1996; Fig. 1). There is a long-standing interest in this regionalized pattern since it may serve as a major molecular basis underlying epididymal physiology. This concept has only recently been summarized in a minireview by Cornwall and Hann (1995a). The authors suggest that it may “result in the sperm encountering a unique and ever changing luminal fluid environment as they progress through the epididymis” (p. 379). The order with which the spermatozoa come into contact with the specific epididymal secretions may be crucial for the resultant physiological effects, with some of the secretory proteins only able to bind, for example, after prior modification of the sperm membrane. Thus, the longitudinal subdivision into morphologically and functionally distinct zones may be fundamental to the process of sperm maturation and storage, suggesting a specific role of the epididymis rather than a general “housekeeping” role (Cornwall and Hann, 1995a). Region-specific gene expression implies regionalization of regulatory mechanisms in the epididymis. These mechanisms, however, are far from being understood. Androgens and nonsteroidal (testicular?) factors have been suggested to be involved, and regional differences in androgen levels, androgen-binding capacity, and the activity of steroid-metabolizing enzymes have been reported (Orgebin-Crist, 1996; Robaire and Viger, 1995). Signaling via tyrosine kinase receptors seems to play an important role in the establishment of regional specificities (Sonnenberg-Riethmacher et aZ., 1996). Moreover, a number of homeobox-containing genes have been suggested to define a proximodistal axis along the developing urogenital tract (Lindsey and Wilkinson, 1996a; Favier and DollC, 1997). In the following section, gene expression in the epididymis will be reviewed in the light of the ontogenetic origin and maintenance of this longitudinal pattern of gene expression as a molecular basis of its vectorial functions.

II. Ontogenesis of Epididymal Gene Expression Pattern Early development of the reproductive system has attracted many investigators owing to the peculiar aspects of its embryology and to the high incidence of congenital anomalies related to its structures. Most of our knowledge comes from developmental studies in rodents and from urogenital malformations in the human. Regarding the high evolutionary conservation of

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a

FIG. 1 Regionalization of specific gene expression in the epididymis. Distinct longitudinal patterns of CEI (a), CE4 (b), and CD5 (c) mRNA expression in the canine epididymis as visualized by low-resolution autoradiography of sagittal sections after in situ transcript hybridization of the three gene probes. Sections were exposed simultaneously to autoradiographic film for the same time period but are not intended to suggest any type of quantitative comparison between different riboprobes. Scale bar = 5 mm [reproduced with permission from Pera, I., Ivell, R., Kirchhoff, C. (1994). Reginnal variation of specific gene expression in the dog epididymis as revealed by in-situtranscript hybridization. lnt. J. AndroL 17,324-3331. (d) Northern blot analysis revealing distinct CEI, CE4, CEYCD52, and CE7/GPXS mRNA levels in various regions (caput, corpus, cauda, and vas) of the canine epididymis. The blot was hybridized, stripped, and rehybridized with two cDNA probes simultaneously; equivalent RNA loading was demonstrated by ethidium-bromide staining of the 18s-and 28s-ribosomal RNA prior to blotting.

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Caput Corpus Cauda Vas

CE1

+ -1.3kb

- 0.6 kb

CD52

FIG. 1 (continued)

many of the genes involved, it may be extrapolated to other placental mammals. Development of the excretory and reproductive systems seems closely related by their common origin from the intermediate mesoderm, and therefore both systems are often grouped under the term “urogenital tract.” Urogenital development proceeds in three stages: In the first two stages, the transient pronephros and mesonephros form along the urogenital ridge. In the third stage, the definitive metanephric kidney forms. The epididymis originates from the Wolffian ducts and mesonephric tubules that develop as a part of the mesonephric kidney. These ducts clearly start to develop before the time of sexual differentiation (George and Wilson, 1994) and thus before the onset of steroid production by the testis. Many studies have postulated regulatory hierarchies of genes that control early differentiation of the gonads (Ramkissoon and Goodfellow, 1996). Similar hierarchies, albeit unknown, may be involved in the differentiation of the reproductive tract primordia.

A. Gene Expression in the Early Wolffian Duct In this chapter various genes or gene families will be discussed that may be operative in the mesenchymal-epithelial interactions leading to morpho-

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genesis in the intermediate mesoderm, the Wolffian duct, and mesonephric tubules. They involve different types of diffusible or membrane-bound molecules as well as specific transcription factors whose role during organogenesis has been disclosed by targeted mutation. The data are highly selective and in part speculative. Important components of mesenchymalepithelial interaction, such as extracellular matrix and cell adhesion molecules (Birchmeier and Birchmeier, 1993), have been omitted since in many instances studies have remained essentially descriptive. 1. Secreted Signaling Molecules In the mouse, hedgehog genes are expressed in epithelia at numerous sites of epithelial-mesenchymal interactions, including parts of the Wolffian duct

(Bitgood and McMahon, 1995). A striking correlation in the expression of hedgehog genes and the bone morphogenetic proteins Bmp-2 and Bmp-4 was observed. Bmps are expressed either in the mesenchyme underlying the site of sonic hedgehog (Shh) expression or in the epithelial cells near or even concominant with the cells that express Shh. Shh expression is detected in the epithelium of the urogenital sinus, and Bmp-4 is observed in the adjacent mesenchyme from as early as 11.5 days postcoitus (pc) (Bitgood and McMahon, 1995). Bmps may be general targets of hedgehog signaling, and the reciprocity of epithelial-mesenchymal interaction could involve a signaling loop between shh and bmp-4 expressing cells in adjacent cell populations (Hammerschmidt et al., 1997). Other possible targets of hedgehog signaling are the mesenchyme forkhead genes (Wu et al., 1998). Interestingly, Shh or a closely related signaling molecule induces expression of the paired-box transcription factor Pax-2 in somites which seems to be a key upstream regulatory gene in urogenital development (see Section II,A,3). 2. Receptor Tyrosine Kinases

Among the earliest genes required for intermediate mesoderm formation and differentiation are the receptor tyrosine kinase c-ret (Schuchard et al., 1994) and its ligand, glial cell line-derived neurotrophic factor (GDNF) (Sanchez et al., 1996; Pichel et al., 1996; Moore et al., 1996; Vega et al., 1996). Targeted mutations (“knockouts”) produce embryos that lack kidneys, ureters, and reproductive tracts. c-ret is expressed in the epithelial structures of pro- and mesonephros at 9.5 days of embryonic development, when the most rostra1 parts of the Wolffian duct are being generated (Pachnis et al., 1993). Targeted mutation of c-ros, a receptor tyrosine b a s e whose ligand has yet to be identified, results in defects specifically restricted to the male

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reproductive tract (Sonnenberg-Riethmacher et aZ., 1996). The temporal and spatial arrangement of c-ros transcripts in the primordia of the urogenital tract is coincident with the development of the Wolffian duct (Sonnenberg et aZ., 1991). Transcripts were first identified during the indifferent stage on Day 11 pc of mouse embryogenesis, and in situ hybridization localizes them in the entire Wolffian duct until Day 13 when they become localized in parts of the developing metanephric kidney (Sonnenberg et al., 1991). There is also a strong epithelial c-ros expression in the epididymides of adult males which is confined to the caput, specifically to the initial segment (Fig. 2), suggesting a function of the receptor-tyrosine kinase in the differentiated organ (see Section IV,D,l). In adult c-ros-’males, the caput epididymidis is aberrant in that the initial segment is lacking (Sonnenberg-Riethmacher et al., 1996). The animals are viable and healthy but completely infertile in vivo (see Section IV,D,l). While the authors suggest that this phenotype is caused by the lack of c-ros expression in the adult mutant epididymis (Sonnenberg-Riethmacher et al., 1996), it is still possible that the transient expression observed around the time of sexual differentiation may be necessary and decisive for the development of the wild-type pattern of epididymal regionalization and, finally, sperm maturation. Immunohistochemical examination and receptor autoradiography suggest that in the mouse embryo the epidermal growth factor (EGF) receptor tyrosine kinase is expressed in the urogenital tract as early as Day 13 of gestation (Bossert et aL, 1990). Several members of the EGF superfamily of growth factors that apparently exert their effects by binding to the EGFR have also been observed simultaneously (Thomson et aL, 1997). To investigate their role during Wolffian duct development, the effects of antiEGF and anti-EGFR antibody were assessed using an in vitro organ culture technique (Gupta et al., 1991; Gupta, 1996). Both types of antibodies prevented Wolffian duct development in a dose-dependent manner and at higher doses also prevented the regression of the Mullerian ducts in the organ cultures obtained from male embryos. 3. “Segmentation Gene” Transcription Factors Soluble or membrane-bound signaling factors, as described previously, seem to induce hierarchies of transcription factors which in turn activate cascades of downstream genes, thereby controlling determination and differentiation of cells. Various families of transcription factors, namely, of the homeobox and paired-box families, have been detected in the primordia of the urogenital system which could elicit Wolffian duct formation and differentiation. For most of them, however, normal target genes are unknown. The homeobox-containing Emx2 gene may be essential in the early

FIG. 2 Expression of c-ros in the proximal part of the mouse epididymis of wild-type (a) and homozygous mutant (c) animals, as analyzed by in situ transcript hybridization and observed in dark field reflectance microscopy. (b, d) Appearance of the sections in bright field microscopy. (e, f ) Control hybridizationwith the corresponding sense c-ros probe. (g, h) Endogenous P-galactosidase activity in the proximal epididymis as visualized by X-gal staining in the epididymis of wild-type (g) and homozygous mutant (h) animals (reproduced with permission from Sonnenberg-Riethmacher d al., 19%).

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development of intermediate mesoderm derivates. Em2-I- mutants fail to develop kidneys, gonads, and genital tracts (Yoshida et al., 1997). In situ hybridization analyses revealed that Emx2 is first expressed in the developing mesonephros by 10 days pc (Pellegrini et al., 1997). A strong epithelial signal is evident at the level of the Wolffian ducts and the mesonephric tubules at 11-13.5 days pc when these structures are at their maximum volume. Pax-2, a member of the paired-box family of transcription factors, is regarded as a key upstream gene involved in intermediate mesoderm differentiation (Torres et al., 1995; Dressler et al., 1990; Dressler and Douglas, 1992). It encodes via alternative splicing two nuclear proteins, Pax-2A and Pax-2B, which bind to specific DNA sequences through the paired-box domain (Phelps and Dressler, 1996).In the mouse embryo, Pax-2 transcripts can be detected in the nephric cord and Wolffian duct at Day 10pc (Dressler et al., 1990). At Day 11 of gestation, both the mesonephros and Wolffian ducts express Pax-2 transcripts. Pax-2-I- mutant embryos lack kidneys and ureters, comparable to Wilms’ tumor-associated gene (WT-l)-’- mutants (Kreidberg et al., 1993). In addition, genital tracts are absent in both male and female Pax-2 homozygous mutant embryos (Torres et al., 1995). The Wolffian duct appears normal at Days 9 and 10 of embryogenesis in these mutants but fails to extend normally as observed in wild-type males. One day later, the mutant ducts begin to degenerate, showing a discontinuous epithelium and enlarged lumen. Remarkably, in Pax-2-I- mutants the testis is completely normal, supporting the view that the effect on the intermediate mesoderm is specific and not caused by degenerative processes within the testis (Torres et al., 1995). Pax-2 responsive genes have not yet been identified. However, in vitro cotransfection studies showed that Pax-2 can transactivate the promoter of WT-1 (McConnell er al., 1997), suggesting that Pax-2 is a modulator of WT-1. Besides the metanephric mesenchyme, WT-1 is also expressed in the mesonephric mesenchyme. However, mesonephric tubules and Wolffian duct form normally in WT-1 mutated mice, indicating that some of the molecular events that underly mesonephric development are different from those in metanephric development (Kreidberg et al., 1993). Expression analyses at later stages of mouse development revealed Pax-2 transcripts also in epithelial derivates of the adult genital tract (Fickenscher et al., 1993; Oefelein et al., 1996, see Section IV,C,2). This biphasic expression pattern suggests that Pax-2 could indeed play a specific role in epididymal development and function. Hox genes, which have long been known to direct the patterning of segmented structures along the embryonic anterior-posterior axes, seem to provide positional information within the developing genital tracts in both sexes (Lindsey and Wilkinson, 1996a; Favier and DollC, 1997). The

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abdominal B class homeobox genes, Hoxa-I0 and Hoxa-11, were the first members of this family of transcription factors implicated in genital tract patterning. Both genes are located at the 5’ end of the Hox gene clusters and are expressed in the posterior domains of the developing mouse embryo, includingthe intermediatemesoderm (Benson et al., 1996;Doll6 et al., 1991). In situ hybridization studies revealed that Hoxa-10 and -11 are expressed in spatially restricted domains of the genital tracts of both sexes. In the male, wild-type Hoxa-I0 expression was observed at Embryonic Day 15.5 with a distinct anterior expression boundary in the midepididymis (Satokata et al., 1995). Hoxa-11 expression was seen in the stromal cells surrounding the distal mesonephric (WoltXan) ducts at Day 14 of gestation (Hsieh-Li et al., 1995). Both male and female Hoxa-10 and - 1 P homozygous mutants were viable but severely hypofertile (Rijli et al., 1995; Satokata et al., 1995). Mutant males exhibited variable degrees of cryptorchidism. Moreover, loss-of-function mutations resulted in characteristic deformations of the genital tracts (Hsieh-Li et al., 1995; Benson et al., 1996), following the principles proposed to govern Hox gene function in segmented organs. From these principles, loss of function of a Hox gene will affect the segment at its anterior boundary of expression and will change its Hox code to that of the next anteriormost segment, leading to an anterior transformation. While the genital tract is not an overtly segmented structure like the vertebral column, the deformations observed in “knockouts” of the Hoxa-10 and Hoxa-11 genes (Hsieh-Li et al., 1995; Benson et al., 1996) support the same paradigms of patterning and suggest that they represent homeotic transformations. Both the distal part of the epididymis and the proximal ductus deferens were abnormal in these mutants, showing partial transformation into a proximal epididymis. The transformation seen in males deficient for Hoxa-11 was partially overlapping that of Hoxa10-deficient males but seemed to extend more distally (Hsieh-Li el al., 1995). To examine whether in Hoxa-IO-’- mutant males the longitudinal expression pattern of region-specific epididymal markers was altered, Benson et al. (1996) investigated their endogenous P-galactosidase activity using X-gal histochemistry (Fig. 3, see color plate). In wild-type animals, strong endogeneous p-galactosidase activity is restricted to the initial segment and the corpus epididymidis (Fig. 2). In the mutant males, however, this regionalized pattern of enzyme activity was destroyed, and the transformed cauda epididymidis as well as the proximal vas deferens both demonstrated @-galactosidaseactivity (Fig. 3). Thus, distal parts of the epididymis had acquired morphological and biochemical features of more anterior segments, and the epididymal duct in the cauda region resembled that of the wild-type corpus (Hsieh-Li et al., 1995; Benson et al., 1996). In addition,

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the cauda epididymidis was enlarged and the proximal part of the ductus deferens remained tortuous as in the cauda. The overall length of the ductus deferens was reduced, suggesting that its proximal part indeed had adopted epididymal-type fate and had been partially incorporated into the cauda region. Since Hoxa-10 and -11 transcripts are expressed in the mesenchymal compartment of the developing mesonephric duct, homeotic transformation in the mutants most probably originates from changes in gene expression in the inductive ductal mesenchyme prior to differentiation of the responsive epithelium. Similarly, in heterotypic combinations of stroma and epithelium from different proximodistal levels of the male reproductive tract, regional specialization of the epithelium did not depend on its own origin but on that of the mesenchyme (Cunha et al., 1992). Recently, it has been suggested that the urogenital tract mesenchyme produces diffusible factors which are responsible for patterning of the duct epithelium (Shima et al., 1995). Hox genes may control the production of such mesenchymal factors in a regionspecific manner. Current data suggest significant degrees of functional redundancy of Hox genes in the genital tract. Besides the described Hoxa10 and -11, other paralogs have been observed in the urogenital system during the same early stages of development. Hoxd-11 is paralogous and functionally equivalent to Hoxu-11; extra doses of Hoxd-11 can phenotypically rescue a Hoxu-11 loss-of-function mutation (Zgkny et ul., 1996). In situ hybridization studies demonstrated expression of Hoxb-13 in the same posterior domain of the urogenital tract at 12.5 days pc (Zeltser et al., 1996).

6 . Androgens and Androgen Receptor Expression [“Virilization”) Androgens are the classical hormones that have long been known to be pivotal in the regulation of adult epididymal function (Orgebin-Crist, 1996) as well as in male genital tract development, named virilization (George and Wilson, 1994). During the early stages described previously, the Wolffian duct presents a single profile in traverse sections, but from Days 17 to 19 of rodent fetal development, numerous profiles appear in traverse sections through the fetus because the duct has grown and become convoluted (Flickinger, 1969). This virilization most probably occurs in response to androgen production by the developing fetal testes. Woman duct convolution in the human fetus seems to proceed in distinct zones which are marked by primary windings of the duct (Holstein, 1969). In the rat, virilization is first detected at Fetal Day 15. Androgen production continues until birth, after which androgen levels fall precipitously (George and Wilson,

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1994). It has long been known that ablation of fetal testes during the indifferent period of sex differentiation inhibits differentiation of the Wolffian duct and urogenital sinus. Androgens induce cell proliferation and growth (coiling) and prevent programmed cell death in the developing Wolffian duct. On a molecular level, however, specific information on the mechanism of androgen action during the early development of the male genital tract is remarkably scarce. 1. The Androgen Receptor Androgen functions are exerted through an intracellular androgen receptor (AR) which in the unliganded state is associated with heat shock proteins and is inactive. As soon as androgen is available, the hormone-AR complex forms and translocates into the nucleus where it binds to specific DNA sequences, the androgen response elements (Tsai and O’Malley, 1994). Steroid autoradiography (Cooke et al., 1991a) and immunohistochemistry (Bentvelsen et al., 1995) detected AR expression in the region of the prospective efferent ductules and epididymis on Fetal Days 13 and 14 of mice and rats, i.e., at approximatelythe time of sexual differentiation.The protein disappears from this region when the indifferent stage has changed into a female direction but continues to be expressed in the developing male (Bentvelsen et aL, 1995). The distribution in the early Wolffian duct, however, differs from that in the genital tract of older animals in that AR is initially present only in the mesenchyme. The appearance of epithelial AR occurs progressively in a proximodistal direction in the reproductive tract over the late fetal and early neonatal periods. In rodents, the region derived from the most proximal part of the Wolffian ducts, i.e., the caput epididymidis, begins to express epithelial AR soon after its initial morphological differentiation (coiling) at Days 18 or 19 of fetal life, whereas in the cauda epididymidis and vas deferens epithelial AR was detected only after birth (Cooke et al., 1991a; Bentvelsen et al., 1995). The markedly different time points of epithelial AR appearance during male reproductive tract development suggest that this event may be controlled locally within the individual parts rather than occurring in response to a systemic signal, although systemic signals, e.g., androgens, may be permissive for initial epithelial AR expression. Since in rats testicular fluid has been reported to reach the epididymis not before 20 days of postnatal age (Vitale et al., 1973;Tindall et al., 1975,this would suggest that a factor(s) from the underlying epididymal mesenchyme controls initial epithelial AR expression. These observations emphasize the importance of “paracrine” interrelationships between the inductive mesenchyme and the responsive epithelium during morphogenesis.

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High fetal androgen levels, early appearance of the AR in the Wolffian duct, and the anomalies observed after testis ablation (George and Wilson, 1994) suggest that androgen-regulated gene expression has already been triggered in the fetus. Candidate androgen-responsive genes, however, are unknown. More than 30 years ago, the epididymis of the human fetus was reported to exhibit specific secretory activity, apparently paralleling the production of androgens by the fetal testis (Zondek and Zondek, 1965). All parts of the fetal human epididymis seem capable of secretion, but secretory activity varies considerably in different areas of the duct. The biochemical nature of the secretory product(s) is not known; periodic acidSchiff-positive staining suggests that it may consist of a glycoprotein(s) produced in response to fetal androgen levels. However, since the secretory activity coincides with maturation processed in other reproductive organs, it may also occur in response to maternal or placental factors. 2. Developmental Pattern of Steroid Sa-Reductase Expression Virilization in mammals is mediated by two steroid hormones, testosterone and the 5a-reduced dihydrotestosterone (DHT). The latter binds the nuclear AR preferentially, indicating that 5a-reduction is a crucial step in androgen action. The developmental pattern of 5a-reductase activity has been most closely studied in humans (Russell and Wilson, 1994), and the inborn error of male phenotypic sexual differentiation, termed 5a-reductase deficiency, provided genetic proof of the important role of DHT. The study of steroid 5a-reductase expression is complicated by the existence of two isoenzymes, encoded by two genes (Russell and Wilson, 1994).The enzymes show different affinities for steroids and are encoded by two different genes in humans as well as in rats. It is not clear why there are two enzymes and two genes and what unique or overlapping roles they play. Targeted mutation of the type I and/or type 2 genes in the mouse and new inhibitors of the 5a-reductases might help to answer these questions. Immunoblotting experiments indicated that most of the activity in the early human embryo can be attributed to the type 2 isozyme (Thigpen et aL, 1993), and male pseudohermaphroditism is due to 5a-reductase type 2 deficiency (Andersson et aL, 1991; Wilson et al., 1993). In the human fetus, enzyme activity is detected in the urogenital sinus several weeks before that in the Wolffian duct derivates (Wilson et aL, 1993), suggesting that expression in the two anlagen proceeds from caudal to cranial and may be regulated by different mechanisms. A similar pattern was observed in the mouse, although the type of isoenzyme was not determined (Tsuji et aZ., 1994). It is possible that the synthesis of DHT by the urogenital sinus brings about expression of 5a-reductase in the Wolffian duct derivates (Silver et aZ., 1994). Moreover, the cranial

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and caudal parts of the Wolffian duct seem to behave differently toward androgens (Tsuji et aL, 1994). Whereas in the cranial portion prevention of programmed cell death may be elicicted solely by testosterone, assessment of the developmental profile of 5a-reductase activity in mice at Days 14.5-16.6 of gestation suggested that DHT may be important for the differentiation of the caudal portion of the Wolffian duct and seminal vesicle, possibly eliciting morphogenesis (coiling) of the epididymis (Tsuji et al., 1991, 1994) which proceeds in distinct sections or zones.

3. Role of Growth Factors in Early Androgen Action (“Aadromedines”) Although androgens are essential for male reproductive development, it is becoming apparent that growth factors modulate AR expression and hormone binding and thus may act as paracrine mediators of androgen action. As shown for androgens, EGF and basic fibroblast growth factor (bFGF) seem to induce normal growth and function in the developing male genital tract. EGF and androgens showed a synergistic cell proliferating effect in primary cell cultures of the fetal mouse urogenital tract (Gupta et al., 1996). EGF mRNA is expressed in the developing genital tract at Day 18 of gestation, and levels were found to be higher in the male than in the female tract. Exposure to testosterone of female fetuses during Days 13-17 of gestation induced EGF mRNA expression and stabilized the Wolffian ducts (Gupta and Singh, 1996). The role of bFGF in the development of the reproductive tracts of the rat was investigated using transplantation and infusion procedures (Alarid et al., 1991). The genital ridges and urogenital sinuses of 16-day-old male and female rat fetuses were grown for 2 weeks on kidneys of adult hosts of the same respective gender. Infused anti-bFGF antiserum had no effect on the urogenital sinus of either sex and no effect in the female, but it had a strong specific inhibitory effect on the growth of the male genital ridge. A severe impairment of epididymal development was visible after 1 week and there was a complete absence of the epididymis after 2 weeks of treatment with anti-bFGF antiserum. In addition to these early effects, bFGF seems to be involved in the regulation of epididymal gene expression in the adult organ (Lan et al., 1998; see Section IV,D,3). Keratinocyte growth factor (KGF) is another andromedin that interacts with androgen receptor signaling in the male genital tract but does not seem to be a direct target of androgen action. KGF has been shown to play an important role in seminal vesicle and prostate development, such as epithelial growth, branching morphogenesis, and cytodifferentiation (Thomson et al., 1997). Whether this is also true for the epididymis is unclear.

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C. Early Expression of Other Members of the Cys4 Zinc Finger Nuclear Receptor Type Superfamily 1. Estrogen Receptors Estrogen receptors (ER) are present throughout the developing male reproductive tract in rodents (Cooke et al., 1991b). Mesenchymal cells of the Wolffian duct showed nuclear localization of [3H]estradiol at 16 days of fetal life. However, its epithelium first labeled at 19 days of fetal life and thereafter (Cooke et al., 1991b). In comparison, mesenchymal and epithelial cells of the efferent ductules showed nuclear localization at the first time point examined and thereafter. Thus, times and initial epithelial ER expression in the epididymis and efferent ductules are similar to those for the epithelial AR in these organs (see Section 11,BJ). Hess et al. (1997a) showed that estrogens regulate the reabsorption of rete testis fluid in the head of the adult epididymis, and that this function is exerted through the ER-a (see Section IV,A,2). The presence of nuclear ER throughout male genital tract development suggests that estrogens might also play some physiological role in the fetal male reproductive tract, in addition to their well-known deleterious effects (Greco et al., 1993). Their specific function, however, remains unclear. Early ER-a expression seems to be dispensable since male reproductive development of estrogen receptor knockout (ERKO) mice seems to proceed normally until puberty (Lubahn et al., 1993; Eddy et al., 1996).

2. Nuclear Retinoid Receptors Vitamin A or its oxidized product, retinoic acid (RA), is required during organogenesis, including the urogenital system. Vitamin A deprivation (VAD) during embryonic development leads to numerous congenital effects, including ureter and male genital tract abnormalities. The molecular mechanism of RA action is complex and involves cellular binding proteins and at least two families of nuclear receptors (Giguere, 1994). Compared to the manifestations of the VAD syndrome, mice carrying targeted mutations in single genes encoding the various receptor types have surprisingly few developmental abnormalities,suggestingthat there is a high redundancy in the functions and expression patterns of these families of nuclear receptors. Unlike single mutants, however, compound RA receptor (RAR) null mutants revealed essentially all the defects characteristic of fetal VAD and led to lethality in utero or shortly after birth (Lohnes et al., 1995). Subtype RARa appears to be an important transcription factor mediating vitamin A action in the developing Wolffian duct (Lufkin et al., 1993) as in the adult epididymis (Costa et al., 1997). The cells of the epithelium

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lining the epididymis and vas deferens express abundant RARa (Akmal et al., 1996). Developmental abnormalitiesof the genital tract were observed in RARary double mutants, suggesting an important role during development. In most of the analyzed cases bilateral agenesis of the epididymis, vas deferens, and seminal vesicles was associated with renal agenesis (Mendelsohn et aZ., 1994). Histological analysis of the corresponding region in these double mutants suggested that RA is most probably required at two stages of male genital tract development, i.e., before 11.5 days pc for the formation of at least the caudal Wolffian duct and after 11.5 days pc for the virilization, i.e., morphogenesisof the epididymis and proximal vas deferens (Mendelsohn et al., 1994). There are a number of candidate RA-responsive genes that are likely involved in the previous processes, including transcription factors, e.g., Hox and N-myc; growth factors, e.g., bone morphogenetic proteins (BMPs), transforming growth factor-/3and platelet-derived growth factor (PDGF); and growth factor receptors, e.g., EGF receptor, PDGF receptor, as well as cell adhesion molecules (Gudas et aL, 1994).

D. Gene Expression in the Juvenile and Peripubertal Epididyrnis

The rat is a convenient animal model for the study of mammalian sexual development, and many of the results obtained in this model may be extrapolated to other species (Ojeda and Urbansky, 1994). Puberty in the rat can be divided into four periods; a neonatal period comprising the first week after birth, an infantile period that extends from Days 8 to 21, a juvenile period that ends about Day 35, the peripubertal period lasting from Day 35 to Day 55. The initial arrival of mature spermatozoa in the vas deferens marks the end of puberty, which in the rat occurs at about 55 days of age (Ojeda and Urbanski, 1994). The epididymal epithelium, however, is in a comparably advanced stage of differentiation prior to sperm arrival, and the adult pattern of gene expression even begins to clearly take shape before the first sperm enter the caput epididymidis.

1. Postnatal Changes in Gene Expression Levels Although the detection of a gene product is a threshold phenomenon, depending on the frequency of the product and the sensitivity of the detection method employed, it appears that a number of epididymal genes are turned on simultaneously with or shortly after initiation of meiosis in the testis. This event is accompanied or followed by an increase of androgen secretion by the Leydig cells and of fluid and protein production by the Sertoli cells (Ojeda and Urbanski, 1994). In the mouse testis, the first

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meiotic events (as recognized by the occurrence of spennatocytes in the leptotene stage) are observed on Day 10 of postnatal development (McCarrey, 1993). Concomitantly, specific epididymal gene expression seems to be switched on. The mRNA encoding the ets-like transcription factor PEA3 starts to accumulate in the epididymides of prepubertal mice at approximately Day 10 of postnatal development (Drevet et al., 1998). PEA3 has recently been shown to be a major factor modulating epididymal gene expression (Xin et al., 1992; Lan et al., 1997; see Section IV,C,l). Its expression precedes the onset of two candidate downstream genes, the secretory glutathione peroxidase (GPXS) and y-glutamyl transpeptidase ( G G T ) .Both genes have been reported to be regulated or modulated by PEA3 binding to specific sites in their promoters (Lan et al., 1997; Drevet et al., 1998; see Section IV,C,l). The GPXS gene is turned on around Day 12 of postnatal development in the mouse, and its mRNA accumulates steadily into adulthood (Drevet et aZ., 1998). Multiple GGT mRNAs were detected at low levels in newborn rats and remained constant until Postnatal Day 8 (Palladino and Hinton, 1994a). From Postnatal Days 8 to 12, GGT mRNA levels transiently decreased, then increased thereafter into adulthood. A large number of other epididymal gene products have been reported to show peripubertal changes in their levels of expression. The mRNA encoding the acidic epididymal glycoprotein (AEG = protein D E ; see Section 111,CJ) was first detected on Northern blots at Days 15-20 of postnatal development in the rat (Charest et al., 1989; Garrett et al., 1990) and reached a maximum at 45 days of age, whereas the protein continued to increase through 120 days, suggesting luminal accumulation (Charest et al., 1989). Likewise, the mRNA encoding an epididymis-specificextracellular RA-binding protein, E-RABP (see Section II,A,4), dramatically increased between Days 20 and 28 after birth and remained high until at least Day 90 (Garrett et al., 1990, Lindsey and Wilkinson, 1996b; Fig. 4). The homeobox-containing transcription factor Pem, which is selectively expressed in male and female rodent reproductive tissue (Lindsey and Wilkinson, 1996a,b; see Section IV,C,3), displayed an ontogenetic expression pattern paralleling that of the previously described secretory proteins. This suggests that Pem may be regulated by the same factors and may be involved directly or indirectly in the regulation of these epididymal proteins (Fig. 4). Another homeobox gene, Hoxc-8, which is expressed at high levels in the postnatal rat epididymis, showed a completely different expression pattern: Hoxc-8 levels were highest in newborn rats and then declined gradually into adulthood (Fig. 4). Although the event of sperm arrival appeared to have little effect on most of the differentiation processes described, it may well induce specific epididymal gene expression. Different from the mRNAs described pre-

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A 10 12 15 18 21 23 26 30 33 36 40 4 4

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FIG. 4 Developmental expression patterns of various mRNAs in the postnatal rat epididymis. (A) RNase protection analysis of total cellular RNA (40 pg) from rat epididymis on the indicated day after birth. A Pem probe was used for hybridization,and the size of the protected band was -210 nt. A GAPDH probe was included in each annealing reaction to verify equal loading. (B) Northern blot analysis of total cellular RNA (10 pg) from epididymides of 5to 90-day-old Sprague-Dawley rats. The same blot was sequentially hybridized with the indicated cDNA probes. [A and B reproduced with permission from Lindsey, J. S., and Wilkinson, M. F. (1996). An androgen-regulated homeobox gene expressed in rat testis and epididymis. Biol. Reprod. 55, 975-983.1 (C) Northern blot analysis of total cellular RNA (10 pg) from epididymides of 15- to 120-day-old Sprague-Dawley rats. The blot was hybridized with a CD52 cDNA probe; equivalent RNA loading was demonstrated by ethidiumbromide staining of the 18s-and 28s-ribosomal RNA prior to blotting.

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viously, epididymal proenkephalin mRNA levels remained very low in the rat epididymis throughout the first 40 days of age. A dramatic increase was observed only at Day 44 (Garrett et al., 1990; Lindsey and Wilkinson, 1996a; Fig. 4), exactly when spermatozoa are first released from the rat seminiferous tubules and enter the caput epididymal lumen (Brooks et al., 1974). Since no other major event in gonaddreproductive tract development occurs during this time frame, spermatozoa or a sperm-associated factor may be directly involved in the regulation of proenkephalin gene expression. This assumption was corroborated by a number of animal experiments, including Busulphan treatment which leads to a reversible loss of germ cells (Garrett et al., 1990). Different from the gene products described, the mRNA for CD.52, a GPI-anchored, major sperm surface glycopeptide (Kirchhoff and Hale, 1996; see Section III,C,2), showed no dramatic developmental changes and was expressed at relatively high levels in infantile epididymides (Fig. 4c; C. Kirchhoff, unpublished results). Although androgen responsive in the adult male (Pera et al., 1997), the mRNA showed only a small and continuous increase during postnatal development, disproving the idea that the expression of a major sperm-bindingprotein in the epididymis would correlate to either a rise of testosterone and nonsteroidal testicular secretions or the initial appearance of spermatozoa at puberty.

2. Developmental “Shift” of Longitudinal Expression Pattern Cytological differentiation of the mouse epididymal epithelium, resulting in the formation of morphologically distinct segments, is obvious before initial sperm arrival. In 15-day-old prepubertal mice, the epididymis is still small and all epithelia appear uniform. During a period of differentiation beginning on Day 16 and ending on Day 44,epididymal segments become distinct (Sun and Flickinger, 1979). This process seems to be accompanied or even preceded by differentiation and regionalization processes on the molecular level, resulting in a shift of gene expression patterns. Immunostaining of the prepubertal mouse epididymis for the GPXS protein was visible on Postnatal Day 20. The characteristicregionalized pattern of GPXS expression within the caput epididymidis was overt from Day 26 onwards, suggesting that the adult pattern of epididymal gene expression develops postnatally (Vernet et al., 1997; Fig. 5). Similarly, a shift in the longitudinal patterns of sulfated glycoprotein-2 (clusterin) mRNA (Cyr and Robaire, 1992; Hermo et al., 1994) and epithelial cadherin mRNA expression (Cyr et al., 1992,1995) was observed in the peripubertal rat before the entry of spermatozoa into the epididymal lumen. Results in the peripubertal calf and boar, describing developmental changes in the spatial distribution of

FIG. 5 Immunohistochemical localization of the GPXS protein during postnatal development of the mouse epididymis. Dark field micrographs of the caput epididymidis after immunogold detection of GPXS antigen showing the efferent ducts (EFF) and caput segments 1-111, respectively, of 20-day-old (a), 26-day-old (b), and 60-day-old (c) mice. Immunolabeling of GPXS is clearly visible at Day 20 (a); however, its regionalization in the various segments is not completed. By Day 26 (b) the GPXS immunolabeling pattern was virtually comparable to that of the adult male. Scale bar = 200 pm [Vernet, P., Faure, J., Dufaure, J.-P., Drevet, J. R. (1997). Tissue and developmental distribution, dependence on testicular factors and attachment to spermatozoa of GPXS, a murine epididymis-specific glutathione peroxidase. Mol. Reprod. Dev. 47,87-98. Copyright 0 1997 WileyLiss, Inc. Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.]

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two abundant epididymal mRNAs (Uhlenbruck et aZ., 1993), indicate a similar pattern. It is often inferred that rising androgen levels bring about the described changes in the pre- and peripubertal epididymis, both in gene expression and at the morphological level. However, this view may be overly simplistic. It should be kept in mind that the most pronounced increase in serum testosterone levels occurs much later, namely, between 50 and 60 days of age in the rat (Ojeda and Urbanski, 1994). During the juvenile period, testosterone is not the primary androgen produced by the testis, and the ability of the epididymis to produce Sa-reduced steroids also changes postnatally. Moreover, besides changes in steroid production by the Leydig cells, the developing testis shows an increase in the capacity of Sertoli cells to secrete certain proteins, including the androgen-bindingprotein (ABP), which specifically binds steroids (see Section IV,A,3). In rodents, ABP declines in the circulation around Day 20, after which it is primarily released into the epididymis.Testicular fluid, containingABP, first enters the epididymal lumen between Days 21 and 26 of postnatal development (Vitale et aZ., 1973; Tindall et aZ., 1975). Lindsey and Wilkinson (1996~)suggest that androgen regulation of Pem expression in the epididymismay be exerted through ABP. Consistent with this idea, W W mutant mice which lack germ cells as well as seminiferous fluid and proteins, including ABP, but have normal androgen levels (deFranca et aZ., 1994), do not express epididymal Pem. Similarly, epididymal expression of 5a-reductase activity has been shown to be critically dependent on a testicular factor secreted directly into the epididymis via the efferent ducts which may be ABP (Robaire and Viger, 1995). Studies on the expression of the Sa-reductase type 1mRNA in male rats of different postnatal ages revealed that the transcript is developmentally regulated. In the caput-corpus epididymidis,mRNA levels slightly decreased between Days 7 and 21 of postnatal development and then rose significantly between Days 21 and 56 into adulthood (Viger and Robaire, 1992). In contrast, no significant age-related changes of the 5a-reductase type 1 mRNA were observed in the cauda epididymidis. A segment-by-segment analysis revealed that the developmental changes in the proximal part of the organ were due solely to changes occurring within the initial segment (Viger and Robaire, 1992).

111. Gene Expression in the Adult Epididymis The secretory and absorptive functions of the epididymal epithelium are directly involved in the creation of the luminal microenvironment in which

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sperm maturation occurs. Thus, numerous investigations have focused on gene expression in the epithelial compartment. In situ hybridization patterns within longitudinal tissue sections of the organ demonstrate in a pictorial manner that gene expression in the epithelium is both complex and highly regulated (Garrett et al., 1991; Pera et al., 1994; Figs. 1 and 6), comprising consecutive subregions of distinct expression patterns suggestive of specialized functions. Moreover, despite a rather uniform epithelial morphology, gene expression in the tubular compartment turned out to be not only region specific but also cell specific. In situ hybridization as well as immunolocalization analyses revealed a characteristic checkerboard expression pattern for many gene products (Orgebin-Crist, 1996). It could be due to a functional mosaicism of cell types of the epithelium which is not reflected by their morphology, with only a few cells being competent to express the specific genes. In comparison, very little is known about gene expression in the intertubular tissue of the adult epididymis. It may be assumed that inductive functions of the underlying mesenchyme are as necessary in the adult organ as they are during fetal and pubertal development (see Sections II,A,3 and II,B,3) to maintain epididymal epithelial structure and function. Thus, the stromal compartment may well play a hitherto unknown role in the regionalization of adult epididymal gene expression. In the followingsections, abundant gene products of the adult epididymis will be described according to the region of their prevailing expression, beginning with the caput region. However, they will be considered only as long as their principal site of gene expression in the male genital tract is the epididymis.Major proteins of the seminal vesicles may also be expressed to a lesser extent in the epididymis as has been reported for one of the semenogelins (Bjartell et al., 1996), but these will not be included here. Rodents have been the primary or only animal models in many investigations, and homologous gene products in other mammals often are difficult to study due to poor evolutionary conservation. It thus remains to be

FIG. 6 Diagrammatic representation of specific patterns of gene expression within the adult rat efferent ductkaput epididymidis as deduced from in situ hybridization patterns. Sites of transcript localization for the proenkephalin, protein D/E, protein BIC, CRBP,SGP-I, and SGP-2 gene products are shown. Dark areas represent regions exhibiting strong in situ labeling intensities, whereas lighter areas represent regions of decreasing labeling intensities. Degrees of shading are intended to reflect relative hybridizationintensities for one particular riboprobe within a tissue section; they are not intended to suggest any type of quantitative comparison between two different riboprobes [Garrett, S. H., Garrett, J. E., Douglass, J. A. (1991). In situ histochemical analysis of region-specific gene expression in the adult rat epididymis. Mol. Reprod. Dev.30,l-17. Copyright 8 1997 Wiley-Liss, Inc. Reprinted by permission of WileyLiss, Inc. a subsidiary of John Wiley & Sons, Inc.].

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., . . . . . . . . . . . ., ........... .,...........

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clarified whether all findings described later can be broadly extrapolated. To cope with the vast amount of literature published, emphasis will be on gene products that have been cloned and analyzed on a molecular level. Still, the selection made is personal.

A. Gene Expression in the Caput Region The caput epididymidishas been found to be most active in gene expression and protein secretion in several animal models (Cornwall and Hann, 1995a; Orgebin-Crist, 1996; Dacheaux et al., 1995), and many gene products are either restricted to this region or exhibit their highest levels of expression in the caput region. A number of them had been previously described as testicular gene products, and the possibility was raised that the proteins detected in the epididymal lumen originated in the testis, only reaching the proximal part of the organ via the rete testis fluid. However, molecular studies localized their mRNAs within the caput epididymidis, revealing that the situation is more complex. 1. Sulfated Glycoproteiu-1 and -2 Sulfated glycoprotein-1 (SGP-1) and SGP-2 are proteins which are expressed in many different tissues, including the epididymis. Both proteins were originally identified and cloned as major secretory products of rat Sertoli cells (Collard and Griswold, 1987; Collard et aL, 1988) and were subsequently shown to be present in the luminal fluid of the rat caput epididymidis (Sylvester et al., 1989, 1991). In situ hybridization studies revealed that they are indeed abundant gene products of the epididymis and that their epididymal expression pattern is distinct despite their ubiquitous tissue distribution (Garrett et aZ., 1991). SGP-1 mRNA was localized in the tubules of the efferent ducts. In the more distal zones of the epididymal duct proper, SGP-1 hybridization signals became weak and diffuse (Garrett et al., 1991;Fig. 6). The SGP-1 cDNA sequence predicts a protein homologous to prosaposin, the polyprotein precusor of small protein activators of lysozymal enzymes degrading glycolipids (Collard et al., 1988; Sylvester et al., 1989). The function of the secreted form, however, is not clear (Morales et al., 1996(a). SGP-2 mRNA is synthesized by the initial segment and the distal caput epididymal epithelium (Garrett et al. 1991; Fig. 6). By radioimmunoassay it was shown that the epididymis contained a 10-fold increased level compared to the testis (Cheng et al., 1990). The epididymal SGP-2 subunits differ from the Sertoli cell products by apparently lower molecular weights

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(Grima et al., 1990; Mattmueller and Hinton, 1991) probably due to posttranslational modification. Interestingly, it is the epididymal form of SGP-2 that binds to sperm during epididymal passage; testicular SGP-2 dissociates from the sperm as they leave the testis (Hermo et al., 1991; Mattmuller and Hinton, 1991) and it is endocytosed by the caput epididymidal epithelia (Morales et al., 1996b). In the distal regions of the epididymis, epididymal SGP-2 dissociates from sperm again and decreases in intraluminal concentration. Molecular cloning disclosed that SGP-2 has frequently been isolated in numerous laboratories working in different areas of molecular biology. Clusterin, SP-40,40, TRPM2, and ApoJ all are different names for the same homologous gene product. Its function, however, is unknown. It is overexpressed in numerous cases of degenerative diseases, in tissue remodeling after injury, and in apoptosis and thus has been suggested to represent a kind of stress protein or extracellularversion of heat shock protein (Michel et al., 1997). It may also play a role in cellular cholesterol homeostasis (Gelissen et al., 1998). In the epididymis, it may facilitate plasma membrane remodeling that occurs during sperm maturation. 2. Neuroendocrine Peptides

Proopiomelanocortin (POMC) was among the first epididymal gene products analyzed at the molecular level (Chen et al., 1984). A major pituitary protein, its mRNA was found in the mouse testis and caput epididymidis but not in the cauda region and the vas deferens. In the pituitary, adrenocorticotropin, &endorphin, and the melanocyte-stimulating hormones are all products of the common POMC precusor. The mRNA of the genital tract was 150 bases shorter than that in the pituitary (Gizang-Ginsberg and Wolgemuth, 1987). It was hypothesized that the POMC-derived peptides may be synthesized in the male genital tract and may exert paracrine and/or autocrine effects in these organs which, however, would require translation of the atypical mRNA. The gene encoding the opioid peptide precursor preproenkephalin is expressed widely within the male and female reproductive systems (KilPatrick and Rosenthal, 1986). The mRNA was found at extremely high levels in the initial segment of the adult rat epididymis (Garrett et al., 1990, 1991; Fig. 6). Within this region, it comprises as much as 5% of the total cellular mRNA, which is the highest concentration of any tissue examined. The size and splicing pattern are the same as those of preproenkephalin mRNA in the brain. Transcripts were localized to the principal cells lining the duct lumen of the initial segment (Garrett et al., 1991),and two different patterns of expression were identified by in situ hybridization: Proximal to the efferent ducts all cells around the circumference of the epididymal duct

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hybridized strongly,whereas more distally only a few cells scattered around the circumferencehybridized strongly while others did not. The observation that proenkephalin expression is abundant and highly restricted in the epididymis suggests that it serves some specific functional role. Functional studies, however, still await the identification of the processed, bioactive peptide product(s) in the epididymis.

3. Cystatin-Related Epididymal and Spermatogenic Gene The cystatin-related egididymal and yermatogenic gene (CRES) was identifiedby a substractive hybridization approach designed to selectively clone genes expressed in the mouse caput epididymidis (Cornwall et aZ., 1992). The cDNA sequence predicts a secretory protein with homology to the cystatin family of cysteine protease inhibitors, suggesting that CRES might modulate the proteolytic processing of sperm surface proteins occurring in epididymal transit. Northern blot and in situ hybridization demonstrated that within the epididymis, CRES transcripts are highly restricted to the very proximal caput region (Cornwall ef aZ., 1992). Additionally, they are stage-specificallyexpressed during spermatogenesis in the testis, albeit at much lower levels (Cornwall and Hann, 1995b). Testicular and epididymal CRES seem to be different in that the testicular protein appears to reside intracellularlyin the elongatingspermatids,whereas in the epididymis it is secreted. Different from other epididymal secretory proteins, CRES has completely disappeared from the lumen in the distal caput region (Cornwall and Hann, 1995b). 4. Retinoid-Binding Proteins

Vitamin A or its metabolite RA are required for maintenance of normal epithelial morphology in the adult epididymis. On the basis of the high levels of various RBPs found in this organ, it has also been suggested to have a particular function in epididymal sperm maturation, although the nature of this function is not known. The intracellular cytosolic retinoidbinding proteins (CRBPs) and cellular retinoic acid-binding protein (CRABP) are among the first gene products whose expression patterns were analyzed in the testis and epididymis of rodents (Porter et aL, 1985). They bind retinol, retinaldehyde, and RA for purposes of protection against decomposition,solubilize them in aqueous medium, render them nontoxic, transport them within cells to their site of action, and present them to the appropriate enzymes and transcription factors. Highest levels of both CRBP protein and its mFWA were found in the caput epididymidis, in the kidney, and in the liver; lower levels were seen in lung, testis, spleen, and small

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intestine (Rajan et al., 1990a). In situ hybridization localized the epididymal CRBP mRNA predominantly in the ductular epithelium of the initial segment and the proximal caput (Rajan et al., 1990b). mRNA levels in this region even exceeded those of the liver. CRBP mRNA was not detectable in the distal portion of the epididymis (Rajan et al., 1990b). Interestingly, a similar regionalization as described for CRBP was observed for the levels of RARa and its mRNA (Akmal et aZ., 1996; see Section IV,B). CRABP, on the other hand, was found in spermatozoa and principal cells throughout the epididymal duct (Porter et al., 1985). In addition to these intracellular RBPs, the rodent epididymis produces high levels of a luminal protein that binds RA at least in vitro (Newcomer and Ong, 1990; Rankin et al., 1992a), suggesting that retinoid trafficking may occur between the epididymidal epithelium and the lumen (Pappas et al., 1993). The extracellular protein had been known for long as an androgen-responsive protein duplett B/C of rat epididymal fluid has been known (Brooks and Higgins, 1980) and was among the first epididymisspecificproteins cloned under various names from epididymal cDNA libraries (Brooks et al., 1986a; Moore et al., 1990). Homologous gene products have been verified only in the mouse (Rankin et al., 1992a,b) and the lizard (Morel et al., 1993). The latter finding suggests, however, that it is conserved among mammals. In situ hybridization analyses in the rat employing a BC riboprobe (Garrett et aZ., 1991;Fig. 6) revealed signals throughout the entire distal caput epididymidis. The mouse protein was immunolocalized in the corresponding region caput region (Rankin et al., 1992b). Following the nomenclature for RBPs, the protein has been renamed extracellular retinoic acid-binding protein (E-RABP) (Newcomer, 1995). Analyses of protein structure (Brooks, 1987b;Newcomer and Ong, 1990) and genomic organization (Girotti et al., 1992) revealed that E-RABP is a member of the lipocalin superfamily to which the blood serum RBP belongs. A specific receptor mediates transfer of retinol from RBP to CRBP (Sundaram et aL, 1998). It is tempting to speculate that a similar transfer mechanism may function in the epididymis, and that E-RABP may bind to sperm via a specific surface receptor (Flower, 1996). 5. y-Glutamyl Transpeptidase

GGT is an ectoenzyme that catalyzes the first step in the cleavage of glutathione and it is another example of an epididymal gene product expressed in a wide range of different tissues. GGT-deficient mice produced by targeted mutation show growth retardation and, interestingly, remain sexually immature (Liebermann et aZ., 1996). Expression in the human is highly complex due to multiple genes located on different chromosomes and expressing multiple mRNAs (Figlewicz et al., 1993). In the rat, a single

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gene gives rise to multiple mRNAs species (Palladino and Hinton, 1994b,c; Hinton and Palladino, 1995;Hinton et al., 1995,1996). Three forms of GGT mRNAs 11,111, and IV)are expressed in the rat epididymis, and each form appears to be under the control of a different promoter (Lahuna et al., 1992, Okamoto et al., 1994). Northern blot analyses revealed a complex regionalized pattern for GGT mRNA expression along the epididymal duct. Expression of the various GGT mRNAs occurred predominantly in the initial segment and caput epididymidis, to a lesser extent in the efferent ducts, and at low levels in the corpus, cauda, vas deferens, and the testis. A decline in GGT activity from the caput to the cauda epididymidis thus would be due to a gradient in mRNA expression. Epididymal GGT is believed to be involved in the protection of spermatozoa from oxidative stress (Hinton et al., 1995,1996). Interestingly, a similar decline from caput to cauda regions in mRNA levels and activity for other enzymes possibly involved in this process has been reported (Faure et a/., 1991). Conversely, mRNA expression for the antioxidant enzyme superoxide dismutase (Perry et al., 1993a) appears highest in rat cauda epididymidis, indicating variable region-dependent mechanisms for protection against oxidative stress (Hinton er aL, 1996). 6. Glutathione Peroxidases

GPX activity in most tissues is mediated by a family of selenium-dependent isoenzymes showing differing substrate specificity and tissue distribution. All these enzymes contain one selenocysteine residue per subunit in their active sites. Their evolutionarily conserved gene transcripts encode the atypical amino acid by an in-frame UGA (opal) codon that normally functions as a termination signal. The extracellular plasma glutathione peroxidase GPX3 is an example of a ubiquitous selenium-dependent isoenzyme that is highly expressed in the genital tract. It was previously reported to be mainly expressed in the kidney but is found throughout the epididymis at significant levels as well (Schwaab et al., 1995). The secretory GPXS, on the other hand, was found to be specific to the caput epididymidis in various mammalian species, including mice (Ghyseliick et aL, 1991), rats and monkeys (Perry et aL, 1992a), boars (Okamura et al., 1997), and dogs (Beiglbock et al., 1998). This adds a member to the GPS gene family which, in contrast to the others, does not contain selenocysteine, or at least does not contain a selenocysteineencoding UGA codon. cDNA sequence analyses showed that the primary structure of the predicted proteins is evolutionarily well conserved. Although it has not yet been proven unequivocally that it acts as a true extracellular peroxidase in vivo, the mouse protein has been tentatively named GPXS. In accordance, recombinant expression of GPXS can protect

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transfected cells against oxidative damage (Vernet et al., 1996). GPXS and its species homologs constitute a major protein of the caput epididymidis in most mammals investigated. It is synthesized by the epithelial cells of the epididymal duct and secreted into the lumen, where it binds to spermatozoa (Vernet et al., 1997). In rodents, its expression in the caput region is highly sensitive to the depletion of androgens and other testicular factors after castration (Perry et al., 1992a; Ghyselinck et al., 1993; see Section IV,A,l). It is noteworthy that a related human gene product has not been described and is not included in the Expressed Sequence Tag (EST) database of human epididymides (Adams et al., 1995).Unlike other mammalian species, the epididymides of humans (as obtained from elderly men suffering prostatic carcinoma) seem not to produce detectable amounts of a corresponding GPXS mRNA (Kirchhoff, 1998). Additional work will be required to reveal the physiological significance of this negative result. 7. Steroid 5a-Reductases %Reduced DHT is the predominant androgen produced from testosterone in the adult epididymis (Brooks, 1990). Expression cloning approaches identified two different Sa-reductase isozymes capable of this reduction reaction, designated types 1and 2 for the order in which they were cloned (Russell and Wilson, 1994). Sa-Reductase type 2 mRNA is preferentially expressed in male reproductive tissues (see Section II,B,2), especially in the epididymis and vas deferens (Normington and Russell, 1992; Thigpen et aZ., 1993). It is expressed in both epithelial cells and mesenchyme of accessory glands (Russell and Wilson, 1994), with the exception of the epididymis in which it is restricted to the epithelium (Silver et al., 1994). Interestingly, the Sa-reductase type 2 gene exhibits a differential methylation pattern in reproductive and peripheral tissues and was found to be more methylated in the testis and epididymis than in the liver (Reyes et al., 1997). Semiquantitative RT-PCR in the nonhuman primate Macaca fascicularis (Mahony et al., 1997) indicated that the Scw-reductase type 1 gene shows a regionalized expression pattern in the male genital tract. It is most abundant in the testis and decreases significantly distally along the epididymis to nearly undetectable levels in the cauda region. Type 2 mRNA, in comparison, was undetectable in the testis but was present throughout the epididymis at uniform levels. The rat epididymis exhibits a gradient of Sa-reductase expression as well, in which the epithelial cells proximal to the testis, especially in the initial segment, express high levels of both enzymes and mRNAs, whereas those distally express lower levels. The expression gradient was first reported for type 1 isoform (Viger and Robaire, 1991) and was subsequently suggested for the more abundant type 2 isoform (Nor-

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mington and Russell, 1992). Robaire and Viger (1995) hypothesize that the initial segment may serve in a “wheelhouse” capacity, converting incoming testosterone to DHT which would prime the rest of the epididymis for optimal androgen action. In the human, the cell-type-specific expression patterns of both enzymes and their corresponding mRNAs are, in general, similar to those of the rat (Russell and Wilson, 19!94), but no such regionalization was observed for either enzyme or mRNAs (Silver ef al., 1994).

8. Metalloprotease-like, Disintegrin-like, Cystein-Rich Proteins

A number of proteins belonging to this family are abundantly expressed in the reproductive organs (Perry ef al., 1995a). While some are specific to the male gametes and have been shown to be directly involved in sperm-egg plasma membrane adhesion (Snell and White, 1996), other members of this family are expressed by epithelial cells of the reproductive tract or have a broader tissue distribution and may therefore play a more general role in integrin-mediated cell-cell recognition, adhesion, or signaling. The first epididymal member of this family, named EAP I, was described by Perry et al. (199213) as an androgen-dependent transmembrane protein synthesized exclusively in the epithelial cells of the caput region of rat and monkey epididymis. Another member, named ADAM7 (a disintegrin and metalloprotease; Cornwall and Hsia, 1997) is expressed i n e caput regon of the mouse epididymis and in the anterior pituitary gonadotropes, with no detectable expression in more than 20 other tissues. 9. Human Epididymis-Specific Gene Products HE2 and HE3

A differential cDNA library screeningapproach designed to clone abundant post festicularly expressed gene products of the human epididymis (Kirchhoff et al., 1990) led to the identification of a series of epididymis-expressed genes, named HE genes (Kirchhoff, 1998). Intriguingly, this approach did not identify any of the gene products described previously (some of which are of course produced by the testis as well), but identified novel (human) proteins that had not been identified previously in animal models. In this context it may be of interest that the human caput epididymidis seems to be structurally dif€erent from that of rodents. Most of the human caput region consists of efferent ducts, and an initial segment (the origin of many of the previously described rodent gene products) is not recognizable as a separate region in the human caput but only as small mosaic areas (Yeung et al., 1991).

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The epididymis-specific HE2 and HE3 cDNAs predict small, abundant secretory proteins of the human caput epididymidis. They have not been found in any other human tissue and show no similarity to known gene products (Osterhoff et al., 1994; Kirchhoff et al., 1994). Research into their function is also hampered by the fact that they seem only poorly conserved among mammals. By Northern blot analysis and in situ transcript hybridization, HE2 mRNA was shown to be specific to the distal human caput epididymidis and barely expressed in the other parts of the organ (Krull et al., 1993). Antipeptide antibodies raised against recombinantly expressed or chemosynthetic HE2 peptides recognized an antigen produced by the proximal epididymal epithelium and shed into the duct lumen. Moreover, antibodies bound to the acrosome and the equatorial region of human ejaculated spermatozoa (Osterhoff et al., 1994). This location of antigen might suggest that HE2 is involved in gamete binding of fusion. However, the outcome of zona binding tests and hamster oocyte penetration tests was not significantlyreduced by any of the HE2 antisera tested (C. Osterhoff and C. Finaz, unpublished results). HE3 transcripts were also found predominantly in the caput region of the human epididymis,but, different from HE2, HE3 seemed to be expressed to a lesser extent in the more distal parts as well (C. Kirchhoff, unpublished results). The situation is complex in that two closely related HE3 mRNAs, HE3-a and -/3, are expressed in the human epididymis which could not be distinguished during in situ transcript hybridization (Kirchhoff et al., 1994). HE3 has only been verified at the nucleic acid level; antibody production designed to discriminate between the two predicted peptides was not successful. 10. Seven Transmembrane-Domain Protein HE6

Different from the previously described HE gene products, HE6 did not predict a secretory protein of the human epididymis. Rather, it encodes a novel transmembrane protein of the epididymal duct epithelium with high homology to the seven transmembrane-domain (Tm7) receptor superfamily (Osterhoff et al., 1997). Moreover, different from the previously described and most other H E cDNAs, HE6 is highly conserved among mammals; homologous gene products showing approximately 90% nucleic acid sequence identity were observed in the epididymides of all mammalian species investigated. Northern blot analyses including 16 different human tissues revealed an epididymis-specific expression pattern. This was confirmed employing various rat tissues. In situ transcript hybridization localized the HE6 mFWA within the epithelial cells lining the epididymal duct. Northern blot analyses suggested a prevailing expression of the mRNA in the caput

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region; however, a fairly strong signal was also observed on cauda RNA extracts (Osterhoff et aZ., 1997). A homology search for related sequences revealed highest similarity of the predicted HE6 protein with the secretinNIP superfamily of G proteincoupled receptors (approximately 30% amino acid identity only within the transmembrane domain). However, a HE6 ligand is hitherto unknown and its involvement in signal transduction remains to be established. The predicted extracellular N-terminal extension was much longer than that in most other members of this receptor superfamily and showed similarity to highly glycosylatedmucin-like cell surface molecules (Osterhoff et aZ., 1997). A number of other multipass transmembrane molecules have been identified that contain similar extracellular modules possibly involved in adhesive/ antiadhesive interaction (McKnight and Gordon, 1996).

B. Genes Expressed throughout the Epididymal Duct 1. Human Epididymal Gene Product HE1

HE1 was the most abundant gene product cloned by the differential screening approach of human epididymal cDNA libraries (Kirchhoff et al., 1990, 1996). Although it is not specific to the epididymis, its mRNA levels in this tissue by far exceed the levels observed in any other human tissue examined. The mRNA was found to be highly expressed by the epithelial cells in all regions of the epididymis, with the exception of the efferent ducts and the distal cauda epididymidis (Krull et aL, 1993), and this spatial pattern seems to be conserved in other mammals (Pera et aL, 1994; Fig. 1). The HE1 cDNA sequence predicts a novel protein domain with a unique structure, showing no close similarity to known mammalian proteins but surprisingly to a group of insect proteins, among them the major mite allergens (Ichikawa et al., 1998).The sequence is well conservedamong mammals; proteins with an identical peptide backbone have been isolated from various primates (Perry et al., 1995b;Frohlich and Young, 1996)and represent approximately 20% of the luminal protein content of cauda epididymal fluid in chimpanzees (Frohlich and Young, 1996). Closely related products were also identified in nonprimate species (Baker et aL, 1993; Ellerbrock et aL, 1994; Syntin et al., 1996). Antipeptide antisera confirmed that HE1 encodes a major secretory glycoprotein of the human epididymis, accumulating in the cauda epididymal fluid and detectable in the ejaculate as a polymorphic component of M , = 20 kDa (Kirchhoff et aL, 1996). In accordance with its postulated function as an epididymal cholesterol carrier (Baker et aL, 1993; Kiuchi et al., 1997),the protein did not bind firmly to ejaculated spermatozoa. Rather

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unexpectedly, the bovine homologous gene product was cloned from the mammary gland and the encoded protein purified from cow milk (Larsen et al., 1997). Its function is not proven in either tissue; however, cholesterol transfer or exchange may represent a common function in mammalian body fluids as different as seminal fluid and milk. From structural comparisons, it is possible that HE1 may be involved in the innate mucosal defense against microbial attack (Ichikawa et al., 1998), a function that may be shared by milk proteins. 2. Putative Secretory Proteinase Inhibitors The HE4 cDNA sequence predicts a novel small, acidic secretory protein of the human epididymis (Kirchhoff et aL, 1991). The positions of halfcysteines suggest that it is a two-domain member of the “four-disulfide core”- or “whey acidic” protein (WAP)-domain proteins with significant structural similarity to, but distinct from, the secretory leukocyte proteinase inhibitor of human seminal plasma, HUSI-I/SLPI (Seemuller et aL, 1986; Kirchhoff et al., 1991). HE4 and HUSI-I/SLPI were mapped in the same region of human chromosome 20 (according to T. Hudson, 1995; dbSTS Database), suggesting that they may have been generated by a common ancestral gene. However, they exhibit different expression patterns within the male genital tract. HUSI-YSLPI is produced by the epithelial cells of the whole genital tract, including testis and prostate (Ohlsson et al., 1995). The HE4 gene product, in comparison, was exclusively posttesticularly expressed by the human epididymal epithelium (Kirchhoff et al., 1991). The principal site of HE4 expression appears to be the epididymis; however, from EST data it appears to be upregulated in a number of carcinomas. Although Northern blot and in situ hybridization analyses of the human epididymis suggested a distal expression (Kirchhoff et aL, 1991), comparative studies in other mammals support a more generalized expression pattern in the epididymis (Pera et al., 1994;Fig. l), excluding the efferent ducts and the distal cauda. Closely related proteins have been cloned from epididymal tissue of other mammalian species (Ellerbrock et aZ., 1994;Xu et aL, 1996);excluding rodents (Kirchhoff et al., 1990). HE4 antipeptide antisera react with an antigen of the epididymal epithelium and duct lumen (Kirchhoff, 1998) and with the surface of ejaculated human spermatozoa. Since it readily dissociates from ejaculated spermatozoa under in vitro capacitation conditions (C. Osterhoff and C. Kirchhoff, unpublished results), the HE4 protein may represent a novel decapacitation factor of human spermatozoa, related to proteinase inhibitors. In this context it might be significant that a bovine mRNA related to HE4 is also expressed in the Fallopian tube (Kirchhoff et al., 1991). Additionally, the WAP-domain proteins may exert

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much broader functions not related to reproduction (Tomee et aL, 1997; Jin et aL, 1997). The acrosin-trypsin inhibitor HUSI-I1 of human seminal plasma is expressed in the human testis and not in the epididymis (MiSritz et aZ., 1991). However, a gene product predicting 78% amino acid identity with HUSII1 is abundantly expressed in the epididymis of M.fmcicularis but barely in the testis of this species (Perry et aL, 1993b). Regional distribution within the epididymis was not investigated. Given the close evolutionary relatedness of this primate and Homo sapiens the difference in tissue distribution of HUSI-I1is surprising. Therefore, it is possible that the longitudinal pattern of proteinase inhibitor expression in the genital tract may differ between species.

C. Genes Expressed in the Distal Part of the Epididymis and in the Vas Deferens 1. Acidic Epididymd Glycoproteiu This abundant epididymal secretory protein has been purified from rodent epididymal fluid under a variety of names: protein D/E (named according to its relative electrophoretic mobility compared to albumin, set as A; Cameo and Blaquier, 1976); acidici qididymal glycoprotein (AEG) (Lea et aZ., 1978); 32-kDa protein (Wong and Tsang, 1982); sialoprotein (Faye et d., 1980); MEW (Rankin et aL, 1992b); 4E9 antigen (Moore et aL, 1994; Xu and Hamilton, 1996; Xu et aL, 1997);and cystein-rich secretory protein1(CRISP-1) (Eberspacher et aZ., 1995). Similar proteins seem to be present in other mammals. Molecular cloning revealed that the protein is specific to the epididymis and beyond this organ is only found in salivary glands (Brooks el aL, 1986b; Mizuki and Kasahara, 1992). In situ hybridization localized the mRNA in the distal parts of the rat epididymis, beginning only in the very distal caput region (Garrett et aL, 1991;Fig. 6); the homologous mouse protein was immunolocalized in the corresponding distal epididymal region (Rankin et aL, 1992b). The encoded protein is a member of the family of CRISPS (Haendler et aL, 1993) that comprises a number of gene products specific to various parts of the male genital tract and salivary glands (Kasahara et aL, 1989; Kratzschmar et aZ., 1996), as well as a venom protein from the salivary secretions of the lizard Heloderma horridum (helothermine, Morrissette et aL, 1995). The rat epididymalsecretory protein has been suggested to bind to sperm and has been implicated in the fertilization process. However, its location on the sperm surface remains questionable. A monoclonal antibody directed against the protein E suggests binding to the sperm tail. Polyclonal antibod-

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ies raised against the purified rat D/E protein, on the other hand, reacted with the postacrosomal region of the rat sperm head and seemed to block fertilization both in vivo and in vitro (Rochwerger and Cuasnicu, 1992; Rochwerger et al., 1992; Perez-Martinez et al., 1995). Two independent research groups (Hayashi et al., 1996; Kratzschmar et al., 1996) cloned a related secretory protein from the human epididymis, designated ARP or CRISP-1, respectively. Since homology screening failed to obtain the human epididymal gene product, the strategy applied included a unidirectional RT-PCR method based on a short conserved amino acid stretch, VGH(Y/H)TQ (Hayashi et al., 1996), common to most members of the CRISP family. Because the human epididymal protein shows only 40% amino acid identity with its rodent counterparts, it may not represent the true genetic 660rtholog.’7 Nevertheless, it is the only human CRISP-like protein that is specifically expressed in the epididymis.

2. The Major “Maturation-Associated” Glycopeptide HE5KD52 A major human epididymal cDNA obtained by the differential cDNA library screening, HE5, was found to be colinear with the lymphocyte CD52 cDNA (Kirchhoff et al., 1993), encoding an unusually small but highly glycosylated glycosylphosphatidyl-inositol(GP1)-anchored cell membrane glycopeptide (Xia et al., 1991,1993;Treumann et al., 1995). Northern analyses revealed that HE5KD52 mRNA is abundant only in two cell types: the epithelial cells of the distal epididymis and vas deferens and blood lymphocytes. mRNA was never found in either spermatogenetic cells or spermatozoa. In situ hybridization showed that the vast majority of the epididymal transcripts were indeed derived from the duct epithelium (Kirchhoff et al., 1993; Krull et al., 1993). Anti-CD52 antibodies raised against the lymphocyte antigen, CAMPATH-1 (Hale et al. 1990), reacted with the corpus and cauda epididymal epithelium, cauda fluid, and epididyma1 but not testicular spermatozoa (Hale et al., 1993; Kirchhoff, 1996). The cross-reactivity persisted in the ejaculate and on ejaculated spermatozoa. These results are consistent with the idea that the antigen is only produced posttesticularly by the genital tract epithelial cells, shed into the lumen with its GPI anchor intact by an unknown mechanism, and incorporated via its GPI anchor into the sperm membrane (Kirchhoff and Hale, 1996; Yeung et al., 1997). Although the mature CD52 peptide backbones are extremely variable among species (Kirchhoff and Hale, 1996), homologous gene products showing highly similar expression patterns have been found in all mammals investigated thus far, including mice (Kubota et al., 1990 Kirchhoff, 1994; Fig. 7) and rats (Zeheb and On,1984; Eccleston et al., 1994; Kirchhoff,

FIG. 7 Localization of the maturation-associated CD52 sperm surface antigen in the murine epididymis as revealed by indirect Cy3 immunofluorescenceemploying the monoclonal “B7” antibody (raised by Kubota et al., 1990, against the corresponding lymphocyte antigen and kindly provided by N. Minato, Kyoto). Strong CD52 expressionstarts distally from the caput in the murine corpus epididymidis.Note the sharpe demarcation of distinct immunostainingpatterns by connective tissue septula (arrows), reflecting different levels of CD52 expression in various epididymal segments. Ca, caput region; Co, corpus region. Scale bar = 200 pm.

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1994,1996).Rat CD52 has long been known as the major galactose oxidasel NaB(3H)4-labeledsperm membrane glycoconjugate (Moore et al., 1989; Eccleston et al., 1994) which appears on the sperm surface in the distal epididymis at the time when the spermatozoa acquire their major physiological properties of maturation. Since the mature CD52 peptides on the sperm surface are so small and diverse, they may provide merely a scaffold for the presentation of a large (conserved?) glycan. A main feature of epididymal CD52 expression in all species is its highly regionalized pattern with maximum mRNA levels presenting in the distal parts of the epididymis. Regionalizationseems to be even more pronounced on the level of the CD52 glycopeptides. Areas of distinct levels of CD52 expression seems to be demarcated by connective tissue septulae (Fig. 7). In the rat epididymis, CD52 mRNA expression revealed a novel aspect of regionalization, namely, a region-dependent variation in the poly(A) tail length (Pera et al., 1997). The rat cauda epididymidis and, to a lesser extent, the deferent duct contained “long” mRNA molecules carrying an extended poly(A) tail. Their occurrence coincided with the occurrence of the principal M , 27 kDa glycopeptide that was inferred to represent the CD52 glycopeptide. This spatial correlation might suggest that only the long caudal CD52 mRNA molecules were efficiently translated, although there is no proof for this assumption. Other mechanisms, i.e., a regionalization of glycosylation (Hamilton et al. 1986), are not precluded.

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N. Factors Regulating Gene Expression in the Adult Epididymis

It is widely accepted that tissue-specific gene expression is mediated predominantly on the level of transcription. Transcriptional regulation would occur via binding of tissue-specific trans-acting factors or via a peculiar combination of more or less ubiquitous trans-acting factors (i.e., transcription factors) onto cis-acting DNA sequences (i.e., response elements) located in the vicinity, most commonly within the promoter region, of the concerned genes. To date, neither true epididymis-specific nor regionspecific trans-acting factors have been characterized, and it may well be possible that they do not exist. In the following sections, a number of transcription factors will be discussed that may be involved in the transcriptional regulation of epididymal gene expression. Moreover, nonnuclear molecules involved in ‘paracrine’signalling and signal transduction pathways of the genital tract will be referred to. Additionally, a very few results point to the possibility that other levels of gene regulation,

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e.g., regulation of mRNA stability and decay, may play a role in the epididymis as well.

A. Steroid Hormone Receptors

1. Androgens and AR Androgen concentrations in rete testis and epididymal duct fluid (Brooks, 1990), as well as in the spermatic vein (Setchell et al., 1994), are much higher than those in peripheral blood, and there is hardly any adult epididyma1function or gene product which has been described that is not responsive to or modulated by androgens or their withdrawal. Androgen functions are believed to be exerted through the ubiquitous AR. The epididymis actually belongs to the small group of organs with reportedly high AR expression (Dankbar et al., 1995). However, different from prostate and seminal vesicles, AR in the epididymis seems barely affected by castration or antiandrogen treatment (Paris et al., 1994). Nuclear AR is found in all regions and most cell types of the epididymis. Of all cell types examined, the principal cells of the duct epithelium showed most intense immunostaining of their nuclei (Takeda et al., 1990, Ruizeveld de Winter et al., 1991; Roselli et al., 1991; Goyal et al., 1997). There is some variation in the literature concerning a regionalized expression of the AR mRNA (Dankbar et al., 1995; Viger and Robaire, 1995; Ungefroren et al., 1997) and the AR protein (Sar et al., 1990; Roselli et al., 1991; Pans et al., 1994; Goyal et al., 1997) within the mammalian epididymis. In most species, however, the caput region seems to express the highest levels. Still, inferences related to quantitative differences in the various regions of the epididymis must be drawn with caution. There is an indication that androgen dependency of a tissue is not necessarily reflected by its AR expression status (Dankbar et al., 1995), and the rather small local differences in epididymal AR expression would not seem to account for most of the observed regional differences of androgen responsiveness of epididymal gene expression (OrgebinCrist, 1996). A few androgen-responsive genes encoding secretory proteins of the epididymal epithelium have been studied on a molecular level. The promoter region of the murine AEGKRZSP-1 gene contains at least 12 sequence stretches with close similarity to the androgen-response element (ARE) consensus sequence, and 2 of these have been shown to bind the AR in v i m as a tandem (Schwidetsky et al., 1997). Additional studies are needed to demonstrate that these sequences represent true ARES. Functional ARE%have been described in the promoter regions of two other

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murine genes, namely, the GPX5 promoter (Ghyselinck et aZ., 1993;Lareyre et aL, 1997) and the promoter of the mouse vas deferens protein (MVDP) gene (Fabre et al., 1994). The levels of induction as observed during in vitro transfection studies, however, were rather low compared to the dramatic in vivo changes observed in the steady-state mRNA levels after castration and testosterone replacement (Manin et aZ., 1992; Rigaudiere et aZ., 1992). This suggests that other factors might act synergistically with the AR on their promoters. Also, nuclear run-on experiments suggested that transcriptional regulation at least of GPX5 mRNA levels would account for only part of their androgen responsiveness (Lareyre et al., 1997). Increased attention has been paid to the regulation of mRNA stability as a control point of gene expression, and steroid hormones represent some of the earliest agents shown to control the stability of a substantial number of mRNAs (Nielsen and Shapiro, 1990). However, the mechanisms by which androgens could govern mRNA stability and turnover in the epididymis are even less well understood. In the rat, epididymal CD52 mRNA levels are affected by androgens in vivo (Pera et aZ., 1997), and testosterone levels act synergistically with temperature and unknown (testicular?) factors in its modulation. An important part of this modulation may take place at a posttranscriptional level. Supporting this idea, CD52 mRNA molecules in the rat epididymis displayed regulated region-specific and androgendependent transitions in their degree of polyadenylation (see Section II,C2). CD52 mRNAs with “short” poly(A) tails are highly responsive to androgen withdrawal and testosterone supplementation, whereas mRNAs with long poly(A) tails are not (Pera et aZ., 1997). Some gene products with a wider tissue distribution, such as GGT, seem to be androgen responsive only in the epididymis (Palladino and Hinton, 1994b),emphasizing the importance of other (tissue-specific?)transcription factors or cofactors in androgen regulation. Moreover, specific genes display a differential response to androgen withdrawal depending on the epididymal segment investigated (Orgebin-Crist, 1996), suggesting the presence of segment-specific combinations of various transcription factors and their cofactors. Again, GGT offers an impressive example of differential androgen responses in the various epididymal regions of the rat depending on different promoters (Palladino and Hinton, 1994b). Although GGT mRNAs types 11-IV disappear from the initial segment after castration, only types I1 and I11 are reinduced by testosterone supplementation. On the other hand, type I1 mRNA, but not type IV, is responsive to androgen in the cauda region. In several cases recovery of caput epididymal mRNA expression was not complete following testosterone replacement therapy (Orgebin-Crist, 1996), suggesting that nonsteroidal testicular factors (Lan et aZ., 1998) and/or region-specific factors from the

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epididymis may be involved specifically in the regulation of gene expression in the caput. Although androgens play a key role in directing stromal-epithelial interactions in the male genital tract (see Sections II,A,2 and II,B3), and nuclear AR is highly expressed in the epididymal mesenchyme, virtually nothing is known about androgen-responsivegenes in the stromal compartment of the epididymis. To address the molecular mechanism of androgen action in the stroma of the prostate, a stromal cell line was developed whose proliferation was significantly stimulated by physiological concentrations of androgens, and differential display PCR was used to demonstrate the presence of androgen-regulated transcripts in these cells (Gerdes et a!., 1996). Such genes should provide novel tools for defining molecular mechanisms of androgen action and elucidating the significance of stromal androgen-regulated genes in stromal-epithelial interactions. 2. Estrogens and ER

Although estrogens are the classical “female” hormones, it is their mechanism of action in the epididymidis that has recently been unraveled. Estrogen concentration can be as high as 250 pg/ml in rete testis and caput epididymal fluid (Ganjam and Amann, 1976; Free and Jaffe, 1979) which is higher than in female peripheral blood. Estrogens regulate sperm concentration by the reabsorption of luminal fluid in the head of the murine epididymis, and this function seems to be exerted through the ERa (Hess et al., 1997a). In the mouse, ERa mRNA is ubiquitously expressed in both sexes, including the reproductive tracts (Couse et aL, 1997). Involvement of the ERa receptor in the regulation of fluid reabsorption was demonstrated by studying the male ERKO (Korach et al., 1996), which is infertile. The reproductive tract in ERKO males contains swollen efferent ducts and a dilated rete protruding into the testis, suggesting that luminal fluid is not being removed by the ductular epithelium. Dysfunction of efferent ducts was shown in an in v i m experiment by surgical occlusion of the ductular ends (Hess et aZ., 1997a).These observationsdefine a physiological endpoint of estrogen action in the male genital tract which is fluid reabsorption and sperm concentration. ER-regulated genes involved in this process have not been described. Candidate genes may be those encoding Na+/K+-ATPase channels, chloride channels, including the cystic fibrosis transmembrane conductance regulator CTFR, and the aquaporins. Different from the AR and also from the ER& the distribution of the ERa receptor followed a very clear regionalized pattern within the male genital tract. In the rat, ERa mRNA expression was greatly enhanced in the caput epididymidis compared to other regions of the male genital tract

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(Hess et al., 1997b). Strongest immunoreactivity of ERa was seen in the epithelia of the ductuli efferentes and the initial segment of the epididymis, whereas the epithelium of the rete testis, the rest of the caput, and the distal epididymis stained less intensively. The vas deferens epithelium was ERa negative. Similar results have been reported for other mammals (West and Brenner, 1990; Goyal et al., 1997; Ergiin et al., 1997). Thus, the spatial pattern of ERa expression correlates with the reported region of fluid reabsorption and sperm concentration in the caput epididymidis. ERP mRNA, in comparison, shows no clear regionalization within the epididymis but is expressed throughout the genital tract. Its role can only be speculated upon at this time, and within the epididymis its role remains unknown. Comparing various mouse tissues, however, ERP seemed to be more specific to the genital tracts. In the male, significant levels of ERP mRNA were observed only in the epididymis and prostate (Couse et al., 1997). 3. Role of the Androgen-Binding Protein Steroids are presented to the epididymal epithelium from the vasculature as well as from the epididymal lumen. DHT concentrations in the caput duct lumen are apparently much higher than are androgen concentrations, mainly testosterone, in the vasculature of the caput (Brooks, 1990; Setchell et al., 1994). The same seems to be true for estrogens (Ganjam and Amann, 1976; Free and Jaffe, 1979). However, bioavailability of steroids in the epididymis seems to be regulated by intraluminal steroid-binding proteins. ABP and steroid hormone-binding globulin (SHBG) are secretory glycoproteins that bind DHT, testosterone, and estradiol with high affinity (Joseph, 1994). Both proteins differ only in their oligosaccharide content. The livers of most adult animals, but not of rodents, secrete SHBG into the blood. ABP is secreted by Sertoli cells into the rete testis and has been described in the genital tract of many mammals, including humans, rats, and mice. Rat epididymal ABP has been studied most intensively (Joseph, 1994).The mouse testis also synthesizes ABP; however, it does so at mRNA and protein levels of only 5% the levels in the rat. It is produced by the Sertoli cells of the testicular seminiferous tubules and is secreted into the rete testis fluid. After transport to the epididymis, it is internalized by the epithelium of the initial segment and caput epididymis by what is thought to be a receptor-mediated process (Joseph, 1994). There has been a longstanding interest in ABP/SHBG function as a steroid carrier (Hammond, 1995), especially in the caput epididymidis (Anthony et al., 1984a,b;Turner and Roddy, 1990; Turner et al., 1994, 1995). In vivo microperfusion was performed in the rat caput epididymidis to study the role of ABP/SHBG

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and other intraluminal factors in the regulation of protein synthesis and secretion (Turner ef al., 1995). Intriguingly, the ABP-homologous SHBG was found to stimulate de n o w protein synthesis, i.e., gene expression in the epididymal epithelium, most efficiently when it was perfused in the absence of androgen (Fig. 8). Overexpressionof ABPISHBG in male transgenic mice, on the other hand, apparently did not af€ect fertility (Joseph ef al., 1997). Knockout mouse models which do not have functional ABP may aid in elucidating its function in the future. 6. Vitamin A, RA, and RARa

Vitamin A or its biology active form, RA, are required for the normal development and maintenance of most epithelia,including the reproductive tract epithelium (see Section I1,C). In the adult epididymis, vitamin A deprivation results in replacement of the normal columnar epithelium by a stratified squamous keratinizing epithelium (Wolbach and Howe, 1925). As described for steroids, it is accepted that RA action at least in part is mediated through the binding of nuclear receptors to response elements in the promoter regions of retinoid-responsive genes (see Section 11,C). Two families, each comprising several members of receptors, have been identified RARs and retinoic X receptors (RXRs) (Giguere, 1994). RARa, although almost ubiquitously expressed, seems to play a critical role in the reproductive tract. In the adult male rat, the cells of the epithelium lining the epididymis and vas deferens express abundant RARa mRNA and protein (Akmal ef aL, 1996). In sifu hybridization as well as immunohistochemistry demonstrated the expression of the RARa to be highest in the initial segment and proximal caput, low in the corpus, and high in the distal cauda. Male transgenic mice expressing a dominant-negative mutation of RARa-1 directed to the genital tract by the murine mammary tumor virus (MMTV) promoter are infertile due to epididymal dysfunction (Costa ef al., 1997). The MMTV promoter was shown to be active only in adult animals (Stewart et aL, 1984), and expression of the transgene was evident in the adult mouse epididymis and vas deferens. Sperm developed normally in the testis but degenerated in the epididymis and vas deferens because inspissated ductal fluid blocked their normal passage (Costa et aL, 1997). The regionalized localization of RARa in the caput and cauda regions of the epididymis suggests that RA may be involved in the transcriptional regulation of epididymalproteins important for sperm maturation processes in the caput and for sperm storage in the cauda. To date, however, it is not clear whether this involves proteins directly conferring fertilizing ability to the sperm or whether this is mediated simply by maintenance of the epididymal epithelium.

STAIN kD

LF

PF TE

AUTORAD LF

PF TE

FIG. 8 Stimulation of de novo protein synthesis in the rat caput epididymidis by steroid hormone-bindingglobulin (SHBG). Proteins synthesizedand secreted into rat caput epididymidis in vivo after 3-h perfusion of tubules with [35S]methioninewere separated by SDS-PAGE. Representative Coomassie blue-stained gels (STAIN) show proteins present in luminal fluid of unperfused lengths of tubule (LF), luminal perfusion (PF), and tubule extract (TJ3). Autoradiograms of these gels (AUTORAD) show total proteins synthesized (TE) and those secreted into both untreated length of tubule (LF) and a length containing perfused lumen fluid (PF). Perfusion fluids were artificial caput fluid (ACF), and the same fluid containing SHBG, dihydrotestosterone (DHT), or SHBG + DHT. SHBG stimulated protein synthesis more than DHT alone or SHBG + DHT [reproduced with permission from Turner, T. T., Miller, D. W., Avery, E. A. (1995). Protein synthesis and secretion by the rat caput epididymidis in vivo: Influence of luminal microenvironment. B i d . Reprod. 52,1012-10191.

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C. Other Epididyrnal Transcription Factors

1. Polyomavirus Enhancer Activator-3 Compared to the previously described ubiquitous members of the ligandactivated nuclear receptor superfamily, PEA3, although clearly not epididymis specific, shows a remarkable degree of tissue restriction. In the adult mouse, this transcriptional modulator was shown to be highly expressed in the brain, in metastatic mammary tumors and several derived cell lines, and also in the epididymis (Xin et al., 1992). It is a highly conserved, E26 transformation-spec (ets-like) transcription factor showing similarity to the PEA3-like factors ER81 and ERM (Monte et al., 1994,1995). Like the other ets-type members, it activates many genes by binding to the PEA3binding elements in their promoter and was actually cloned by using its DNA-binding motif (5’-AGGAAG-3’) as a probe (xin et aZ., 1992). In the rodent epididymis, PEA3 seems to be expressed in a regionalized manner, showing highest expression in the caput region at both mRNA and protein levels (Lan et al., 1997, Drevet et aZ., 1998).Numerous PEA3-binding motifs are found in the promoter region of two caput epididymidis-expressed genes, i.e., in the promoter of the murine GPXS gene (Ghyselinck et al., 1993) and in promoter IV of the rat GGT gene (Lahuna et aZ., 1992; Okamoto et al., 1994). Gel shift assays revealed specific PEA3 binding to these motifs (Lan et aZ., 1997; Drevet et al., 1998), and in vitro promoter studies using cotransfection with PEA3 showed modulation of transcriptional activity (Drevet et al., 1998). Thus, PEA3 was suggested to be an important epididymal transcriptional modulator of these caput-expressed genes. The PEA3 transcription factor gene in the rodent caput epididymidis was proposed to be regulated by androgens (Drevet et aZ., 1998) and testicular factors, specikally bFGF (Lan et aZ., 1998). In other systems, DNA binding activity and transactivation activity of PEA3 has been shown to be under the regulation of phorbolesters, suggesting an involvement of the PKC pathway, and growth factors, including bFGF, via ras-raf mitogenactivated protein b a s e signal transduction pathways (O’Hagan et al., 1996). Recent studies in human breast tumors revealed that PEA3 is a downstream target of the HER2Neu receptor tyrosine kinase, and that HER2/Neu upregulates transcriptional activity of PEA3 (O’Hagan and Hassell, 1998). A constitutively activated allele of the HER2Neu receptor that was, among others, targeted to the epithelial cells of the male reproductive tract (Stocklin et al., 1993; Guy et ale, 1996) caused hyperplasia and hypertrophy in the epididymis, vas deferens, and seminal vesicles. The transgenic males were infertile. It is not known,however, whether this might be caused by PEA3 overexpression in the epididymides of these animals.

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2. Segmentation Gene Pax-2

Segmentation genes have recently been shown to dictate positional and cellular differentiation in the developing mesonephros and Wolffian duct derivates (see Section II,A,3). A number of them are still transcribed during postnatal development and adulthood; however, the significance of these observations is unclear. Transcription in adult tissues of genes with identified roles in embryogenesis has been observed but may not result in a functional protein. Sry transcripts, for example, are circular in adult testes, do not associate with polysomes, and thus are unlikely to be translated (Cape1 et al., 1993). Pax-2 expression is critical for the early development of epithelial components in the intermediate mesoderm (Torres et al., 1995; see Section II,A,3). In addition, there seems to exist a second phase of Pax-2 gene activity, mainly in the adult kidneys and urinary bladder and in the male and female genital tracts. In the mouse, high Pax-2 expression at the transcript level was shown in the adult epididymal duct but not in the testis (Fickenscher et al., 1993). This finding strengthens the hypothesis that Pax-2 might be a specific transactivator of gene expression in the adult genital tract as well. There are, however, currently no known in vivo DNA recognition sequences nor epididymal genes known to be regulated by Pax-2. The authors speculate that the uteroglobin gene, showing a similar posttesticular pattern of gene expression in the epididymis, might be a target gene for Pax-2. Using a polyclonal antiserum, the Pax-2 protein was localized within the epithelial compartment of the murine epididymal and deferent duct at all time points investigated from birth to adulthood (Oefelein et al., 1996). Neither developmental nor regional variations in Pax-2 immunostaining intensity were reported. However, final conclusions must await a more detailed and explicit analysis. 3. Homeodomain Transcription Factor Pem

The Pem homeobox gene also shows a selective pattern of tissue distribution in rodents. While it seems to be restricted to extraembryonic tissue during early murine development (Lin et aZ., 1994), in adult mice and rats Pem transcripts are present predominantly in reproductive tissues, namely testis and epididymis in the male and ovary and placenta in the female, but not in most other tissues investigated, including thymus, spleen, bone marrow, brain, liver, intestine, pancreas, lung, kidney, and heart (Lindsey and Wilkinson, 1996~).Gene transcription is initiated at two alternative promoters depending on tissue, and Pem transcripts undergo alternative splicing events, again depending on the tissue investigated (Maiti et aZ., 1996). Rat epididymal and testicular Pem transcripts are distinct from “female” or “somatid’ transcripts, exhibiting differences in their 5’ ends and using a

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different, possibly androgen-dependentpromoter. Interestingly,the epididymis expressed much higher levels of male-specific, androgen-dependent

Pern transcripts compared to the testis and at earlier, prepubertal stages, whereas in the testis these transcripts accumulated only postpubertally (Lindsey and Wilkinson, 1996c; Maiti et aL, 1996). In situ hybridization and RNase protection assays localized Pern transcripts mainly in the apical cells of the distal corpus and proximal cauda region of the rat epididymis (Lindsey and Wilkinson, 1996b). This distal localization is congruent with the regionalized expression pattern of a number of epididymal gene products, namely, the acidic epididymal glycoprotein D/E (Rankin et aL, 1992b; Fig. 6), and the major maturation-associated glycoprotein CD52 (Krull et aL, 1993; Pera et aL, 1994; Figs. 1and 7). Future studies should reveal whether Pem transcription factors specifically bind to the promoter regions of epididymis-expressed genes. A processed Pem gene, possibly generated by reverse transcription of spliced Pern mRNA, has been identified at a novel site in the rat genome that is transcriptionally active (Nhim et aL, 1997). This processed gene, r.Pem2, is expressed at high levels in the rat epididymis but not in any other tissues that express the Pern gene, including the testis. The r.Pem2 gene exhibits differential developmental regulation, and r.Pem2 transcripts accumulate in the caput and cauda regions, excluding the initial segment (Nhim et aL, 1997). Different from the Pern transcripts, r.Pem2 expression in the epididymis seems to be independent of androgen since hypophysectomy and testosterone supplementation had no effect on its transcript levels. However, several putative PEA3-binding sites exist near the transcription start site of r.Pem2 (Nhim et aZ., 1997).

D. Nonnuclear Protooncogenes and “Paracrine” Factors 1. Receptor Tyrosine Kinase c-ros

The epithelial receptor tyrosine kinase c-ros has been shown to control regionalization and differentiation in the mouse caput epididymis (Sonnenberg-Riethmacher et aL, 1996). The gene was originally identified in mutant form as an oncogene. The protooncogene encodes an orphan receptor tyrosine kinase homologous to the sevenless tyrosine kinase receptor and expressed only in a small number of epithelial cell types, including the epithelial cells of the caput epididymidis (SonnenbergRiethmacher et aL, 1996). Targeted mutations revealed an essential role in male fertility. While female homozygous mutant animals displayed normal fertility, male c-rosd- mutants were sterile. Sperm generation and storage were not affected. Rather, the primary defect was located within the epidid-

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ymis (Sonnenberg-Riethmacher et al., 1996). Histological analysis revealed the absence in mutant males of the characteristic tall columnar epithelial cells of the initial segment of the epididymis. c-ros, comparable to Pax-2,is expressed in both the early Wolffian duct anlage during embryogenesis (Sonnenberg et al., 1991; see Section II,A,2) and in the adult caput epididymidis of the mouse (Sonnenberg-Riethmacher et al., 1996; Fig. 2). Expression during these different time periods suggests two possible models for c-ros function in male fertility. First, loss of embryonic expression in c-ros-l- mutant males around the time of male sexual differentiation (see Section I17A,2)may result in a developmental “switch” in signal transduction cascades, finally transforming the prospective initial segment into a distal caput-like structure. This transformation could explain the observed morphologic deformation of the epididymis, followed by multiple molecular changes during later development. Infertile sperm would result from these early developmental defects. A second model of male c-ros-l- infertility proposes that it has a more direct function during sperm maturation in the adult. Loss of c-ros signaling in the epididymal epithelium would directly result in changes in the sperm properties within the caput region, rendering sperm infertile. Although these two models are not necessarily exclusive,the observation that spermatozoa in c-ros-l- mutant males are bent and show slightly reduced motility (Yeung et al., 1998) seems to support the second model of a more direct effect of c-ros in male fertility. Sperm from c-ros-l- males can fertilize in vitro, and the fertilized eggs develop without apparent defects (SonnenbergRiethmacher et ul., 1996), suggesting that even very subtle defects are sufficient to completely knock out sperm function in vivo. 2. Protooncogene A-raf

The protooncogene A-rufis expressed at high levels in a variety of steroidresponsive tissues, including epididymis (Winer et al., 1993). It belongs to a family of cytoplasmic proteins with associated serinekhreonine kinase activities and is involved in a number of second messenger signaling pathways. Evidence from other systems suggests that raf kinases are involved in regulating both expression and activity of transcription factors in the nucleus. In the mouse epididymis, A-raf exhibits a regionalized pattern of expression (Winer et ul., 1993; Winer and Wolgemuth, 1995). The highest levels are observed in the caput epididymidis. Within the caput, A-ruf mRNA levels are not uniform: Maximum levels are found in the proximal caput, lower levels in the initial segment, and intermediate levels in the distal caput. The high levels of A-raf mRNA expressed in the epididymis and other androgen-dependent organs, as well as the presence of a potential steroid response element in the 5’-flankingregion of the human A-raf gene,

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suggest that A-raf expression might be androgen dependent. Castration abolished A-raf expression throughout the epididymis, and nonsteroidal testicular factors seemed to modulate the androgen response within the initial segment (Winer and Wolgemuth, 1995). Interestingly, the transcription factor PEA3 (see Section IV,C,l) is activated by two distinct mitogenactivated protein kinase (MAPK) cascades acting through the Ras signal transduction pathways: one of them involving v-raf (Wasylyk et aZ., 1989) and MAPK family members being substrates for c-raf kinase activity (O’Hagan et al., 1996). 3. Basic Fibroblast Growth Factor

bFGF has been suggested to represent an important testicular factor regulating gene expression in the initial segment and possibly also in other parts of the adult epididymis (Lan et al., 1998). A signal transduction pathway was hypothesized (bFGF + bFGFR + Ras + raf +. MAF’K + PEA3) that might activate a number of epididymal genes via PEN-binding motifs in their promoters, i.e., GGT (Lan et aZ., 1997), GPXS (Ghyselinck et aZ., 1993; Drevet et al., 1998) and others. bFGF-like proteins were detected in rete testis fluid, and rete testis fluid was able to restore GGT activity and protein levels in the initial segment of the rat epididymis (Lan et al., 1998). bFGF among other growth factors seems to be produced by the testis (Mullaney and Skinner, 1992) but possibly also by the epididymis. According to earlier studies, bFGF is involved in the fetal development of the epididymis and may act as a mediator of androgens in mesenchymalepithelial interaction (Alarid et al., 1991; see Section II,B,3). In the prepubertal 20-day-old rat, bFGF mRNA was detected in the seminal vesicle, prostate, epididymis and, at low levels, in the testis (Mullaney and Skinner, 1992). In whole testis, bFGF seemed predominantly expressed during early prepubertal development and decreased with sexual maturity. Still, it may be active in the adult organ.

E. Direct Effect of Temperature on Epididymal Gene Expression

In scrotal mammals, physiological scrotal hypothermia is necessary for normal fertility, and testicular descent is complete either at birth or shortly after birth in most species. An elevated temperature adversely affects not only testicular but also epididymal functions (Bedford, 1978, 1991, 1994). In the cauda epididymidis of the rat, sperm storage functions as well as its ability to support sperm survival are severely impaired at abdominal temperature, even when this situation is associated with a normal scrotal

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testis (Foldesy and Bedford, 1982). Elevation of caudal temperature in vivo is accompanied by a dramatic change of cauda epididymidal morphology (Fig. 9a), of cauda epididymidal fluid proteins (Esponda and Bedford, 1986), and of water and ion flux across the epithelium (Rasweiler and Bedford, 1982; Wong et al., 1982). As postulated for the testis, however, the influence of temperature seems to be selective and per se does not disturb all epididymal functions. Indeed, sperm maturation in the proximal part of the organ does not seem to be directly affected by the elevated temperature (Bedford and Yanagimachi, 1991). Little is known about the possible mechanism(s) of direct temperature action in the epididymis. In the rabbit, body temperature specifically reduced de novo synthesis of two epididymal proteins in vivo, an effect which could not be reversed by androgen treatment (Regalado et aZ., 1993). In order to investigate temperature effects on the epididymis independently of effects on the testis, primary cell culture systems of the epididymal epithelium were studied (Pera et al., 1996; Carballada and Saling, 1997). Cultured canine epithelial cells (Pera et aZ., 1996) endogenously expressed high levels of a specific mRNA homologous to the human CD52DYE.5 mRNA (see Section III,C,2). Exposure to a culture temperature of 37°C (correspondingto abdominalbody temperature), compared to 33°C (corresponding to a mean scrotal temperature), dramatically and specifically reduced the levels of this mRNA but not those of others (Fig. 9b). The temperature effect was also observed during exposure to transcriptional inhibitors, suggesting that it operates on a posttranscriptional level and selectively reduces the half-life of CD52 mRNA (Pera et al., 1996). Similar effects on CD52 mRNA stability were observed during in vivo experiments in the rat (Pera et al., 1997)that would support a direct effect of temperature on epididymal gene expression. Interestingly, temperature effects in vivo seemed to correlate to the length of the poly(A) tail of the CD52 mRNA, again indicating a posttranscriptional effect. It is well established that the half-lives and decay rates of specific mRNAs are dependent on poly(A) tail length and may be altered in response to physiological signals, such as changing levels of hormones and blood metabolites (Ross, 1996).Although little is known about the perception and transduction of an external temperature signal by animal cells, exposure of the epididymal cells to varying temperatures may act by a similar mechanism. F. Alterations of Epididymal Gene Expression Pattern Related t o Aging

Numerous studies have described maturational changes occurring in the gene expression pattern of the epididymis during puberty (see Section

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FIG. 9 Direct temperature effects on morphologyof cauda epididymidis (a) and on epididymal gene expression as revealed by comparison of mRNA levels (b). (a) Histological sections through the center of the cauda epididymidis of a 450-gmale Sprague-Dawley rat (top; control) whose contralateral cauda had been reflected in the abdomen 3 months previously (bottom, crypt-epididymd side). Note that on both sidesthe epididymisremained in continuity with a normal scrotal testis and that the number of spermatozoa in the caput segment was comparable both on the control and crypt-epididymal sides [reproduced with permission from Foldesy, R. G., and Bedford, J. M. (1982).Biology of the scrotum. 1. Temperature and androgen as determinants of the sperm storage capacity of the rat cauda epididymidis. Biol. Reprod. 26, 673-6821. (b) Downregulation of CD5YCE5 mRNA levels by elevated culture temperature (37"C), as shown by Northern analysis. Epididymal cells were grown in parallel wells for 5 days at either 33 or 37°C in different media, as indicated for each pair of lanes. Equal amounts of total RNA (5 pg) from parallel wells were loaded per lane. (Lanes 1-8) RNA was from cells grown in standard medium. (Lanes 9-12) RNA was from cells grown in minimal medium. Both media were supplemented as indicated for each pair of wells (FCSI BSA, fetal calf serum replaced with bovine serum albumin after 24 h +T, supplemented with androgens) (reproduced with permission from I. Pera, R. Ivell, and C. Kirchhoff. Body temperature (37°C) specificallydown-regulates the messenger ribonucleic acid for the major sperm surface antigen CD52 in epididymal cell culture, Endocrinology 137,4451-4459,1996, 0 The Endocrine Society).

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+FCS/ +BSA +FCS+T +FCS +FCSIBSA BSA+T n n - m n

33"37" 33"37" 33"37" 33"37" 33"37' 33" 37"

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b FIG. 9 (continued)

11,D);however, very little information is available on how aging affects the epididymis. The most common finding has been a gradual decline in the concentration of epididymal spermatozoa, most probably reflecting a defecit in testicular sperm production. In humans and rodents, male reproductive aging is accompanied by a decrease in testosterone production, decreasing serum testosterone levels, and atrophy of the seminiferoustubules, resulting in a germ cell loss (Wang et al., 1993; Wright et al., 1993; Zirkin et al., 1993; Gruenwald et al., 1994). Histologically, there is a report that principal cells from the aging rabbit epididymis show cytoplasmic vacuolization and accumulation of lipofuchsin pigment (Cran and Jones, 1980). Viger and

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Robaire (1995) showed, in a pioneering study, that in the Brown Norway rat, which has proved a useful model for the aging male (Wang et al., 1993), epididymal gene expression is specikally altered, and that the age-related effects vary depending on which region of the epidymis is studied. Expression of six different epididymal marker mRNAs was studied in rats ranging from 6 to 30 months, and the relative mRNA concentrations in the proximal and distal epididymides were assessed by Northern blot analysis (Viger and Robaire, 1995). No significant age-related changes in either segment were observed in the mRNA levels for the androgen receptor. Similarly, the mRNA levels of secretory proteins B/C = E-RABP and acidic epididymal glycoprotein AEG = DW did not change. In comparison, Sa-reductase type 1and type 2 mRNA levels decreased significantly in the caput/corpus region but not in the cauda of aged animals. SGP-2 mRNA levels, on the other hand, showed no age-related changes in the caputl corpus region but a sharp decrease in cauda sectionsfrom aged rats between 18 and 30 months. Interestingly, proenkephalin mRNA was detected only in the caputkorpus epididymidis of young adult males at the age of 6 months. Future studies should clarify whether these changes in epididymal gene expression are indeed important for fertility. Despite significant differences in serum testosterone and follicle-stimulating hormone levels, no differences in semen parameters of older (>50 years) compared to younger men (<30 years) were observed (Rolf et al., 1996).

V. Concluding Remarks The regionalized pattern of epididymal gene expression which becomes overt at puberty may originate in early fetal development when positional information is first provided in the mesonephros and Wolffian duct by a hierarchy of diffusible and membrane-bound signaling molecules, inducing specific transcription factors, e.g., members of the Hox gene family. At puberty, differentiation of the epididymal epithelium precedes sperm arrival, and the adult pattern of gene expression begins to develop clearly before the first sperm enter the caput epididymidis. Tests of the functional state of discrete sperm populations taken from successive levels of the adult epididymis have provided region-related profiles of developing fertility (Orgebin-Crist, 1969; Bedford and Hoskins, 1990), indicating that sperm gradually and progressively attain their full fertilizing ability. Transport of spermatozoa through the eutherian epididymis requires a mean period of about 6-14 days, depending on species. Net forward movement is generated through segmental contractions of the duct wall, and sperm are moved back and forth within a particular segment for a while, a process that is

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supposed to chum the duct contents and to expose all spermatozoa to the mileu of each epididymal region traversed (Bedford, 1975; Jaakola and Talo, 1983). These observations suggest on a physiological level that the epididymis consists of a number of consecutive functional subunits. Data presented previously showing region-specificdifferences of epididymalgene expression reflect this at a molecular level and also suggest that functional subunits of the epididymis might represent regulatory subunits. Although a number of important epididymal transcription factors have already been identified, organ-specific as well as region-specifictrans-acting factors of the epididymis have not yet been found, and it may well be possible that they do not exist. However, from the various studies on the regulation of epididymal gene expression it is becoming apparent that the caput region, and especially the efferent ducts and the initial segment, may be under the control of factors different from the rest of the epididymis. Estrogens and the ERa, receptor tyrosin kinase c-ros, as well as specific testicular factors, including ABP and bFGF, may play important regulatory roles mainly in the caput region, and future experiments may identify additional factors. Much less is known about regulatory mechanisms specifically controlling gene expression in the distal parts of the epididymis. Transcription factors, such as the Pem factors, which are predominantly expressed in the corpus and cauda regions may be involved. Also, the few results on temperature effects support the idea that direct effects of elevated temperature are operative mainly in the distal parts of the epididymis. A large number of epididymis-expressed mRNAs and proteins have been shown to respond to androgen withdrawal or supplementation in vivo (Orgebin-Crist, 1996). However, there is little information regarding direct transcriptional control, i.e., AWARE interactions in the promoters of androgen-responsive genes. Part of the regulation of epididymal gene expression may take place on a posttranscriptional level, and future studies may identify (steroid-dependent?) RNA-binding proteins involved in the control of RNA stability and translation. In most mammalian species investigated, physiologically significant numbers of fertile spermatozoa seem to appear first in the mid- to lower corpus epididymidis (Bedford, 1966; Orgebin-Crist, 1967; Bedford and Hoskins, 1990). Thus, it has been speculated that genes expressed in the proximal segments of the organ, especially in the caput epididymidis, are more or less directly related to maturational events, whereas genes expressed in the more distal parts may be involved in sperm maintenance and storage functions (Cornwall and Hann, 1995a). Although this idea is appealing, the current data concerning the functions of specific epididymal gene products are not adequate to support this. Functions of individual gene products as summarized previously appear to be more compatible on the whole with a role in the maintenance of sperm function and fertility rather than with the

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acquisition of novel functionsrequired during gamete interaction. However, functions of epididymal gene products are mostly inferred from structural similarities to proteins of known functions in other organs, and we have only a basic molecular understanding of epididymal functions. Targeted mutations of specificepididymal gene products, above all secretory proteins, may reveal hitherto masked functions. Also, we cannot exclude that a number of less frequent but functionally important gene products may have escaped our cloning approaches and may be identified in future studies. Acknowledgments The author is grateful to coworkers for their support and patience during the writing of this review. I am indebted to Richard hell and Ralph Telgmann, IHF,for improving the manuscript. I also thank the many collegues who supplied pertinent discussions of their work, prepublication results, antibodies, and illustrations-especially J. Michael Bedford, Rosa Carballada, Joel Drevet, Geoffrey Hale, Barry T. Hinton, Nagahiro Minato, Terry T. Turner. I apologize that much valuable information could not be cited. Work from the author’s laboratory was funded by Grants Ki 31715-1 and Iv 7/43 from the German Research Association and by Grant 01Ky9103 from the German federal ministry for science and technology.

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Waites, G. M. H., and Setchell,B. P. (1990). Physiologyof the mammalian testis. In “Marshall’s Physiology of Reproduction. Vol. 2 Reproduction in the Male” (G. E. Lamming, ed.), pp. 1-105. Churchill Livingstone, Edinburgh, UK. Wang, C., h u n g , A., and Sinha-Hikim, A. P. (1993). Reproductive aging in the male BrownNorway rat: A model for the human. Endocrinology W3,2773-2781. Wasylyk, C., Flores, P., Gutman, A., and Wasylyk, B. (1989). PEA3 is a nuclear target for transcription activation by non-nuclear oncogenes. EMBO J. 8,3371-3378. West, N. B., and Brenner, R. M. (1990). Estrogen receptor in the ductuli efferentes, epididymis and tesitis of rhesus and cynomolgus macaques. Biol. Reprod. 42,533-538. Wilson, J. D., Griffin, J. E., and Russell, D. W. (1993). Steroid 5-a-reductase 2 deficiency. Endocr. Rev. 14,577-593. Winer, M. A., and Wolgemuth, D. J. (1995). The segment-specificpattern of A-raf expression in the mouse epididymis is regulated by testicular factors. Endocrinology l36,2561-2572. Winer, M. A., Wadewitz, A. G., and Wolgemuth, D. J. (1993). Members of the raf gene family exhibit segment-specific patterns of expression in mouse epididymis. Mol. Reprod. Dev. 3516-23. Wolbach, S. B., and Howe, P. R. (1925). Tissue changes following deprivation of fat soluble A vitamin. J. Exp. Med. 42,753-777. Wong, P. Y. D., and Tsang, A. Y. F. (1982). Studies on the binding of a 32k rat epididymal protein to rat epididymal spermatozoa. Biol. Reprod 27,1239-1246. Wong, P. Y. D., Au, C. L., and Bedford, J. M. (1982). Biology of the scrotum 11: Suppression by abdominal temperature of transepithelial ion and water transport in the cauda epididymidis. Biol. Reprod. 26,683-689. Wright, W. W., Fiore, C., and Zirkin, B. R. (1993). The effect of aging on the seminiferous epithelium of the Brown Norway rat. J. Androl. 14, 110-117. Wu, S. C., Grindley, J., Winnier, G. E., Hargett, L., and Hogan, B. L. (1998). Mouse mesenchyme forkhead 2 (Ma): Expression,DNA-binding and induction by sonic hedgehog during somitogenesis. Mech. Dev. 70,3-13. Xia, M. Q., Tone, M., Packman, L., Hale, G., and Wddmann, H. (1991). Characterization of the CAMPATH-1 (CD52) antigen: Biochemical analysis and cDNA cloning reveal an unusually small peptide backbone. Eur. J. Immunol. 21,1677-1684. Xia, M. Q., Hale, G., Lifely, M. R., Ferguson, M. A., Campbell,D., Packman, L., and Waldman, H. (1993). Structure of the CAMPATH-1 antigen, a glycosylphosphatidylinositol-anchored glycoprotein which is an exceptionally good target for complement lysis. Biochem. J. 293,633-640. Xin, J. H., Cowie, A., Lachance, P., and Hassell, J. A. (1992). Molecular cloning and characterization of PEA3, a new member of the Ets oncogene family that is differentially expressed in mouse embryonic cells. Genes Dev. 6,481-496. Xu, W., and Hamilton, D. W. (1996). Identification of the rat epididymis-secreted 4E9 antigen as protein E. Further biochemical characterization of the highly homologous epididymal secretory proteins D and E. Mol. Reprod. Dev. 43,347-357. Xu, W. D., Wang, L. F., Miao, S. Y., Zhao, M., Fan, H. Y., Zong, S. D., Wu, Y. W., Shi, X. Q., and Koide, S. S. (1996). Identification of a rabbit epididymal protein gene. Arch. Androl. 37,135-141. Xu, W., Ensrud, K. M., and Hamilton, D. W. (1997). The 26kD protein recognized on rat cauda epididymal sperm by monoclonal antibody 4E9 has internal peptide sequence that is identical to the secreted form of epididymal protein E. Mol. Reprod. Dev. 46,377-382. Yanagimachi, R. (1994). Mammalian fertilization. In “The Physiology of Reproduction” (E. Knobil and J. D. Neill, eds.), pp. 189-317. Raven Press, New York. Yeung, C. H., Sonnenberg-Riethmacher, E., and Cooper, T. G. (1998). Receptor tyrosine kinase c-ros knockout mice as a model for the study of epididymal regulation of sperm function. J. Reprod. Fertil., in press.

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Yeung, C.-H., Cooper, T. G., Bergmann, M., and Schulze, H. (1991). Organization of tubules in the human caput epididymidk and the ultrastructure of their epithelia. Am. J. Anat. 191,261-279. Yeung, C.-H., SchrOter, S., Wagenfeld, A., Kirchhoff, C., Kliesch, S., Poser, D., Weinbauer, G. F., Nieschlag,E., and Cooper, T. G. (1997). Interactions of the human epididymalprotein CD52 (HE5) with epididymal spermatozoa from men and monkeys. Mol. Reprod Dev. 4% 267-275. Yoshida, M., Suda, Y., Matsuo, I., et al. (1997). Emxl and Emx2 functions in development of dorsal telencephalon. Development U4,101-111. ZBkBny, J., Gerard,M., Favier, B., Potter, S. S.,and DuboulB, D. (19%). Functional equivalence and rescue among group 11 Hox gene products in vertebral patterning. Dev. BioL 174, 325-328. Zeheb, R., and Orr, G. A. (1984). Characterization of a maturation-associated glymprotein on the plasma membrane of rat caudal epididymal sperm. J. Biol. Chem 259,839-848. Zeltser, L., Desplan, C., and Heintz, N. (1996). Hoxb-13: A new Hox gene in a distant region of the HOXB cluster maintains colinearity. Development 122,2475-2484. Zirkin, B. R., Santulli, R., Strandberg, J. D., Wright, W.W.,and Ewing, L. L. (1993). Testicular steroidogenesis in the aging Brown Norway rat. J. Androl. 14,118-123. Zondek, L. H., and Zondek, T. (1%5). The secretory activity of the maturing epididymis compared with maturational changes in other reproductive organs of the foetus, infant, and child. Acta Paediatr. Scand. 54,295-304.

Roles of Reactive Oxygen Species: Signaling and Regulation of Cellular Functions 1. A. Gamaley and 1. V. Klyubin Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia

Reactive oxygen species (ROS) are the side products (H202,0;-, and OH')of general metabolism and are also produced specifically by the NADPH oxidase system in most cell types. Cells have a very efficient antioxidant defense to counteract the toxic effect of ROS. The physiologicalsignificance of ROS is that ROS at low concentrations are able to mediate cellular functions through the same steps of intracellularsignaling, which are activated by natural stimuli. Moreover, a variety of natural stimuli act through the intracellularformation of ROS that change the intracellular redox state (oxidationreduction). Thus, the redox state is a part of intracellularsignaling. As such, ROS are now considered signal molecules at nontoxic concentrations. Progress has been achieved in studying the oxidative activation of gene transcription in animal cells and bacteria. Changes in the redox state of intracellularthiols are considered to be an important mechanism that regulates cell functions. KEY WORDS: Reactive oxygen species, Redox state, Transcriptional factors, lntracellular signaling, Thiols. o isa ~cademiiPress.

1. Introduction Reactive oxygen species (ROS) is a term used to indicate products which are generated as intermediates in the redox (oxidation-reduction)processes leading from molecular oxygen to water. Every cell type is known to produce a small amount of ROS either by normal physiological processes or due to effects of exogenous factors which occur naturally in the biosphere. Generation of a large amount of ROS such as superoxide anion and hydroInternational Review of Cytology, VoL 188

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Copyright 8 1999 by Academic Press. All rights of reproduction in any form reserved.

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gen peroxide by the NADPH oxidase complex of phagocytic cells has been studied since 1933 when Baldrige and Gerard found an extra respiration during phagocytosis. This cellular function is known to be an important defense mechanism against invading pathogenic agents. Since the early 1980s, increasing evidence has been presented that other cells and tissues can also produce small amounts of ROS by the NADPH oxidase-like enzyme. It has become increasingly clear that effects of ROS are not restricted to a protective role. The generation of ROS might be an important factor that triggers a variety of cellular functions (cell growth, development, protein synthesis, secretion, and locomotion). From the increasing evidence for the essential role of ROS inside the cell, two important questions arise: What is the molecular mechanism by which ROS are recognized by the cells and how are redox signals transduced into changes in gene expression? We intend to summarize the effects which ROS cause in the cell and to discuss molecular mechanisms underlying these effects.

II. Mechanisms Inducing Generation of ROS by Cells A. Steps in Reduction of Oxygen

ROS are any species that are more reactive than the ground state oxygen molecule that does not react directly (by a noncatalytic mode) with most organic substances. Most of the molecular oxygen consumed by aerobic cells during cellular metabolism is tetravalently reduced to H20. Such a reduction of O2to H20occurs in mitochondria in which cytochrome oxidase transfers four electrons to 02.However, when oxygen is partially reduced, it becomes “activated” and reacts readily with a variety of substances. This partial reduction occurs in one-electron steps, by addition of one, two, and four electrons to 02, which leads to successive formation of ROS. The scheme of these reactions is

o25 0;-e-\ op 5 0-5 0 2 .1

.1

.1

.1

‘02

H@

H202

OH’

4 2H+ H20

These are five possible species: superoxide anion 02’-, hydroperoxyl radical HO;, peroxide ion H02-, hydrogen peroxide H202,and hydroxyl radical OH’ (Green and Hill, 1984). The scheme shows that the first reduction product is 02’-.It is a base with the equilibrium with its conjugate acid, the hydroperoxyl radical HO;. In aqueous solution, at neutral or slightly

205

ROLES OF REACTIVE OXYGEN SPECIES

acid pH, 0;- is a relatively nonreactive species and dismutases to H202. This reaction either occurs spontaneously or is catalyzed by intracellular enzyme superoxide dismutase (SOD). It has been proposed that 02'-, owing to its unreactivity, can diffuse a long way from its site of production (Bielski et al., 1983; Gus'kova et aZ., 1984). However, at the site of low pH in the cell, it becomes protonated (H02*) and, hence, reactive. The lifetime of 02'in the water cellular environment is approximately s (Pryor, 1986). In any system producing 02*-, substantial amounts of H20z are formed. The H202is decomposed, although not readily, to HzO and 02. H202is a stable molecule. H202,like most peroxides, is very sensitive to decomposition by the species that react with it. The reaction is catalyzed by redoxactive metal complexes, of which catalase and peroxidase are the most effective exponents. Metal ions have a strong effect on chemistry of dioxygen and its reduction product. If Fe2+comes into contact with H202,the well-known Fenton reaction producing the OH' radical can be initiated (Fenton and Jackson, 1899): Fez+ + H202+ Fe3+ + OH'

+ OH-

Ions of Cu, Co, and Ni can also participate in a similar reaction. Another reaction resulting in formation of OH' is the metal-catalyzed Haber-Weiss reaction of 02'-with H202(Haber and Weiss, 1934): Fe2+

02.-

+ H202 + 0 2 + OH' + OH-

The hydroxyl radical OH' is highly reactive. It can react with practically any molecule present in cells. For this reason it is short-lived. This insufficient stability does not allow it to diffuse some distance in the cell; therefore, it reacts with an organic substrate at the sites or near the sites of its formation. s (Pryor, 1986). It does not survive for The half-life of OH' at 37°C is more than a few collisions after its formation and its reactions are "site specific" (Borg and Schaich, 1987). Such a short time of the radical and ion life makes them too difficult a species for investigation by conventional methods. Properties, characteristics, formation, and reactions of oxyradicals and related species are described in detail in several reviews (Brunori and Rotilio, 1984; Green and Hill, 1984; Pryor, 1986). 0;- is the simplest peroxyl radical. It can be generated by various systems present in animal and plant organisms. The most important source of 02'and H202 is oxidative enzymes, among which xanthine oxidase and NADPH/NADH oxidase are the most powerful producents (Cross and Jones, 1991). All the oxidases possess flavin or transition metals (Fe, Zn, and Cu) which serve as donors of electrons (Parks and Granger, 1986; Mohazzab and Wolin, 1994). Autooxidizing small molecules such as leucoflavins, catecholamines, tetrahydroproteins, quinones, and a cycle of xe-

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nobiotics also leads to formation of 02*-. Mitochondria produce 1 or 2% partially reduced ROS (02*-, H202,and OH') as electrons leak out of the electron transport chain during respiration (Hassan and Fridovich, 1979). In red blood cells, 0;- can reduce methemoglobin (if it is formed) to finally produce HO' (Fridovich, 1986). Thus, the ROS formation is a property shared by a great number of redox component in biochemical pathways of the cell.Various types of ROS can interact in redox cycles of cellular metabolism. The resulting product depends to a large extent on the transition metal ion and on the protein or enzyme containing this ion. It should be noted that ROS can give rise to secondary reactive products such as lipid peroxides (Pryor, 1986). H202is a substrate for the generation of hypochlorite (HOCl) by myeloperoxidase inside the cell. Analysis of the details of O2 chemistry, on one hand, and of physiological conditions in living cell, on the other hand, permits the conclusion that only three ROS are relevant for biological consideration: 02*-, H202, and OH'. It is these three species that are the subject of numerous literature in the field.

6. Regulation of ROS Production by NADPH Oxidase ROS are generated by specialized phagocytic cells (neutrophils and macrophages) as cytotoxic agents to fight invading microorganisms, a process known as the respiratory or oxidative burst. For this purpose, phagocytes use the membrane-bound NADPH oxidase complex which catalizes the one-electron reduction O2 to 0;- according to the reaction: NADPH

+ 202 + NADP+ + 202'- + H+

The 02*produced dismutases to form H202. The structural analyses and biological importance of NADPH oxidase of professional phagocytes have been the subjects of many studies (Henderson and Chappell, 1996). In the past few years, it has become evident that other cells and tissues can also produce a small amount of ROS by the NADPH oxidase-like enzyme (Jones, 1994). This growing list includes B lymphocytes,fibroblasts, kidney mesangial cells, endothelial cells, and osteoclasts. The physiological and pathophysiological role of this extraphagocyticsuperoxide production has only recently begun to be addressed. 1. Phagocytic Cells

The NADPH oxidase of phagocytic leukocytes is a multicomponent electron transport system. It transfers electrons from NADPH at the cytosolic side of the membrane to molecular oxygen at the other side of the mem-

207

ROLES OF REACTIVE OXYGEN SPECIES

brane. The NADPH oxidase contains plasma membrane cytochrome b558 and at least four cytosolic factors. In resting cells the enzyme is dormant. Activation of phagocytes in response to appropriate stimuli leads to the assembly of the active NADPH oxidase complex at the plasma membrane (Fig. 1).The superoxide production in phagocytic cells is induced by different types of molecules, including complement anaphylatoxin C5a, chemotactic peptides, leukotriene B4,platelet-activating factor, and many others (Morel et al., 1991). Both the time interval between contact of the cell with specific stimuli and a rise in superoxide production and duration of the response varied from a few seconds to several minutes and from a few minutes to hours, respectively. These kinetics parameters of the NADPH oxidase activation can also be controlled by a number of priming agents, including tumor necrosis factors, lipopolysaccharide, interferon-y, and in-

0;-

store

0,

\

FIG. 1 The model of phagocyte NADPH oxidase activation. In resting cells, cytochrome b558 subunits gp91-phox and p22-phox, and possibly small G proteins of the rac family, are located in the plasma membrane. After exposure to a stimulus,the agonist-receptor complex stimulates phospholipaseC (PLC) via Gprotein (G). Breakdown of phosphatidylinositol4,5-bisphosphate by PLC results in generation of inositol1,4,5-trisphosphate(IP3) and of diacylglycerol(DAG). IP3 induces Ca2+release from intracellular stores. DAG and Ca2' activate protein kinase C (PKC), which in turn phosphorylates p47-phox that leads to translocation of complex p47-phox and p67-phox to cytochrome bsss in plasma membrane to form the active NADPH oxidase.

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1. A. GAMALEY AND 1. V. KLYUBIN

terleukin-1, which prepare phagocytes for a more rapid and more intense response. On the contrary, substanses such as prostaglandins & and I2 attenuate phagocyte response to NADPH oxidase activators (Thelen et al., 1993).The redox component of the NADPH oxidase complex is an integral membrane heterodimer cytochrome b558which consists of two subunits, p22-phox and gp91-phox (Thelen et al., 1993; Henderson and Chappell, 1996). This protein contains a fzavin and two heme redox centers. In resting phagocytes, this flavocytochrome is associated with membranes of specific granules. Upon activation, fusion of these organelles with the plasma membrane leads to reallocation of the oxidase. In addition to the cytochrome b558,components from cytosol appear to be required for NADPH oxidase activation (Leusen et al., 1996). At least four such components have been identified and have been shown to be functionally essential, two of which are p47-phox and p67-phox. Upon activation,p47-phox becomes phosphorylated and, together with p67-phox, is translocated to cytochrome b558to form the active enzyme. Two low-molecular-weight GTP-binding proteins are involved in the activation of the NADPH oxidase, Rap 1A and p21rac. Moreover, there is evidence that other factors also regulate activity of the NADPH oxidase. The classical signal transduction cascade (Morel et al., 1991; Thelen et al., 1993) involved in the NADPH oxidase activation is initiated by binding of ligands to their specific plasma membrane receptors. The agonistreceptor complex via pertussis toxin-sensitive G proteins stimulates phospolipase C. Breakdown of phosphatidylinositol4,5-bisphosphateby phospholipase C results in generation of two second messengers, inositol1,4,5trisphosphate (IP3) and diacylglycerol.IP3induces Ca2’ release from intracellular stores. Diacylglycerol and Ca” activate protein kinase C , which in turn phosphorylates p47-phox which induces translocation of all cytosolic factors to cytochrome b558 in plasma membrane to form the active NADPH oxidase complex. NADPH oxidase can also be activated by receptorindependent stimuli such as long-chain unsaturated fatty acids and phorbol 12-myristate13-acetate (PMA). In parallel with the typical signal transduction mechanism described previously, there are other pathways leading to oxidase activation involving phospholipase A2 (Dana et al., 1998),phospholipase D (English, 1996), and tyrosine kmases (Dusi et al., 1996). The real situation is likely to be much more complex, and the alternative involvement of one of the signaling pathways of the NADPH oxidase activation depends on the nature of the activator and on priming the cells. Release of 0;- by the phagocytes is vital for the host. However, an excessive production of ROS may induce serious injuries in tissues and lead to a variety of clinical disorders. This dual role of products of the NADPH oxidase explains the necessity for a careful regulation of the enzymatic activity.Probably, for these reasons, the organizationof NADPH

ROLES OF REACTIVE OXYGEN SPECIES

209

oxidase is very complex, and the phagocytic cell has different mechanisms for regulation of this enzyme activity. The importance of NADPH oxidase in the human host defense is illustrated by patients suffering from chronic granulomatous disease (CGD). CGD is an immunodeficiency syndrome characterized clinically by severe recurrent bacterial and fungal infections (Smith and Curnutte, 1991). CGD is also characterized by the inability of phagocytes to generate ROS due to genetic defects of NADPH oxidase. CGD patients have phagocytes lacking different components of NADPH oxidase. Characterization of mutations that lead to CGD is important for treatment of CGD patients but it also provides a better understanding of the functional domains in the oxidase components.

2. Other Animal Cells A variety of mammalian cell types are able to produce ROS after a specific stimulation. In these cells there exists an enzyme that is similar to NADPH oxidase of phagocytes. The properties of the NADPH oxidase-like enzymes in nonphagocytic cells are summarized in Table I. The list of the cell types in which the ROS-generating enzyme has been identified is currently growing. The enzyme systems of nonphagocytic cells, which are responsible for the ROS production, were called NADPH oxidase-like because these systems are functionally similar but genetically and structurally distinct to the phagocyte NADPH oxidase. Thus, while neutrophils of CGD patients demonstrate a reduced activity of NADPH oxidase, which correlates with the decreased content of cytochrome b558, fibroblasts of the same individuals show no impaired production of ROS and have a normal cytochrome b558 content (Meier et al., 1993; Emmendorffer et aL, 1993). On the other hand, experiments on reconstitution of NADPH oxidase show that the fibroblast cytosol can initiate ROS generation when mixed with membranes of resting human neutrophils (Jones et al., 1994). This may be due to many but not all components of NADPH oxidase of phagocytes appearing to be expressed in other cell types or because NADPH oxidase components of nonphagocytic cells have high homologous sequences to those of phagocytes. Another main distinction between the phagocytic NADPH oxidase and the ROSgenerating system in other cells is the rate of oxidant production. Nonphagocytic cells produce a low level of ROS. It reaches only 1%of the ROS generation of activated phagocytes. Despite the fact that in various cells the ROS-generating systems are distinct, it is important that many cell types or tissues are able to respond to specific stimuli under normal but not pathophysiological conditions by a transient rise in the ROS production. The physiological reason for expression of the HADPH oxidase-like enzymes in nonphagocytic cells will be discussed later.

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I. A. GAMALEY AND I. V. KLYUBIN

TABLE 1 Cellular Systems in Which ROS Were Demonstraied to Be Produced by an Enzyme That Is Similar to NADPH Oxidase of Phagocytes

Enzyme components cell type

Cytochrome b subunits

Cytosolic factors

Reference”

Fibroblasts Endothelial cells

p22-phox

Smooth muscle cells

gp91-phox, p22-phox

Chondrocytes

gp91-phox, p22-ph0~ p47-ph0~,p67-phOX

10,11

@91-ph0~,p22-ph0~ p47-ph0~ p47-ph0~,p67-ph0~

12 13,14

Osteoclasts Mesangial cells

B lymphocytes Carotid body cells Podocytes Proximal tubular epithelial cells Adipocytes Mast cells Thyroid cells Cardiac myocytes Microglial cells Spermatozoa

gp91-phox, p22-phox

@91-ph0~,p22-phox gp91-phox, p22-phox

p47-phox, p67-phox, Racl p47-phox, p67-phox ?b

p47-phox, p67-phox, RaplA

gp91-phox, p22-phox p47-phox, p67-phox g~91-ph0~, p22-ph0~ p47-ph0~,p67-ph0~ ?

1-4 5,6 7-9

15-17 18-20 21

?

22

?

23 24

?

?

?

?

25,26 27

?

?

?

?

? ?

p47-ph0~

28 29

1, Meier et al. (1991); 2, Meier et aL (1993); 3, Jones et aL (1994); 4, Sundaresan et al. (1996); 5, Zulueta et al. (1995); 6, Jones et al. (19%); 7, Marshall et al. (1996); 8, Ushio-Fukai et al. (1996); 9, De Keulenaer et aL (1998); 10, Hiran et al. (1997); 11, Moulton et al. (1998); 12, Steinbeck et al. (1994); 13, Radeke et al. (1991); 14, Jones et al. (1995); 15, Kobayashi et al. (1990); 16, Maly et al. (1994); 17, Chetty et nl. (1995); 18, Cross et al. (1990); 19, Kummer and Acker (1995); 20, Youngson et aL (1997); 21, Greiber et aL (1998); 22, Cui and Douglas (1997); 23, Krieger-Brauer et al. (1997); 24, Fukuishi et d.(1997); 25, Deme et al. (1994); 26, Gorin et al. (1997); 27, Mohazzab-H et aL (1997); 28, Klegeris and McGeer (1994); 29, Aitken et al. (1997). ?, not known.

3. Plant Cells

The respiratory burst in plants was first demonstrated much later than in mammals (Doke, 1983). Recently published data have indicated that ROS generation in plant cells similar to that in neutrophils serves as a protective mechanism against invading pathogen infection. In plants, ROS are routinely generated at low levels in nonstressed cells in chloroplasts and mito-

ROLES OF REACTIVE OXYGEN SPECIES

21 1

chondria and also by cytoplasmic, membrane-bound, or exocellular enzymes involved in redox reactions. Particularly, two species, H202 and 0 2 * - , are discussed in connection with ROS effects (Low and Merida, 1996; Wojtaszek, 1997). The following are possible sources of ROS in the oxidative burst in plant cells to be discussed. First is the NADPH oxidase system analogous to that of animal phagocytes. In the same way as various ligands activate NADPH oxidase of phagocytes, elicitors (or pathogens) induce the oxidative burst in plant cells. The NADPH oxidase complex has not been purified from plant cells but several of the neutrophil NADPH oxidase complex subunits were identified in plants: p22-phox (Tenhaken et al., 1995), p47-phox, and p67-phox (Dwyer et al., 1996). The gene rbox A, similar to the gene coding the mammalian gp91-phox, was also identified (Keller et al., 1998). Apart from this, the presence of NADH oxidase and NADH-dependent cytochrome c reductase in plant cells was demonstrated (Vera-Estrella et al., 1994). Furthermore, many components of the signal transduction pathway in neutrophils were also found to play a role in the oxidative burst in plants. There is strong evidence for participation in the signaling pathway of phospholipases, H+/K+exchange, Ca2+influxes, protein kinases, phosphatases, as well as GTP-binding proteins (Chandra el al., 1996; Low0 and Merida, 1996). An alternative model of generation of H202is the pH-dependent cell-wall peroxidase (Bolwell et al., 1995).This model, in contrast to NADPH oxidase activated as a result of intracellular signaling events, emphasizes an importance of proexisting components, especially those present in plant exocellular matrix. The existence of the third system for ROS generation in plant cells is suggested hypothetically. It is a g e d o x a l a t e oxidase system (Zhang et al., 1995). This oxidase is not related to the oxidative burst but produces H202and C 0 2 from oxalic acid in response to pathogens. Wojtaszek (1997) presents a long list of plants generating ROS in response to the effect of different elicitors. The source of ROS and the major ROS vary depending on the plant species and the type of elicitor used.

111. ROS Homeostasis in Animal Cells A. Defense Enzymes and Antioxidants

Being oxidants, all ROS are agents which at high concentrations are toxic to cells. To avoid the damage of cellular components, several biochemical safety mechanisms (defense enzymes and antioxidants) developed inside the cell. The first line of defense against 0;- and H202-mediated injury are antioxidant enzymes: SODS, peroxidases, and catalase.

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I. A. GAMALEY AND I. V. KLYUBIN

SODSare a family of metalloenzymes that converts 0;- to H202according to the following reaction: 0;-

+ 0;-

2H

4 HZOZ SOD

+0

2

SOD is the most important enzyme because it is found in virtually all aerobic organisms. There are four families of SOD: Cu-SOD, Cu-Zn-SOD, Mn-SOD, and Fe-SOD. Human SOD is the Cu-Zn enzyme. The transition metal of the enzyme reacts with Oz*-, taking its electron. 0;- is the only known substrate for SOD. Each type of SOD has its own peculiarities; however, all types of the enzyme have similar properties (Fridovich, 1975; Oberley and Oberley, 1984). SOD is localized in the cytosol, chloroplasts, mitochondria (Bannister and Bannister, 1980), and peroxisomes (Del Rio et aL, 1990). All animal cells, aerobic microorganisms, and many species of plants contain this ubiquitous enzyme. Catalases and peroxidases decompose Hz02.Catalases and peroxidases (glutathione or thioredoxin dependent) are heme-containing proteins. The mechanism of their action is identical: catalase 2H202 + 2HzO

+02

General properties, molecular structure, and mechanism of catalase and peroxidase action have been reviewed in several papers (Pryor, 1976,1986; Vuillaume, 1987). Catalase was found to act 104 times faster than peroxidase. Catalases and peroxidases are localized mainly in mitochondria and in subcellular respiratory organelles, peroxisomes, of almost all animal cells and organs, in aerobic microorganisms, and in plant cells. In all cells, glutathioneperoxidase removes not only Hz02but also many organic hydroperoxides convertingreduced glutathione to oxidized glutathione. Oxidized glutathione is regenerated by glutathione reductase (Meister, 1983;Meister and Anderson, 1983). Apart from antioxidant enzymes, all cells contain a variety of substances to scavenge ROS. These reductants are as follows: vitamins C, A, E, and K, lipoate, thiols, urate, and ubiquinone (Packer et aZ., 1979, 1995; Burton et al., 1983; Gutteridge, 1994; Sies, 1997). Vitamins C, A, and K keep metal-containing catalysts in the reduced state. Uric acid acts by binding to metals. Vitamin E (a-tocopherol) is a membrane antioxidant owing to its lipophility. Even a small amount of a-tocopherol is capable of preventing formation of lipid peroxides in cellular membranes. It occurs due to oxidation of a-tocopherol by primary peroxyl radicals (Briviba et aL, 1996). The most important intracellular reductant is a thiol-containing peptide, glutathione (GSH). This tripeptide consists of glycine, cysteine, and glu-

ROLES OF REACTIVE OXYGEN SPECIES

213

tamic acid moieties (Meister and Anderson, 1983). Bacteria, green plants, and fungi contain GSH in lesser amount than animal cells. Other important dithiol proteins are thioredoxin and glutaredoxin. These small proteins (molecular weight around 12 kDa) have been known since the 1960s (Holmgren, 1985). Different types of thioredoxin have a similar amino acid sequence in its active site: cysteine-glutamine-proline-cysteine(Cys-GluPro-Cys). Two known types of glutaredoxin also have the identical active center (Holmgren, 1989). GSH and thiol-containing proteins participate in redox reactions via the reversible oxidation of cysteine SH groups in active site to disulfide bridges (SS). Considerable attention has been recently paid to a-lipoic acid as an antioxidant. Lipoate, or its reduced form, dihydrolipoate, reacts with all the ROS, with hypochlorous acid, and even with singlet oxygen (Packer et al., 1995). Its functional group is cyclic disulfide. Extracellular fluids contain neither catalase nor glutathione peroxidase and just a small amount of SOD. The list of extracellular reductants includes ceruloplasmin, vitamins, uric acid, and metal chelators, transferrin, albumin, metallotionein, Cu-chelatin, and others. A ceruloplasmin, Cu-containing protein, plays a role for SOD in plasma. The role of extracellular antioxidants is not to remove 0;- or H202but to prevent or to stop their chemical reactions leading to formation of the destructive radical OH'. As mentioned previously, ionic iron is very active in stimulatingthe OH' formation. Therefore, the amount of transferrin is much higher than the amount of iron that is transported. B. Control of lntracellular Redox State (Oxidant-Antioxidant Interaction)

Antioxidant enzymes, together with the substances that are capable of either reducing ROS or preventing their formation, form a powerful reducing buffer which affects the ability of the cell to counteract the action of catalase and peroxidase oxygen metabolites. All reducing agents thereby form the protective mechanisms which maintain the lowest possible levels of ROS inside the cell. On the other hand, each cell needs a certain redox state to be used for a necessary charge in intracellular compartments and for the normal action of hormones. The redox state of the cell may be expressed as the ratio of the concentration of oxidizing equivalents to the concentration of reducing equivalents (Chance et al., 1979; Meister, 1983; Allen, 1991; Sies, 1997). The amount of the oxygen species depends on the power of the reducing buffer inside the cellular compartment. GSH functions as the main redox buffer. Its concentration in animal cells varies in the range of 1-10 mM and depends on subcellular site and cellular type

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(Sobol et aZ., 1995).A surprizing example is the redox state of the endoplasmic reticulum which is more oxidative than that of the cytosol; the ratio of GSH to its oxidized form GSSG inside the endoplasmic reticulum varies from 1:1to 1:3, whereas the overal cellular GSWGSSG ratio varies from 31 :1 to 100: 1 (Hwang et aZ., 1992). Any cell responds to excess oxidants by an elevation of reducing agents, with SOD and glutathione being the first (Allen, 1991). Oxidation of thiols to disulfide retains many intracellular SH proteins in the reduced state. Optimal concentrations of thiol and disulfide are required for rapid and complete refolding of many proteins (Koivu and Myllyla, 1987; Hwang et aZ., 1992). Regulation of enzymes and cell functions via reversible oxidation of dithiols to disulfides will be discussed in Section VI.Wayner et al. (1985) evaluated contributions of various scavengers to the total antioxidant capacity of plasma and found that they were 5% for a-tocopherol, 15% for ascorbate, 25% for urate, and 50% for thiol-containing proteins. Recently, it has been shown that the lipoate-dihydrolipoate couple also may be considered to be an effective exogenous regulator of the cellular redox status (Packer et aL, 1995; Sen and Parker, 1996). It should be emphasized that different scavengers may act synergistically.Thus, vitamin E is repaired by vitamin C (Doba et aZ., 1985). Radicals formed in redox reactions with vitamins E and C can be recycled by glutathione or dihydrolipoic acid. Oxidized thiols (glutathione, thioredoxin, or lipoate) are regenerated by NADPH- and NADH-dependent enzymes (Packer et aZ., 1979,1995). Since 0;- and Hz02 are integral compounds of aerobic metabolism, they are unavoidable. However, it is rather difficult to estimate the concentration of 0;- or HZO2at a moment inside the cell because they are scavenged both enzymatically and nonenzymatically. Nevertheless, it is not unlikely that H202formed in biochemical pathways before reacting with a substrate might diffuse both along intracellular compartments and across the membrane to the extracellular medium (Schubert and Wilmer, 1991; Uchida et aZ., 1992, Mathai and Sitaramam, 1994;Makino et aZ., 1994). Due to the small reactivity at physiological pH this may also be true for the short-living 0;-(Bielski et aZ., 1983; Gus’kova et aZ., 1984). Hence, the effects of H202and 0;- on cellular functions are of great interest. In summary, it is important to note that relationships between the 0 2 * - generating system and antioxidant enzymes may be very complicated because 0;-itself affects directly the activity of catalase and peroxidase (Kellogg and Fridovich, 1977; Kono and Fridovich, 1982). IV. Cytotoxic Effects of

ROS

The cellular damage caused by ROS is referred to as “oxidative stress.” The discovery of oxidative burst in mononuclear phagocytes has stimulated

ROLES OF REACTIVE OXYGEN SPECIES

215

intense investigations of the cellular injury induced by effects of H202 and 02*-. In vivo the first targets of ROS are endothelial cells, fibroblasts, and smooth muscle cells. These cells are directly affected by exposure of ROS due to inflammatory reactions in the blood, which are accompanied by ROS formation. Therefore, these cells are the most common objects of studies on ROS-induced cell damage.

A. Mechanisms of Cellular Damage by ROS Another kind of irradiation, thermal degradation of organic materials, and other processes include the formation of a variety of radicals and ROS. H202is believed to be an important agent of cellular damage in the course of these processes (Basaga, 1990; Huie and Padmaja, 1993; van Wyngaarden and Pauwels, 1995). The list of human diseases, which correlates with the high level of H202or 02'-in blood, is long. It includes cancer (Cerutti, 1985; Sun, 1990), arthritis, atherosclerosis, liver cirrhosis, cataracts, and many others (Pryor, 1986). H202may be formed by autooxidation of polyphenols, such as catechol and hydroquinone in cigarette smoke. This event may be of great significance in the initiation of certain steps of tobacco carcinogenesis (Nakayama et al., 1984). The loss of functional properties and activity of biopolymers during generation of radicals and ROS (Harman, 1982; Saul et al., 1987) or storage of only 02*(Sawada et al., 1992) are the bases of the free radical theory of aging. There are many speculations about the relationship between ROS and aging; however, the involvement of ROS and different radicals in the aging process remains obscure. The direct effect of high concentrations of H202and 02'-cause DNA damage which can be seen in the form of a base damage (depurination or depyrimidination),single-strand breaks, double-strand breaks, cross-linking between DNA, and chromosomal aberrations (Cadet and Berger, 1985; Cadet et al., 1986). The strand breaks result in activation of the DNA repair enzyme poly(ADP-ribose) polymerase. Ultimately, this enzyme forms ADP-ribose polymers which are linked to various nuclear proteins (Carson et aZ., 1986; Schraufstatter et al., 1986a,b, 1987). High doses of ROS cause a decrease in intracellular NAD, GTP, UTP, and CTP and finally the depletion in ATP (Spragg et al., 1987; Schraufstatter et al., 1986a,b, 1987). ROS action also causes a decrease in the rate of glycolysis as a result of inactivation of aldehyde dehydrogenase and a decrease in lactate content (Spragg et al., 1987). The exhausted pool of ATP leads to cell death (O'Donnell-Torney et al., 1985). In vitro H202is able to induce cell lysis (Nathan et al., 1979; Simon et al., 1981).

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Apart from enzymatic disturbances, an alteration of Ca2+homeostasis occurs, which leads to a nonphysiological elevation of intracellular Ca2+ concentration (Hinshaw et al., 1986; Orrenius et al., 1988; Elliott et al., 1992; Klyubin et al., 1996). Disturbances in the Ca2' homeostasis may affect the Ca2+-activatedenzymes and cytoskeletal network. Finally, effects of ROS cause an alteration of microfilaments via depolymerization of the actin network and morphological changes in plasma membrane (formation of blebbing) (Nicotera, 1986a,b; Orrenius et al., 1988). Data from Hastie et al. (1998) indicate that the H202-inducedphospholipase D activation may mediate the filamin redistribution and F-actin rearrangement. Another possible event caused by ROS is lipid peroxidation of cellular membranes. The excess of ROS is involved in peroxidation of the membrane which in turn causes polyunsaturated fatty acids, especially c20:. and GZ6, cell injury. Lipid peroxidation is a process in which a polyunsaturated fatty acid radical reacts with oxygen to form a fatty acid peroxy radical that then undergoes various complex reactions. The steps of lipid peroxidation are quite different but ultimately they induce a group of complex degradative reactions. The secondary products of lipid peroxidation (hydroperoxides, hydroxy derivativesof arachidonicacid, aldehydes,products of metal catalysis such as ethane, pentane, and malonaldehyde, and many others) produce a wide range of biological effects which lead to cell injury. Myeloperoxidase was found to promote peroxidation of phospholipids in the presence of H202and under other acidic conditions (Carlin and Djursater, 1988). Details of the problem of lipid peroxidation can be found in several reviews (Halliwell and Gutteridge, 1984; Vaca et al., 1988). A prolonged reaction of hemoglobin (Hb), the major heme protein of red blood cells and reticulocytes, with H202leads to Hb damage and cellular injury, both in v i m and in vivo (Davies and Goldberg, 1987; Giulivi and Davies, 1990). The investigationsof photodynamic therapy (PDT) of cancer are of great interest. The essence of this treatment is that a photosensitizer localized extranuclearly inside malignant cells forms several radicals including ROS under irradiation of tumor by light at a certain wavelength (Jori et al., 1993; Hamblin and Newman, 1994; Zarebska, 1994). It is singlet oxygen (lo2in the reaction I) which is suggested to be responsible for necrosis of tumor cells during PDT (Henderson and Dougherty, 1992). It is essential that the lifetime of this species in vivo is of the order of several tens of nanoseconds (Moan and Berg, 1991). Interestingly, apoptosis has been considered a result of an oxidative stress induced by the disturbance in redox balance inside the cell. The inability to neutralize the oxidative stress resulted in the apoptosis of lymphoid cells under L-cysteine and GSH-depleted conditions (Iwata et al., 1997). The events that occur during ROS damage are summarized in Fig. 2.

217

ROLES OF REACTIVE OXYGEN SPECIES

R0s

-

cytoskeleton disruption

NUCLEUS FIG. 2 Events that occur during cell injury under the effect of ROS. PM, plasma membrane; LP, lipid peroxide.

B. Toxicity of ROS Analysis of data in the literature shows that the actual mechanisms by which ROS damage the cells are unknown. Figure 2 is not intended to explain that mechanism of the ROS-induced cellular injury. It only demonstrates the known events which occur during cell injury. The data on cytotoxicity and specificity of the ROS (OZ*-,Hz02,and OH') are controversial; although, in vitro any oxygen species in high concentration kills the cell.

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1. A. GAMALEY AND I. V. KLYUBIN

Scavengers of OH' and 0;- were not able to protect the cells against ROS injury in several studies (Nathan et aL, 1979; Simon et al., 1981). Another set of experimental studies shows that 0;- directly affects some intracellular enzymes, changing their activities. Examples are epinephrine and creatine phosphokinase (McCord and Russell, 1988), lactate dehydrogenase-bound NADH (Bielski and Chan, 1973), Ca2+-ATPaseof the vascular smooth muscle sarcoplasmicreticulum (Suzuki and Ford, 1994), aconitase (Gardner and Fridovich, 1992), 6-phosphogluconate dehydratase (Gardner and Fridovich, 1991), and a,P-dihydroxyisovaleratedehydratase (Kuo et aZ., 1987). Interestingly, the antioxidant enzymes, catalase (Kono and Fridovich, 1982) and peroxidases (Metodiewa and Dunford, 1989),may be inactivated in the presence of the Oz*--generatingsystem. As shown in Section III,A, the cells are able to protect themselves against the effect of oxidants with a powerful redox buffer. The rate of ROS generation and the site of their formation, on the one hand, and the location and content of the antioxidants, on the other hand, are important in determining targets of the ROS action and the ultimate cellular damage. There is no convincingevidence for cellular injury through an intracellular oxidative stress by ROS or by other radicals. It is important to realize that in both in vivo and in vitro experiments the cellular damage begins when ROS affect the cells from extracellular medium. The only example in which the ROS-induced (if ROS are formed) or another radical-induced damage is considered as the only intracellular event is the effect of PDT. However, this effect is too complicated to be explained with certainty. The higher the rate of metabolic activity, the greater activity of antioxidant enzymes. Thus, it has been concluded from experiments in vivo that ROS injury of liver occurs endogenously. Therefore, although the capacity of endogenous antioxidants is high, it is lower than the capacity of the intracellular buffer (Jaeschke, 1991a,b).It is suggested that oxidants diffuse into the extracellular medium to be destroyed by endogenous antioxidants. The extracellular oxidant stress is more important than intracellular stress in producing cell injury in the liver, endothelium cells, or neutrophils (Jaeschke, 1991a). It might be assumed that pathogenesis of an ischemic and toxic cellular damage in the organism (diseases of blood, heart, lungs, liver, and kidney) is related to the excess of intracellular ROS secreted into the extracellular medium to start their toxic effect. Large amounts of xanthine oxidase and xanthine dehydrogenase are suggested to be released into circulation after hepatic ischemia so that the formed ROS could produce a widespread liver and endothelium injury (Yokoyama et al., 1990). In this context it should be mentioned that experiments by Makino et al. (1994) showed that Hz02 added to cultured fibroblasts was decomposed rapidly by GSH peroxidase at an Hz02concentration lower than 10 p M ; a rise in H202concentration led to increased activity of catalase and removal of HzOzfrom the cells.

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219

To induce cellular damage, high concentrations of H202 (of the mM order) are necessary. Schraufstatter and coworkers (1986a,b) showed that depletion of the cell energetic pool under the effect of H202in small doses (250 p M and lower) was reversible. There is neither elevation of Ca2+nor fragmentation of DNA due to endonuclease activity in hepatocytes under the effect of oxidant in small doses (25 p M menadione) (Orrenius et al., 1988). HzOZ at a concentration lower than 50 p M does not damage the cellular functions at all (Oosting et aL, 1990; Varani et aL, 1990; Ikebuchi et al., 1991). It has been demonstrated that the cell responds to oxidative stress by synthesis of GSH and by an increase of SOD activity (Allen, 1991). Moreover, SOD is considered to be a stress protein which is synthesized in response to oxidative stress (McCord, 1990). However, from the results of experiments with ROS and antioxidant enzymes, some authors conclude that elevation of intracellular SOD increases the cell damage, allowing more Hz02 to be generated (Kellogg and Fridovich, 1977; Simon et aZ., 1981). The physiological role of stress proteins in prevention and repair of damage as well as the relationships between the stress protein induction, oxidative state, proliferation, and cell death are discussed by Marini et al. (1996). Interestingly, acatalasia, an inheritable human disease due to the absence or a very low activity of catalase, does not result in a severe disturbance in the organism (Delgado and Calderon, 1979) in contrast to chronic granulomatous disease due to defects of NADPH oxidase. An increased detoxification of ROS by an increased activity of antioxidant enzymes may be related to the resistance of tumor cell lines to the effect of anti-cancer agents. However, some findings indicate that many tumors appear to have a decreased level of expression of antioxidant enzymes (Baker et al., 1997). The Fenton reaction generating destructive OH' is the only chemical mechanism which describes cellular damage. However, for these reactions to occur, H202 or 0;- and iron must be present at the same plasma or intracellular membrane site. Studies on initiation of lipid oxidation in or on membrane raise the question as to how readily these reactions are initiated. Since both H202 and 02'-diffuse easily across membranes, the microenvironment in the developed cytotoxic reactions must be taken into consideration (Borg and Schaich, 1987). A cell may recover after losing 5% of every enzyme activity, but it may not recover after losing 95% of some, particularly vital, enzyme activity. Reactivity and toxicity are to a large degree inversely related (McCord and Russell, 1988). The ability of the cell to resist an oxidative stress permits the conclusion to be made that 02'-and H202are not highly reactive species (McCord and Russell, 1988;Elliott et aL, 1992;Suzuki and Ford, 1994).This suggestion is supported by the fact that 0;- and H202 have a low oxidative potential

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(Aust ef aZ., 1985; Fridovich, 1986; Wood, 1988; Gutteridge, 1994). Experiments with antioxidant enzymes show that H202, rather than 02'-, is the more essential species to induce cell injury. However, an increase in the glutathione peroxidase activity of endothelial cells had no protective effect against the hyperoxia-induced inhibition of DNA synthesis (Junod, 1987). Possibly, tissue damage might be connected with some intermediate products appearing under effects of high ROS concentrations. Thus, a rapid and spontaneousinteractionbetween 0;- and nitric oxide (NO) to generate strong oxidants, peroxynitrite and peroxynitrous acid, has been suggested to represent an important pathway by which tissue may be injured during inflammation (Beckman and Crow, 1993; Miles ef aZ., 1996).

V. Regulation of Cell Functions by ROS A. Cell Responses to the Direct Effects of ROS Independent studies in recent years have clearly demonstrated the ability of small (noncytotoxic) doses of H202 and 0;- to activate or modulate various cellular functions. The surprising results of these studies are given in Table 11.The concentration range of HzOz,in which it causes an acceleration of cellular functions is rather small (1-50 p M ) . Interestingly, bovine pulmonary artery endothelial cells are more resistant to oxidant damage than other endothelial cells because they express higher levels of antioxidant enzymes (Vercellotti et aZ., 1988). These cells survive the effect of 1-10 mM H202without significant injury and display H202-inducedtimeand dose-dependent increases in cytoskeletal actin and myosin (Zhao and Davis, 1998). The intracellular events occurring during activation of cellular functions under effect of small doses of ROS will be discussed in Section VI. The previous examples raise the question as to why the small doses of ROS affect the cells despite the powerful intracellular reducing buffer. The sensitivity of the cell plasma membrane to ROS regarding signals and its ability to respond to these signals by activation (or modulation) of the same cellular functions that are induced by natural activators reflects the intracellular sensitivity to slight changes in the redox state.

6. Redox Responses during Growth and Development A variety of data indicate that the processes of cell proliferation and differentiation are activatedmediated by ROS. Despite an attempt to determine H202, and OH'), the results obtained by involvement of all ROS (02'-,

221

ROLES OF REACTIVE OXYGEN SPECIES TABLE II Activation/Modulation of Cell Functions by ROS

Cell type

Cell response

Reference"

Amnion cells

Arachidonate release

1

Cardiac cells

Adrenergic receptor modification

2

Cartilage

Proteoglycan synthesis

Endothelial cells

Endocytosis

3 4

Endothelial cells

Nitric oxide release

5

Endothelial cells

Actin and myosin reorganization

6

Endothelial cells

ATP hydrolysis

7

Endothelial cells Fibroblasts

Prostaglandin & and I2 synthesis

8

Proliferation

9

Fibroblasts

Apoptosis

9

Mast cells

Histamine release

10

Macrophages Macrophages

Oxidative burst

11 12 13

Organic anion efflux

Smooth muscle cells

Chemotaxis

Smooth muscle cells

Re1axation

14

Leukocytes Neutrophils

Adhesion

15-17

Oxidative burst

18

Neutrophils

Locomotion

19

CHO cells

Insulin's bioeffects

20

Platelets

Aggregation

21-23

Platelets

24

Spermatozoa

Arachidonic acid metabolism Hyperactivated motility

Thyroid cells

Hormone synthesis

26-28

25

1, Ikebuchi et al. (1991); 2, Kaneko et af. (1991); 3, Homandberg et al. (1996); 4, Liu and Sundqvist (1995); 5, Madge and Baydoun (1994); 6, Zhao and Davis (1998); 7, Gamer ef aL (1984); 8, Ager and Gordon (1984); 9, Burdon ef al. (1996); 10, M a g i et af. (1994); 11, Gamaley et af. (1994); 12, Gamaley et al. (1991); 13, Sundaresan et al. (1995); 14, Burke and Wolin (1987); 15, Suzuki et al. (1991); 16, Ding et al. (1992); 17, Gaboury et al. (1994); 18, Winn et al. (1991); 19, Klyubin et al. (1996); 20, Heffetz et al. (1992); 21, Pratico et al. (1992);22,Yaoetal. (1993);23, Ikedaetnl. (1994);24, Pignatellietal. (1998);25, De Lamirande and Gagnon (1993); 26, Deme et al. (1994); 27, Gorin et aL (1996); 28, Carvalho et al. (1996).

several researchers argue in favor of HzOz(Rao, 1996). Thus, it has been shown that Hz02stimulates proliferation of smooth muscle cells (Rao and Berk, 1992; Gong et aZ., 1996; Li et al., 1997; Nishio and Watanabe, 1997), mesangial cells (Gonzalezrubio et d,1996),fibroblasts (Burdon et aZ., 1995, 1996), and human leukemic cells (Yang et aZ., 1996). On the other hand,

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overexpression of SOD and elevation of H202in glioma cells either inhibited cell proliferation or increased cell death by oxidative agents (Zhong et al., 1996). Dependence of cellular response (proliferation, apoptosis, or features of senescence) on H202 concentration was demonstrated in fibroblasts. Thus, 1 p M of H202 resulted in proliferation of the cells, whereas larger concentrations of H202(100-400 p M ) induced senescence or even apoptosis (Burdon et al., 1996;Bladier et al., 1997). H202at 10 nM to 100 p M concentration was able to induce proliferation of human aortic smooth muscle cells, and this mitogenic H202effect was mediated by an increase in the affinity of basic fibroblast growth factor for its receptor (Herbert et al., 1996). Increased 0;- production of endothelial cells during proliferation (Amal et al., 1996) and involvement of this species in proliferation of smooth muscle cells (Bhunia et al., 1997) and B lymphocytes (Morikawa and Morikawa, 1996)were demonstrated. Gamberini and Leite (1997) were able to show that the fibroblast proliferation was mediated by OW and depended on simple iron complexes. It has been hypothesized that the developmental stages of life are associated with lower antioxidant defense levels than those present in postnatal phases of life (Allen, 1991; Allen and Venkatraj, 1992). This hypothesis implies that development is accompanied by metabolic gradients which may alter the cellular environment and stimulate variations in the rate of generation of ROS by metabolic pathways. Both ROS and redox status can affect gene expression through transcriptional factors and ultimately affect the protein products of the genes (Keogh et al., 1996b; Furth et al., 1997). It was shown that activities and mRNA abundance of H202 metabolizing enzymes (glutathione peroxidase and catalase) were higher in skin fibroblast cultures from postnatal donors than in fetally derived cultures; however, there were no significant differences in these parameters in cell lines from postnatal donors of different ages (Keogh et al., 1996a). The activity and mRNA abundance of another ROS metabolizing enzyme, SOD, were significantly lower in cell lines derived from fetal skin than in lines from postnatal skin (ages 17-94 years) (Allen et al., 1995). The ratio of the enzyme activities that control electron entry into, and exit from, the electron transport chain in fetal cell lines varied directly with 0;- generation and inversely with H202generation, which indicates a difference in predominant ROS generated during fetal and adult stages of life (Allen et al., 1997). The ability of mammalian spermatozoa to generate ROS is thought to be of significance in the regulation of sperm function (Aitken el al., 1997). A concentration effect of ROS on in vitro development of bovine embryos has also been demonstrated (Fujitani et al., 1997). The plasma membrane redox system as an important element controlling the cell growth has been discussed for many years (Crane et al., 1985).

ROLES OF REACTIVE OXYGEN SPECIES

223

Currently, oxidants are considered early growth signals because they have been shown to activate a number of pathways that are also stimulated by growth factors ( h i et al., 1996; Sun and Oberley, 1996). The intensive studies on involvement of ROS in the cellular signal transduction will be discussed.

VI. ROS as Signal Molecules Signal transduction is a series of conversions of external stimuli into chemical signals and then into cellular responses. Second messengers are the critical links in the signal transduction pathways. The important criterion for second messengers is a transient increase in their concentration inside the cell in response to an extracellular stimulus. Is there reason to suggest that ROS serve as signal molecules? As discussed previously, every cell has an enzyme system that generates ROS in response to a specific stimulation (see Section 11) as well as an intracellular redox buffer that rapidly destroys ROS (see Section 111). Therefore, ROS may fit the definition of the second messenger because their concentrations in the cell after the action of physiological stimuli are able both to increase and to decrease rapidly. Many hormones, growth factors, and cytokines act by increasing the intracellular concentration of ROS (Table 111).Based on analysis of a number of experimental results, the rise in ROS production is suggested to be involved in receptor-mediated signal transduction systems. In confirmation of this suggestion, ROS may directly affect effector molecules both from the extracellularmedium and from the sites of the intracellular interior where they are generated, and antioxidants are able to prevent receptormediated cellular responses to appropriate stimuli. The signaling role of ROS has been found to extend to a wide variety of agonists mediating many cellular responses. A. ROS in Receptor-Mediated lntracellular Signaling

Attention began to be paid to ROS as second messengers when it was found that an agonist-stimulated activation of the transcription factor NF-KBwas accompanied by ROS production (Schreck er al., 1991). Many different stimuli (PMA, lipopolysaccharide, and inflammatory cytokines) which are not known to use the common signaling pathway can activate the factor. Several lines of evidence indicated that ROS, particularly Hz02, act as second messengers in the activation of NF-KB.Treatment of cells with many structurally unrelated antioxidants or overexpression of antioxidative

224

I. A. GAMALEY AND 1. V. KLYUBIN

TABLE 111 Agents Which Induce ROS Production in Various Cells"

Agonist

Cell type

Angiotensin I1

Smooth muscle cells

Arachidonic acid ATP

Epithelial cells, mast cells, platelets Podocytes, thyroid cells Cerebral arterioles, endothelial cells, keratinocytes Endothelial cells

Bradykinin C5a Calcium ionophores

Carbachol Collagen Epidermal growth factor Fibroblast growth factor Insulin Interferon-y Interleukin-1

oxytocin Phorbol esters Platelet-derived growth factor Thrombin Thyrotropin Transforming growth factor Tumor necrosis factor

B lymphocytes, chondrocytes, fibroblasts, keratinocytes, mast cells, platelets, thyroid cells Thyroid cells Platelets A431, fibroblasts, keratinocytes 3T3 L1 cells, chondrocytes 3T3 L1 cells, fat cells Osteoclasts B lymphocytes, chondrocytes, fibroblasts, keratinocytes Fat cells B lympohcytes, fibroblasts, platelets 3T3 L1 cells,fibroblasts, smooth muscle cells Platelets Thyroid cells Endothelial cells, fibroblasts, osteoblasts B lymphocytes, endothelial cells, fat cells, fibroblasts, mesangial cells, smooth muscle cells

Referenceb 1 2-4

5,6 7-9 10 4,9,11-15

15 16 9, 17, 18 19,20 19,21 22

11, 13, 18, 23, 24 25 4, 13,26 18, 19, 27, 28 4 6, 15 29-31 10,11,18,25,32, 33

a The table is restricted to nonphagocytic cells. Activators of phagocyte NADPH oxidase are discussed in Section 11,BJ. 1,Ushio-Fukaiel al. (1996); 2, Cui and Douglas (1997); 3, Fukuishi et al. (1997); 4, Maresca et al. (1992); 5, Greiber et aL (1998); 6, Bjorkman and Ekholm (1994); 7, Sobey et al. (1997); 8, Sundqvist (1991); 9, Goldman et aL (1998); 10, Murphy et aL (1992); 11, Hancock et al. (1990); 12, Hiran et aL (1997); 13, Meier et aL (1991); 14, Tsinkalovsky and Laerum (1994); 15,Raspe and Dumont (1995); 16, Pignatelli et al. (1998); 17, Bae et aL (1997); 18, Sundaresan et al. (1996); 19, Krieger-Brauer and Kather (1995a); 20, Lo and Cruz (1995); 21, KriegerBrauer and Kather (1992); 22, Darden et al. (1996); 23, Lo et al. (1998); 24, Turner et al. (1998); 25, Krieger-Brauer and Kather (1995b); 26, Hancock et aL (1989); 27, Sundaresan el al. (1995); 28, Marumo et aL (1997); 29, Thannickal et al. (1993); 30, Thannickal and Fanburg (1995); 31, Ohba et al. (1994); 32, Satriano et al. (1993); 33, De Keulenaer et al. (1998).

ROLES OF REACTIVE OXYGEN SPECIES

225

enzymes inhibited NF-KB activation (Schreck et al., 1991; Schmidt et al., 1995). Moreover, addition of exogenous HzOz activated the transcription factor (Los et al., 1995). To date, NF-KBhas been identified as a target for the receptor-driven ROS production in various cells and tissues, such as macrophages (Kaul and Forman, 1996), microglial cells (Ferrari et al., 1997), endothelial cells (Weber et al., 1994), and mesangial cells (Satriano et al., 1993).The role of ROS in regulation of transcription factors is not restricted to NF-KB activation. Results from in v i m assays have established that activity of a growing number of transcription factors, including AP-1, Myb, and Ets, is regulated by ROS (Myrset et al., 1993; Wasylyk and Wasylyk, 1993; Rao et al., 1996). These transcription factors participate in activation of a diverse range of genes involved in inflammation, immune response, differentiation, growth control, and development. ROS can affect gene expression in different ways. The mitogenic signals, many of which are probably mediated by generation of ROS, activate early response genes such as c-fos, c-jun, c-myc, and egr-1 (Rao and Berk, 1992; Rao et al., 1996;Nose and Ohba, 1996). Interestingly,these genes encode transcription factors whose activity is also regulated by the oxidant-antioxidant balance (Abate et al., 1990). In recent years research has concentrated on the role of ROS in mitogenic stimulation of the cell. Some of these studies dealt with the involvement of ROS in growth factor-induced signal transduction mechanisms. In rat vascular smooth muscle cells, HzOzwas shown to act as a signal-transducing molecule in platelet-derived growth factor-induced proliferation (Sundaresan et al., 1995). In addition, the epidermal growth factor and transforming growth factor-pl use ROS to perform their mitogenic activities (Ohba et al., 1994; Bae et al., 1997). Furthermore, it has been shown that NIH 3T3 fibroblasts transformed by overexpression of oncogenic Ras protein produced ROS constitutively and that the cells were able to progress through the cell cycle in the absence of growth factors (Irani et al., 1997). Growth factors initiate multiple signaling pathways, including those mediated by phospholipase c,various families of protein tyrosine kinases, and mitogen-activated protein (MAP) kinases; this leads to activation of several transcription factors. The growth factor-stimulated signal network is also controlled by small G protein Ras. Moreover, growth factor receptors have tyrosine kinase activity which plays an integral role in generation of second messengers. Currently, the mechanisms by which ROS convey mitogenic signal are still uncertain. However, in recent years, ROS have been shown to regulate numerous steps of growth factor signaling. In fact, ROS are involved in epidermal growth factor receptor tyrosine b a s e activity (Bae et al., 1997) and in nonreceptor tyrosine activity (Brumell et al., 1996; Yan and Berton,

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1996).In some systems studied, it also appears that ROS activate phospholipase C (Goldman and Zor, 1994) and MAP b a s e s (Tournier et al., 1997). Much data indicate that many other signal transduction systems, such as regulation of gene transcription, operate ROS to convert an extracellular signal into the intracellular effector system. Ion signals that include changes in membrane potential and an increase of cytosolic free Ca2+concentration are known to initiate a variety of cellular responses. Most ion signals are mediated by activation of receptor-operated ion channels located in the plasma membrane as well as in membranes of intracellular compartments such as endoplasmic reticulum. In recent years, several publications have demonstrated that ROS at low concentrations, in contrast to the ionic perturbation associated with an oxidative damage in response to large amount of oxidants, can induce ion signals that are similar to receptormediated ones, including a transient rise in intracellular free Ca2+(Ikebuchi et al., 1991; Klyubin et aL, 1996; Volk et al., 1997), activation of potassium channels (Kuo et al., 1993; Wei et al., 1996; Wang et al., 1996), plasma membrane hyperpolarization (Gamaley et al., 1994; Krippeit Drews et al., 1994; Bauer et al., 1997), and changes in intracellular pH (Ikebuchi et al., 1991). Furthermore, several researchers proposed that the ion signals produced by ligand-receptor interactions were ROS mediated (Sobey et al., 1997; Gamaley et al., 1998). Detailed studies of biological effects of ROS on intracellular ion signaling have shown that several classes of both potassium and calcium channels, ion pumps, and transporters are redox sensitive (Campbell et al., 1996; Taghalatela et al., 1997; DiChiara and Reinhart, 1997; Klusener et aL, 1997) and that even very low doses of oxidants have remarkable specific effects on different types of ion channels (Sen et al., 1995). There is at least one additional possible scenario for a role of ROS as activatorsof ion channels. ROS have been reported to induce IP3production (Schieven et aL, 1993).IP3promotes release of calcium from internal stores, and Ca2+serves to activate Ca2+-dependention channels. Alternatively, ROS may release Ca2+from mitochondria (Richter et aZ., 1992). However, the exact mechanism by which ROS affect ion channel activity (direct activation of the channels or an effect through generation of additional second messengers) remains to be determined. The demonstration of involvement of ROS in intracellular Ca2+homeostasis extends the abilities of ROS to regulate intracellular signalingbecause many cellular processes are Ca2+dependent. It is still poorly understood whether ROS can affect Ca-controlledintracellular effectorswith or without Ca2+involvement. It has been shown in various experimental systems that ROS activate phospholipaseA2(Goldman et al., 1992;Rao et al., 1995)and phospholipase D (Natarajan et al., 1993; Ito et al., 1997) and stimulate tyrosine as well as serinehhreonine phosphorylation (Siflinger Birnboim et aL, 1992; Vepa et al., 1997), possibly by inhibition of protein phosphatases (Sullivan et al.,

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1994) and by a direct stimulation of protein kinases (Fialkow et al., 1993; Konishi et al., 1997). Regarding the analogy between ROS and classical second messengers, it is important to emphasize that not only are the added exogenous ROS able to stimulate cellular functions (Table 11) but also the same functions can be mediated by intracellular ROS production in response to appropriate stimuli. In addition to proliferation, growth, and development discussed previously, the list of these functions includes platelet aggregation (Pignatelli et al., 1998),chemotaxis (Sundaresan et al., 1995;Nishio and Watanabe, 1997), thyroid hormone synthesis (Dupuy et al., 1991), bone resorption (Darden et al., 1996), vasodilatation (Sobey et al., 1997), and apoptosis (Sandau et al., 1997). By analysis of the mechanisms of ROS signaling, several contradictions are evident; they depend on the particular cell type used in experiments and even on the agonist used for the same cell. Thus, a number of reports on various tissues describe effects of the elevated CAMP level on ROS production in ROS-mediated cellular responses. In neutrophils (see Section II,B,l), microglial cells (Colton and Chernyshev, 1996) and Ehrlich ascites tumor cells (Salimath and Savitha, 1992) the elevated CAMPlevel has been shown to inhibit generation of ROS. Similar findings were reported for tumor necrosis factor-stimulated mesangial cells. In these cells, pretreatment with agents that increase intracellular cAMP level decreased the degree of the ROS-mediated NF-KBactivation (Satriano and Schlondorff, 1994). In contrast to the previous effects of CAMP, the cAMP cascade in thyroid cells has been proposed to act as an activation mechanism of the NADPH-dependent HzOz-generatingsystem (Bjorkman and Ekholm, 1994). The multiple effects of CAMPon ROS production are also revealed in mesangial cells in which an increased cAMP is associated with a decrease in generation of ROS upon action of inhibitors of phosphodiesterase-4 but not of phosphodiesterase-3 (Chini et al., 1997). It has been suggested that in the same cell type, different cAMP signaling pathways can exist. With respect to activation of ROS production, there is also a contradiction in interrelation between signals which initiate generation of ROS and signals which participate in ROS-mediated cellular responses. In some cell systems, CaZf,arachidonic acid, and G proteins, including the Ras family of small G proteins, are necessary for activation of ROS production (Table 111; see Section II,B,l). On the other hand, surprisingly, it has been shown that ROS can be involved in the pathways leading to arachidonic acid release (Tournier et al., 1997), in G protein activation (Lander et al., 1993), and in an increase in cytosolic free Caz+concentration (Volk et al., 1997). It is yet to be determined whether these signals have different targets for regulation in different systems. As evident from the previous discussion, describing universal principles of the ROS signaling is a difficult task. However, as

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more studies have been carried out, it is apparent that the ROS transduction system is highly organized (see Section I1,B) and is involved as a step of the receptor-mediated intracellular signaling in many cellular processes (Fig. 3).

B. Involvement of ROS in Cellular Mechanism of Oxygen and Mechanical Stimuli Perception In animals, many types of cells are normally affected by mechanical stimuli These stimuli are well known and changes in oxygen partial pressure (PO,). to cause a variety of effects on the structure and function of the cells. Nevertheless, it is still poorly understood how the cells perceive the stimuli which do not act through the traditional interaction of ligands with receptors. A potential candidate for an 02-sensingprotein in the carotid body

ca2+store

-

)

-

-

'Ca2+-

/

gene trmcription

1

FIG. 3 ROS in signal transduction pathways. R, receptor; G, G protein, RTK, receptorassociated protein tyrosine kinase; 0, ROS-producing oxidase; IC, ion channel; PLA,, phospholipase A2;PLC, phospholipase C; PLD, phospholipase D; DG, diacylgylcerol;IP3,inositol 1,4,5-trisphospate;MAP, mitogen-activated protein kinase; STPK, serine/threonine protein kinase; TF,transcription factor; TK,tyrosine protein kinase.

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is the NADPH oxidase (Lopez-Barneo et al., 1993; Kummer and Acker, 1995; Youngson et al., 1997). It has been shown that the NADPH oxidasedependent ROS production positively correlates with the pericellular P02. Changes in the cellular content of 0;- and H202may modulate activity of hypoxia-inducible factor 1 through 02-sensitive K+ channels and, as a consequence, the expression of genes that are under its control. Likewise, endothelial and smooth muscle cells respond to shear flow (Hsieh et al., 1998) and to pulsatile stretch (Hishikawa et al., 1997), respectively, by an elevation of intracellular ROS. Furthermore, ROS seem to be involved as second messengers in mechanisms of gene induction by mechanical forces via activation of transcription factors NF-KB and AP-1 (Hishikawa et aL, 1997; Wung et al., 1997; Hsieh et al., 1998).

C. Mechanisms of Activation of lntracellular Targets for ROS 1. Redox Modifications of Proteins All the data presented previously demonstrate that ROS may affect (i) directly the plasma membrane from extracellular medium and (ii) the activity of a wide range of intracellular proteins from the intracellular sites where they are generated. What kind of sites in the previous protein targets are sensitive to the ROS effects? Because the main intracellular antioxidants are thiol-containing substances, whose active site cysteine SH groups are oxidized to disulfide bridges, it is commonly believed that effects of ROS are mediated by a covalent modification of critical protein sulfhydryl residues. The involvement of the thiol status in regulation of intracellular enzymes under the effect of oxidative stress has been demonstrated in many studies. Thus, H202or menadione induced activation of heat shock transcription factor-1, accumulation of heat shock protein (Hsp) 70 mRNA, and increased synthesis of Hsp 70 (Huang et al., 1994; Liu et al., 1996; McDuffee et al., 1997). Such activation is followed by intracellular protein thiol oxidation and formation of disulfide-linked aggregates of cellular proteins, which in turn are linked to the heat shock transcription factor-1 and Hsp transcriptional activation. Thiol status is believed to be one of the key factors of the apoptotic pathway. Reduction in intracellular glutathione (GSH) increased apoptosis of polymorphonuclear leukocytes; this effect could be reversed with N-acetylcysteine (a precursor for glutathione synthesis and an antioxidant), suggesting that apoptosis may be caused by depletion of GSH (Watson et al., 1996).N-acetylcysteine also decreased the extent of cartilage

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proteoglycan depletion caused by H202 or 0;- (Homandberg et aZ., 1996). The overexpression of protooncogene bcl-2, which inhibits both apoptosis and necrotic cell death of many cell types including neural cells, shifts the cellular redox potential (mainly the GSH redox state) to a more reduced state through a decrease in ROS generation (Ellerby et al., 1996). However, Sat0 et al. (1995) suggest that thiols other than GSH may play more important roles in apoptotic pathway. Some findings indicate that sulfhydryl groups on one or more proteins may be essential for modulation of ROS generation. Thus, activation of NADPH oxidase in neutrophils, which results in ROS generation (oxidative burst), is mediated by S-thiolation (formation of mixed disulfides between sulfhydry1 groups of proteins and low-molecular-weight thiols): Generation of intracellular oxidants was increased many times by the effect of direct thiol oxidation (Moriguchi et al., 1996). A similar conclusion was made for the H202-dependent synthesis of thyroid hormones (Gorin et al. 1997). During respiratory burst in monocytes, low-molecular-weight thi01s (GSH and cysteine) can bind to specific cytosolic proteins, including glyceraldehyde-3-phosphatedehydrogenase (Ravichandran et al., 1994), resulting in the enzyme inactivation. In muscle (Vaidyanathan et aL, 1993) and endothelial cells (Schuppe Koistinen et aL, 1994), this enzyme is also inhibited by ROS-induced S-thiolation. NO was also shown to inactivate glyceraldehyde-3-phosphatedehydrogenase in neurons through an activesite thiol modulation (Brune and Lapetina, 1995). It is possible that Sthiolation of cytosolic proteins serves to modulate cellular metabolic events. In experiments with application of different thiols it has been shown that the resulting effect (prevention or acceleration of lipid peroxidation) depends on the ability of thiols to scavenge or generate free radicals (Benov et aL, 1992). Pan et al. (1995) consider GSH a novel modulatory system for the differential regulation of inhibitory neurotransmission in the mammalian retina. Phospholipase D in myocardial sarcoplasmic reticular membranes, in which it takes part in regulation of Ca2+movements, is regulated by H202 through a reversible modification of associated thiol groups (Dai et al., 1995). The role of the cysteine residues in regulation of function of the p53 tumor suppressor was demonstrated in several studies (Hainaut and Miller, 1993; Rainwater et aL, 1995). However, only certain cysteins may cooperate to modulate the structure of the DNA-binding domain (Rainwater et aL, 1995). Oxidation of SH groups of rabbit muscle creatine b a s e by H202 seems to be the major mechanism of inactivation of the enzyme (Suzuki et al., 1992). This enzyme in heart mitochondria may also be inhibited by ROS through modification of its SH groups (Yuan et al., 1992).Interestingly, oxidation of SH groups of cysteine and bovine serum albumin by H202

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and 02*in human polymorphonuclear leukocytes is not modified by NO (Koshida and Kotake, 1994). It was shown that endopeptidase (EP24.15), involved in the regulated metabolism of a number of neuropeptides, displayed a unique activation through cysteine residues: The formation and disruption of disulfide bonds may be a mechanism by which the EPR24.15 activity is regulated through changes in intra- and extracellular redox potential (Shrimpton et al., 1997). ROS may alter ion channel activity through an oxidative modification of thiols. Thus, the role of SH groups for nonselective cation current in ventricular myocytes was demonstrated by Jabr and Cole (1995). The function of the Na+/K+-pumpin these cells may be related to both protein and nonprotein sulfhydryl status because the sulfhydryl oxidation and depletion of GSH was responsible, at least partly, for depression of pump activity during oxidative stress (Haddock et al., 1995). Activity of ryanodine receptors at the skeletal sarcoplasmic reticulum, which are essential for normal Ca2+channel function, involves redox cycling of functionally important hyperactive sulfhydryls (Liu and Pessak, 1994). ROS was found to depress the heart sarcolemmal Ca2+pump activity and to increase permeability of sarcolemmal membranes to Ca2’ (Rowe et al., 1983; Kaneko et al., 1989). Because adrenergic receptors are known to control entry of Ca2+into the cardiac cells, of great interest is the finding of modification of a- and padrenergic receptors by direct effect of ROS (Kaneko et al., 1991). The mechanisms by which ROS may modify these receptors are not clear. Surprising data were obtained by Hwang et al. (1992). These authors studied the redox state of GSH in endoplasmic reticulum (ER) to show that the secretory pathway was more oxidative than that of cytosol. How the ER is maintained in a much more oxidative state is not known. One mechanism appears to be the preferential transport of GSSG (oxidized form of GSH) from cytosol into the ER lumen; other mechanisms could include a NADPH-dependent oxidase system (Ziegler et al., 1979), the vitamin K redox cycle (Vermeer, 1990), and the sulfhydryl oxidasecatalyzed formation of disulfide bonds (Janolino and Swaisgood, 1987). It was found that H202, as well as NO, was a potent relaxant of the pulmonary vascular smooth muscle. The relaxing mechanism involves the H202activation of soluble guanylate cyclase, which results in the formation of the intracellular mediator of relaxation, cyclic guanosine 3’5’monophosphate; hypoxia inhibits production of H202 and NO, causing vasoconstriction (Burke and Wolin, 1987;Monaco and Burke-Wolin, 1995). ROS may oxidize not only cysteine SH groups but also the sulfur-containing amino acid residues of methionine. However, in contrast to cysteine, the specific functions of the methionine residues are not known (Levine et al., 1996). Nevertheless, it was demonstrated that the effectiveness of calmoduline as an activator of Ca2+-ATPasedepended on oxidative modification

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of the methionine residues to the corresponding sulfoxide in calmodulin (Michaelis et al., 1996). Activation of methionine synthase may be NADPH dependent (Michaelis et al., 1996). A search for mechanisms by which ROS affect the cells have stimulated investigations of gene expression under the effect of ROS and substances that affect the intracellular interior redox state. Progress was achieved by studying the oxidative activation of transcriptional factors, NF-KB, and activator protein-1 (AP-1). NF-KB, in contrast to many other transcriptional factors, is present in an inactive form in the cytoplasm of most cell types. In the cytoplasm, NF-KB (a member of the Re1 family of proteins) is associated with inhibitor protein IkB (for structure and functions of Re1 and IkB families of proteins, see Baeuerle and Henkel, 1994; Thanos and Maniatis, 1995). Due to proteolytic processing of IkB proteins after activation, NF-KB (Re1 protein complex p50-p65) is translocated to the nuclei and binds there to a DNA active site. Although the mechanisms of the NF-KBactivation by ROS are not completely understood, some steps which may be regulated by ROS are known. Note that NF-KBmay be activated by a variety of stimuli and not only by ROS. The first step of the NF-KB activation is a degradation of the bound IkB protein. Dissociation of IkB from NF-KB may be caused either by ROS directly or by various effector molecules of intracellular signaling pathways (inositol phosphatides, phosphatases, kinases, and Ca2+).Taken together, data from the literature suggest that changes in the intracellular Ca2+concentration are essential for the NF-KB activation (Sen and Packer, 1996). The second step of the NF-KB activation is binding to DNA. An in vitro biochemical analysis has shown that the cysteine residue of NF-KBwas involved in its interaction with the target DNA sequence. It was reported that the DNA-binding activity was regulated by a cellular reducing catalyst thioredoxin (Trx). It turned out that among various reductants, either physiological or artificial Trx showed the greatest effect in activating the DNA-binding activity of NF-KB(Hayashi et al., 1993). It was also found that cysteine at amino acid position 62 in p50 protein of NF-KB was the target of redox regulation (Mathews et al., 1992; Hayashi et al., 1993). Thus, Trx has a regulatory role in controlling NF-KBactivity. A transient oxidative stress inside the cell caused by H202 or by other stimuli may induce production of Trx, and this induced Trx would then activate NF-KBthat was oxidized by ROS (Hayashi et al., 1993). Regarding the redox regulation of Fos and Jun DNA-binding activity, a particular role of the single cysteine residue in a highly conserved triamino acid sequence (Lys-Cys-Arg) was shown in the DNA-binding domains of the two proteins and AP-1 factor. The conversion of this cysteine residue to reversible oxidation products, such as sulfenic (RSOH) or sulfinic(RS02H) acids, could contribute to the regulation of DNA-binding activity of Fos and Jun (Abate et al., 1990). However, in vitro experiments indicate that

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the redox regulation of activity of these protein differs from regulation of NF-KB.Nuclear Ref-1 protein (product of ref-l gene) was identified as a reductant for Fos and Jun proteins. Ref-1 stimulates DNA binding of the proteins by acting on the regulatory cysteine residue of Lys-Cys-Argin the active site. It is important to note that Ref-1 protein has a low efficiency in reducing NF-KB(Xanthoudakis et al., 1992). Recently, it was reported that AP-1 transcriptional activity could be regulated by a direct association between Tpx and Ref-1 in the nucleus (Hirota et al., 1997). It is possible that various intracellular reductants might have differential roles in the redox regulation of different target proteins (Hayashi et al., 1993). Finally, ROS act as a simple on-off switch that conveys information to the components of the intracellular redox buffer. 2. Metal-Containing Proteins and Redox State The signal transduction roles in iron sulfur centers in connection with the redox state are discussed for the regulator of gene expression, an ironresponsive element (IRE)-binding protein in mammalian cells (Hentze et al., 1989;Paraskeva and Hentze, 1996). IRE-binding protein has been found to be sensitive to oxidation in vitro (Hentze et al., 1989). This protein, a regulator of ferritin expression in human cells, requires free SH groups for binding to IRES. It has been shown that the IRE activity can be induced by iron deficiency, NO, and H202; the pathways of regulation for IREbinding protein by ROS and NO have been characterized (Bouton, 1996; Pantopoulos et al., 1996). Thus, the cells perceive the presence or absence of iron by modulating reduction or oxidation of the IRE-binding protein despite the constant cytosolic redox buffer. It is not unlikely that activities of other regulators of gene expression are also modulated by the redox processes.

D. Redox Regulation of Transcriptional Activity in Bacteria The effect of ROS on bacterial cells leads to production of novel proteins with protective functions. The defense activities in bacteria in response to the oxidative stress result from the transcription of genes initiated by regulatory proteins. These proteins have been well-defined. The oxyR protein activates transcription of oxidative stress-inducible genes (oxyR) in Salmonella typhimurium and Escherichia coli. Treatment of bacterial cells with low doses of H202results in synthesis of at least 30 proteins, and the cells become resistant to subsequent doses of HZO2that would otherwise be lethal (Demple, 1991; Christman et al., 1995). The expression of nine of the proteins induced by the effect of H202is under the control of the

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oxyR gene (Christman et al., 1989,1995). These proteins include catalase, alkyl hydroperoxide reductase, glutathione reductase, and a protective DNA-binding protein (Ding and Demple, 1997). Another transcriptional or NOactivator, soxR protein, controls the cellular response to the 02*-generating system in E. coli. The soxR transcriptional activity increases expression of SOD, glucose-6-phosphate dehydrogenase,fumarase C, aconitase, and the DNA repair enzyme endonuclease IV (Demple, 1996). Thus, bacteria are able to protect themselves against the oxidative burst which occurs when bacteria encounter professional phagocytes. Interestingly, OxyR autoregulates its own expression. In vitro experiments have shown that the oxidized, but not reduced, OxyR activates transcription of oxyR regulon. Interconversion of the oxidized and reduced forms of OxyR was revealed to be readily reversible. Both forms are able to bind to three diverse sequences upstream of OxyR-regulated promoters, although DNA binding of these two forms of OxyR is different. The oxyR expression was reduced substantially in the presence of both oxidized and reduced OxyR. However, oxidized OxyR was more efficient than the reduced form in repressing its own expression. OxyR is a thiol-containing activator. Conformational changes caused by direct oxidation of the OxyR protein transduce an oxidative stress signal to RNA polymerase. It has been proposed that one essential cysteine may be reversibly oxidized to a sulfenic acid (RSOH), possibly by direct binding of ROS (Storz et al., 1990). It was reported recently that the oxyR gene may also be activated by Snitrosothiols. Signaling by S-nitrosylation can extend to the level of transcription and thereby contributes to bacterial adaptation to “nitrosative stress” (Hausladen et al., 1996). Dimeric SoxR protein in E. coli acts both as a sensor and as a transcriptional activator of the gene SOXS.SoxR contains a pair of redox active iron sulfur centers [2Fe-2S]. Experiments in vivo using electron paramagnetic resonance (EPR) spectroscopy (Ding and Demple, 1997) have shown that oxidation and reduction of [2Fe-2S] clusters of SoxR can reversibly switch on and switch off the transcription of SOXS.The SoxR [2Fe-2S] clusters were in the completely reduced state during normal aerobic growth but were rapidly and completely oxidized under the effect of 02*-. The oxidized clusters were readily re-reduced after removal of oxidant. EPR analysis has shown that the electron-transfer processing involving the [2Fe-2S] centers is fast, and that SOXStranscription is directly proportional to the amount of oxidized SoxR. The kinetic analysis also indicated that an oxidative stress-linkeddecrease in SOXSmRNA contributedto the rapid establishment of a new steady state after SoxR activation. However, stability returns to the nonstress level after the stress is removed. There are some strain-tostrain variations in the metabolism of redox-cycling agents and in reducing activities specific to the SoxR [2Fe-2S] centers (Gaudu and Weiss, 1996;

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Gaudu et al., 1997). The experiments described permitted researchers to conclude that a redox stress-related change in sox$ mRNA stability might represent a new level of biological control. It is likely that such easy redoxregulated iron sulfur switches might also have other biological roles (Ding and Demple, 1997).

E. ROS as Useful Metabolites Since the redox potential of 0;- in a lipid environment makes it a very good electron donor (Peover and White, 1966), Mailer (1990) demonstrated that 02*could induce electron flow from 02*to the electron transport chain in isolated heart mitochondria under normal physiological conditions resulting in oxidative phosphorylation of ADP and formation of ATP. These data are confirmed by experiments in which ATP was formed from ADP and O,*- in a nonaqueous environment in the absence of any biological material (Lippman, 1982). Because the endogenous SOD is a matrix enzyme, the position of the 0;- generator is of crucial importance. H202is also considered an energetic agent; the energy of one H202molecule (25 kcal) is sufficient to produce about two molecules of ATP (Buvet, 1982; Buvet and Le Port, 1984). Thus, the H20z-mediated ATP formation with ADP and P o d - as necessary substrates in mitochondria and cytosolic extracts of insect (Pieris brassicae) and green phytoflagellate (Euglenagracilis) was demonstrated by Vuillaume et al. (1982) and Calvayrac et al. (1985). The authors concluded that the energy of photoproduced or experimentally added H202could be converted by a catalytic-type system into energy-rich ATP bonds.

VII. ROS in Plants Data on the role of ROS in functioning plant cells are much poorer than similar data for animal cells and bacteria. Therefore, in this review we will briefly outline this problem in the plant physiology. During their life cycle, plants have to react to various threats from the environment. Plants respond to a pathogen attack by activating a variety of defense reactions, such as generation of ROS, induction of pathogenesis-related genes, biosynthesis of phytoalexins, low-molecular compounds with antimicrobial activity, cell wall reinforcement, and generation of a hypersensitive response (the death of small number of cells at the site of infection). It is well documented that the oxidative burst resulting in large amounts of ROS is one of the earliest observable aspects of the plant defense strategy. However, the intrinsic

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differences in anatomy, morphology, and physiology between the animal and plant kingdoms are reflected in the character of defense responses. Among other peculiarities, the quite different ability to move and the presence of a structural exocellular compartment (cell wall) distinguish the plant cell from the animal one. The nature of the elicitor and receptor molecules participating in the recognition events, the elements and the arrangement of the signaling pathways, and the sources of ROS in the oxidative burst in plant cells are only partially elucidated; the data on biochemical reactions that occur both during and after oxidative burst in plants are controversial. In mammals, the phagocytes generating ROS mostly remain alive after killing the microorganism. In plants, the successful restriction of the spread of pathogen is often accompanied by a hypersensitive response and death of cells; cell death may possibly result from the effect of ROS (Low and Merida, 1996; Wojtaszek, 1997). Observations from many experiments on different plant species suggest that there are some peculiarities in the components of the ROS generating system in comparison with animal cells. Thus, of particular interest is the finding that mannitol dehydrogenate is one of pathogenesis-related proteins; mannitol can serve as an antioxidant, and changes in its content can affect the extent of internal ROS production in response to pathogens (Williamson et al., 1995). A correlation between ROS production and biosynthesis of phytoalexins in response to the effect of elicitor is not observed in all experiments. Obviously, the results depend, on the one hand, on the cell type, and, on the other hand, on the recognition and signaling system (Jakobek and Lindgren, 1993). However, in leguminous plants, whose flavonoids and isoflavonoids act as both phytoalexins and antioxidants, the relative timing of ROS production and secretion of phenolic compounds toward the site of infection may be of crucial importance to the final magnitude of the oxidative burst (Wojtaszek, 1997). Systematic acquired resistance (SAR) is one of the consequences of the plant defense response. In question here is the relationship between ROS generation and accumulation of salicylic acid (SA) in the site of infection. SA inhibits catalase in plants, and this effect results in an elevated level of H202at the infection site; this elevated H202has been proposed to act as a second messenger of SA in the signal transduction pathway leading to SAR and gene activation for pathogenesis-related proteins (Klessig and Malamy, 1994). Also in question is the causal link between the oxidative burst and immobilization of cell wall proteins (Bradley et al., 1992; Wojtaszek, 1997). In plants, rapid immobilization of proteins may strengthen cell walls, thus slowing down invasion of pathogen. The immobilization of cell wall proteins and cross-linking of these proteins are suggested to be HzOz driven (through the peroxidase-catalase reaction in the wall) and pH dependent (Iiyama et al., 1994). However, another mechanism,

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pH-independent and lipid peroxide driven, has also been shown (Wojtaszek, 1997). Recently, another role of H202was proposed as the key component in the formation of hypersensitive cell death (Levine et al., 1994). It has been suggested that H202and O2.-, while acting directly as antimicrobial agents, may serve as second messengers or catalysts in plants to activate a more diverse set of defense responses. Thus, H202is able to initiate expression of genes encoding cellular protectants such as glutathione S-transferase or glutathione peroxidase. Interestingly, the gene activation occurred at the lower concentration of H202(2 mM) than the cell death (4-6 mM) (Levine et al., 1994).An increasing amount of data on a correlation between the H202 production and cell death permit the conclusion that ROS from the oxidative burst are necessary but not sufficient for triggering the hypersensitive cell death (Jabs et aZ., 1997). We now discuss the role of ROS in the activation of gene expression for cellular protectants in cells adjacent to the site of infection in plants, possibly via induction of nuclear transcription factors similar to the NF-KBor AP1 factors in animal cells (Wojtaszek, 1997). Interestingly, the role of 02.-, but not of H202, in gene activation to phytoalexin synthesis in parsley has been demonstrated (Jabs et aZ., 1997). Regulators in chloroplasts and mitochondria, the sites of electron transport associated with photosynthesis and oxidative phosphorylation, may also be sensitive to oxidation or reduction. Similar to animal cells, sulfhydryl redox status plays a critical role in the maturation and assembly of globulins in seeds (Jung et al., 1977). The study of Sanchez-Fernandez et al. (1997) indicates an important role of glutathione for redox regulation of cell division in the roots. Thus, accumulating evidence supports the concept that ROS are involved in pathogen defense-related signaling in plants.

VIII. Concluding Remarks ROS has long been the subject of great interest. Briefly, these substances which pose an evolutionarily ancient threat to all cells and organisms, have been determined to be necessary transmitters in intracellular signaling. Practically all cells have a specialized enzyme system for ROS formation. This means that ROS are not only side products of general metabolism but also play an important role in the performance of cellular functions. New concept and new experimental approaches emerged from the discovery that ROS at subtoxic concentrations activate cellular functions through the same signal transduction pathways as do natural activators. In this case, however, the concentration range of ROS is very small and depends on the cell type. The next experimental boom started when ROS (predominantly

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H202) were found to be formed in the cell under effects of quite different stimuli, especially growth factors. The observation that intracellular H20z formation is required for realization of mitogenic effects of growth factors and other agonists has made possible the conclusion that H202can function as a second messenger in activation of gene transcription and, as a result, in the control of cellular responses. At high concentrations, ROS are toxic to cells; therefore, they are unusual, even surprising,candidates for the role of signal molecules. However, it is no longer surprising that Ca2+and NO, two well-known messengers, are also harmful to cells at high concentrations. The ROS effects, similar to those of Caz+,in intracellular damage mechanisms involve participation of the same effector molecules as those in the activation process. Also, when comparing Ca2+and ROS, it should be noted that the realization of the regulatory role of ROS necessitates, apart from specialized systems of their formation, the antioxidants capable of removing ROS and preventing damage. Progress has been made toward the understanding that ROS act via changes in the cell redox state, especially via oxidation of thiol groups of proteins and glutathione. Nevertheless,much remains to be learned about mechanisms of ROS action on cells and their involvement in intracellular signaling. However,it is clear that the delicate intracellular balance between oxidizing and reducing equivalents is an important tool in the regulation of cellular processes. Acknowledgments We express cordial thanks to Dr. K. M. Kirpichnikova and Dr. L. Z. Pevzner for help in preparing the manuscript. Our studies included in this review were supported by Grant 9704-49614from the Russian Basic Research Foundation and this review was made possible in part by The Physiological Society under its Support Scheme for Centers of Excellence in Eastern Europe and Third World Countries.

References Abate, C., Patel, L., Rauscher, F. J., 111, and Curran, T. (1990).Redox regulation of Fos and Jun DNA-binding activity in vitro. Science 249,1157-1161. Ager, A., and Gordon, J. L. (1984). Differential effects of hydrogen peroxide on induces of endothelial cell function. J. Exp. Med. 159,592-603. Aitken, R. J., Fisher, H. M., Fulton, N., Gomez, E., Knox, W., Lewis, B., and Irvine, S. (1997).Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine. Mol. Reprod. Dev. 47,468-482. Akagi, M.,Katakuse, Y., Fukuishi, N., Kan, T., and Akagi, R. (1994). Superoxide anioninduced histamine release from rat peritoneal mast cells. Biol. Phorarncol. Bull. 17,732-734.

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High-Density Lipoprotein: Multipotent Effects on Cells of the Vasculature Gillian W. Cockerill* and Stephen Reedt *Department of Cardiovascular Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Hammersmith Hospital Campus, London W12 O N N , United Kingdom; and ?School of Biosciences, University of Westminster, London WIM WS,United Kingdom

The epidemiological evidence showing a strong inverse correlation between the level of plasma high-density lipoprotein (HDL) and the incidence of heart disease suggests that HDL has a protective effect against cardiovascular disease. The mechanism of this protective effect has been the raison d'efre for much research. The ability of HDL to mediate cholesterol efflux from peripheraltissues has been used to explain the cardioprotective effect of HDL. However, there is little direct evidence to suggest that in subjects with low plasma levels of HDL the rate of cholesterol efflux from peripheral tissues is significantly reduced. This observation suggested that HDL may be mediating its protective effect through other mechanisms. This review provides an account of the burgeoning evidence that HDL has many effects on cellular processes, in addition to the effects on cholesterol efflux, and will illustrate the multipotency of this lipoprotein. KEY WORDS: HDL, Atheroclerosis, Inflammation, Apolipoproteins, Thrombosis, Vasoactive agents, Acute phase reactants. o 1% Academic PW.

1. Introduction A. Structure of High-Density Lipoproteins

High-density lipoproteins (HDLs) are normally considered to consist of those plasma lipoprotein particles which fall into the density range of 1.0631.21 g/ml. Within this range there is a large spectrum of species (Albers el aZ., 1984). The two major species, designated HDL, and HDL3, fall within the density ranges 1.063-1.125 and 1.125-1.21 g/ml, respectively. The genInrematiom1 Review of Cytology, Vol. 188

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era1 structure of the lipoprotein particle is globular. Within the outer part of the lipoprotein are the more polar lipids, phospholipids, and free cholesterol, with their charged groups pointing out toward the aqueous environment. Protein components are embedded in the outer layer. The more hydrophobic lipids, such as the esterified cholesterol and triglycerides, reside in the core of the particle. These hydrophobic lipids form a central lipid droplet into which are anchored the surface-coating molecules of phospholipid, cholesterol, and protein (Soutar and Myant, 1972). Newlyformed nascent HDL particles lack this central lipid droplet and appear as disc-like bilayers composed of phospholipids and proteins (Fig. 1). The most abundant apolipoprotein in HDL is apo A-I, with smaller amounts of apo A-11, apo A-IV, C-111, apo D, apo E, and apo J. Various other proteins also reside on the HDL particle, such as lecithin-cholesterol acetyl transferase, PAF-acetyl hydrolase, and paraoxonase. HDLs are dynamic components of blood plasma, able to interact with cells and other lipoproteins. Although it would be naive to consider HDLs as a single group of lipoprotein particles, in order to understand their function, in vitro analysis often involves the use of discrete subfractions or synthetic reconstituted particles prepared with a restricted number of proteins and lipids. In this chapter we will present and discuss the in vitro effects of HDLs and examine the possible in vivo signiscance of these findings. 6. Evidence Supporting the Protective Effect of HDL against Heart Disease

Following the original observation that the level of plasma HDL cholesterol was inversely proportional to the incidence of coronary artery disease (CAD) (Miller and Miller 1979), several large epidemiological studies substantiated this relationship (Gordon et aZ., 1977, 1989). Recently, a study of Japanese men, whose plasma total cholesterol levels were relatively low, showed an inverse relationship between HDL cholesterol and CAD incidence (Kitamura et d.,1994). Compelling evidence in support of the

FIG. 1 Possible structure of spherical and discoidal HDL.(Top) The possible structure of the spherical native HDL particle. The central lipid core containing esterilied cholesterol and triglycerides is surrounded by the more polar lipids, phospholipids, and free cholesterol into which are embedded the apolipoproteins. (Bottom) The possible structure of nascent and discoidal HDL particles. These particles lack a central lipid core and are formed as a lipoprotein bilayer with the amphipathic helices of protein components arranged around the edge of the disc. TG, triglyceride; CE, cholesteryl ester; PROT, protein, PL,phospholipid; FC, free cholesterol) (Adapted from Soutar and Myant, 1972).

HIGH-DENSIPI LIPOPROTEIN Transverse section TG

PROT

PL

FC

0

@ 0

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Overhead view

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protective effects of raised HDL levels has come from studies in which murine HDL levels have been induced by the transgenic insertion of the human apolipoprotein A-I (apo A-I) gene. Elevation of circulating HDLs by approximately two-fold above the normal level in such mice confers a dramatic reduction in the extent of fatty streak formation in response to an atherosclerotic diet (Rubin et d,1991; Chajek-Shaul et al., 1991). In addition, the insertion of the human apo A-I transgene into apo-E knockout mice, which develop spontaneous atheromatous lesions as a result of high circulating levels of chylomicron particles, significantly inhibited the rate and extent of spontaneouslesion formation (Paszty et al., 1994).From these studies it was clear that the human apo A-I protein was able to incorporate into murine HDL particles and was an effective antiatherosclerotic agent. These studies demonstrated that the species of origin of the apolipoprotein had no effect on the ability of the particle to mediate its protective effect. Finally, Badimon and coworkers (1990) demonstrated that bolus injections of homologous HDL into rabbits ameliorated the fatty streak formation and progression followingcholesterol feeding. Using a similar experimental approach, recombinant apo A-Imo h e r s were also shown to be able to inhibit neointimal thickening (Soma et aZ., 1994). C. Cellular Mechanisms Involved in Atherogenesis

The initial “response to injury model” has been significantly modified since its inception (Ross, 1993). There is now general agreement that fairly modest changes in the normal phenotype of the endothelial cell can result in the sequence of events resulting in the development of atheroma (Fig. 2). Cytokines, endotoxins, oxidized lipoproteins, and shear stress have all been shown to alter the permeability, adhesiveness, and thrombogenicity of the endothelium. This altered phenotype is able to support the adhesion of mononuclear leukocytes, the earliest observable cellular event in the genesis of atheroma (Gerrity et d,1979; Davies et aL, 1988) (Fig. 2B). Following transmigration of these leukocytesinto the subendothelial space, the cells differentiate and are able to accumulate oxidized low-density lipoprotein (LDL) and express a range of peptides able to affect the phenotype of medial smooth muscle cells (SMCs). These cells assume a “foam cell” morphology (Gemty, 1981; Fig. 2C). Subsequent migration of synthetic-state SMCs into the intima form the diffuse intimal thickening which is often the precursor lesion of the atheromatous plaque (Velican and Velican, 1980). Further accumulation of lipid by both the macrophages and the SMCs within these thickenings generates a fatty streak lesion. These raised lesions progress to the state of the complex plaque by further intimal hyperplasia, lipid accumulation, and modification of matrix subse-

A

EC EL

SMC

C

D

FIG. 2 Cellular processes involved in atherogenesis. Diagram illustrates that inflammatory mediators (cytokines,shear stress, and high cholesterol) can alter the (A) normal nonadhesive, nonthrombogenic endothelial layer upon a medial layer of contractile smooth muscle cells into (€3) an altered endothelial phenotype which is now able to support the binding and transmigration of leukocytes. Once the leukocyte has arrested in the subendothelial space, its own phenotype will change, and it is now able to produce chemokines to attract further leukocyte infiltration, accumulate oxidized lipids, and allow platelet activation (C). Production of growth factors, metalloproteases, and cytokines by the differentiated leukocytes supports smooth muscle cell migration and proliferation in the intima, generating a raised lesion which ultimately develops into a complex plaque with a grumous core (I)).EC, endothelial cell; EL, elastic lamellae; SMC, smooth muscle cell; PL, platelet.

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quent to the interaction of further leukocyte infiltration, the formation of microthrombi, and fibrinolytic events (Fig. 2D). Many of these processes will be discussed with respect to HDL in the following sections.

II. In V

i Effects of HDL on Endothelial Cells

A. Morphology In the absence of pathology the vascular endothelium forms a compact monolayer of cells with one of the slowest rates of cell turnover in the body. Human umbilical vein endothelial cells (HUVECs) in culture mimic this and form a tight monolayer of pavement-like cells. In vivo the monolayer is under the constant force of the flow of blood, which is both pulsatile and either laminar at sites of low resistance or turbulent, usually at sites of bifurcation. The monolayer of endothelium under laminar flow forces appears to be polarized, a feature not seen in static culture. Interestingly, when native HDL is added to static cultures of HUVEC the cells appear to undergo a shape change resulting in a monolayer of cells which are polarized and have more discrete cell-cell junctions (Fig. 3). Little is known about the effect of lipoproteins on cell junctions. However, using vital dye transfer, it has been shown that native LDL must undergo oxidative modification before being able to influence intercellular communication with cultures of human smooth muscle cells. Similar to the effects observed with antioxidants used in these experiments, HDL was able to diminish oxidized LDL-induced inhibition of cell-cell communication. The authors suggest that the balance of lipoprotein could modulate gap junctionmediated intercellular communication, which could subsequently influence pathogenesis (Zwijsen et aL, 1991).

B. Proliferation The maintenance of an intact endothelium is important in preserving a nonthrombogenic vessel surface. The demonstration that HDL could be mitogenic for endothelial cells (ECs) provided a further mechanism for its atheroprotective mechanism (Tauber et aZ., 1980, 1981; Fig. 4). HDL was observed to maintain proliferation of low-density ECs in serum-free conditions with transferrin when culture dishes were coated with extracellular matrix. The mechanism of this proliferative effect is not completely understood. Recent studies have shown that HDL is able to induce a rapid and maintained increase in intracellular pH, inositol phospholipid breakdown,

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H 50pm FIG. 3 Effect of HDL on morphology of endothelial cells in culture. Confluent monolayers of human umbilical vein endothelial cells were incubated overnight in the presence (top) or absence (bottom) of native HDL at normal physiological concentration (1 m g / d apo A-I). The phase-contrast photographs shown here are typical of the morphological effect observed. In the presence of HDL (top), the cells appear to organize in an elongated aligned pattern, more typical of endothelium under shear stress in vivo.

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" 1 3

0.8

TIME IN CULTURE (DAYS) FIG. 4 HDL is mitogenic for human umbilical vein endothelial cells in culture. Human umbilical vein endothelial cells were seeded at 2 X 103 celld200 pl/%-well microtiter plate and grown in the presence (solid bars) versus the absence (stippled bars) of HDL (1 m g / d apo A-I) in medium 199 containing 2% fetal calf serum in the presence of endothelid cell growth factors. At Days 3, 5, and 7 cell numbers were estimated by fixing and staining the cells in 1%methylene blue. The graph demonstrates the ability of native HDL to substitute for the presence of fetal calf serum in the presence of exogenousendothelial cell growth factors.

calcium flux,and stimulatiton of protein kinase C (PKC) (Tamagaki et aL, 1995), intracellular events similar to those observed following receptor activation. These intracellular responses to HDL were postulated to play a key role in the proliferative response of the endothelium to the lipoprotein. In particular, Tamagaki and coworkers (1995) have shown that the alkalinization-inducedproliferative response to HDL is blocked by amiloride, suggesting a dependence on stimulation of the Na+/H+antiporter system. In vivo, intimal thickening, due to the modulation, migration, and proliferation of medial smooth muscle cells into the intimal region of the vessel, is important in the development of these precursor lesions of the atheroscleroticplaque (Wissler, 1984).The effects of HDL on SMC proliferation have mainly been studied under culture conditions in which either lipoprotein-deficient serum or exogenous growth factors, such as insulin, transferrin, epithelial growth factor, fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), would be present (Fischer-Dzoga et al., 1974, 1976; Ross and Glomset, 1973; Libby et al., 1985; Chen et al., 1986; Bjorkerud and Bjorkerud, 1994). The results from these studies vary, suggesting a stimulatory (Ross and Glomset, 1973; Libby et al., 1985; Bjorkerud and Bjorkerud 1994), inhibitory (Hessler et aL, 1979), or no effect (Fischer-Dzogaet al., 1976; Fischer-Dzoga and Wissler, 1976). These

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studies do not reveal whether the lipoproteins have intrinsic growthpromoting properties or whether they potentiate other factors. However, Resink and coworkers (1995) have shown that both HDL and LDL demonstrated growth-promotingeffects on omental arteriolar SMC in the absence of exogenous factors. Since neither lipid peroxidation nor inhibition of scavenger receptor binding were able to affect the response to lipoproteins in this study, the authors suggested that the mitogenic potential of lipoproteins was dependent on direct activation of replication-coupledsignal transduction pathways. To what extent the mitogenic response to lipoproteins is dependent on cell type is very difficult to evaluate since a wide variety of cell types have been used in studies to date. In rat mesengial cell cultures the mitogenic potential of HDL was demonstrated to be dependent on apo A-I binding and involved tyrosine kinase activation but was independent of PKC (Neverov et al., 1997). Since modulation of protein phosphorylation plays an important role in the early biochemical events which occur during mitogenesis (Kaur et al., 1989; Regazzi et al., 1988), the effect of HDL upon this process has been examined. Total native HDL was shown to induce phosphorylation of a 28-kDa protein in bovine vascular ECs, the level of phosphorylation being synergistically induced with bFGF (Darbon et al., 1988). Interestingly, although the lipid-free apoprotein was able to induce this response, the protein-freelipid component of HDL was not. Studies using apo-E-depleted HDL3-induced mitogenesis of A549, a human adenocarcinoma cell line, have identified the importance of a Ca2+-dependentphosphorylation of a 20-kDa protein. The downregulation of PKC by 24-h incubation of the cells in phorbol myristate acetate (PMA) prevented phosphorylation of a 24- and 28-kDa protein without affecting the 20-kDa protein phosphorylation, thus suggesting that the kinase activity identified is not modulated by PMA and could be a calmodulin kinase or an isoenzyme of PKC not dependent on PMA (Favre et al., 1993; Tazi et al., 1995). What are the effects of lipoproteins on immediate early response genes? Egr-1, a transcription factor belonging to a family of immediate early response genes, is expressed upon growth and/or differentiation signals in a wide variety of cell types and as such is a reasonable marker of proliferation (Sukhatme et al., 1988; Cao et al., 1990; Sukhatme, 1990). Incubation of rat aortic SMCs with either HDL, LDL, or very low-density lipoprotein (VLDL) was shown to induce a rapid Ca2+-dependentincrease in the expression of Egr-1 (Sachinidis et al., 1993). The direct mitogenic effect of HDL upon SMCs in vitro is not supported by studies in which HDL was given as a bolus after the initiation of experimental intimal thickening in vivo (Badimon et al., 1990). In these studies increasing plasma levels of HDL prevented the formation of intimal thickening and thus inhibited either the migration or the proliferation of SMCs

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into the intima. These effects may be due to the ability of HDL to inhibit adhesion and infiltration of leukocytes through the vascular endothelium, which will be discussed in Sections I11 and IV.

C. Cytotoxicity It is interesting to note that, in several studies, LDL (both native and oxidized) has been shown to be cytotoxic for endothelial cells (Zhao et aL, 1994,1995). This cytotoxic effect of LDL is thought to be attributed to its triglyceride component (Speidel et aL, 1990). However, the addition of HDL can rescue this response and prevents the cytotoxic effects of LDL (Evensen, 1979). The relevance of this observation in vivo is not known.In subjects with inherently low levels of circulating HDL, such as in Tangier’s disease, the extent of atherosclerosis is not grossly accelerated, suggesting that if the cytotoxic effects of LDL are important in vivo, then even very low levels of HDL can prevent their apparent cytopathic effects. It may be that the cytotoxic effects of LDL have more significancein the interpretation of in vitro experiments.

D. Apoptosis A most important feature of the mature atheroscleroticplaque is the cellular dynamics of these lesions. Lesions with a reduced number of SMCs, myofibroblasts, and fibroblasts are likely to have a weak friable cap and are prone to rupture (Davies, 1995; Libby et aL, 1996; Rajagopalan et aZ., 1996). These lesions are known as unstable plaques and are clinically the most life-threatening. Stable atherosclerotic plaques have a thick cap and contain a high proportion of mesenchymal cells which establish and maintain this clinically “benign” lesion. The processes governing the cellular dynamics of the mature plaque are not well understood but must include control of the rate of cell proliferation, necrosis, and apoptosis, all processes identified in the mature plaque and during wound healing (Geng and Libby, 1995; Han et aL, 1995; Des-Mouliere et aL, 1997). Products of lipid peroxidation and oxysterols, found in atherosclerotic plaques, have been shown to induce apoptosis (Bennet et aL, 1995). In addition to apoptotic cells being isolated in mature plaques, in vitro studies have demonstrated that IL-1 and transforming growth factor-a (TNF-a) in combination can induce nitric oxide (NO)-mediated upregulation of Fas in vascular SMCs (Fukuo et al., 1997). These two lines of research suggest a mechanism and provide evidence that apoptosis can play a role in plaque development. Recently, it has been shown that HDL can inhibit oxidized LDL-induced apoptosis of SMCs

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(SUCet aL, 1997). The mechanism of this antiapoptotic effect of HDL in vascular cells is unclear. The demonstration that ligand binding of Fas does not appear to be involved in EC apoptosis suggests an alternative mechanism may be involved in apoptosis of ECs (Richardson et aZ., 1994).

E. Migration It is very rare that extensive denudation of the vascular endothelium occurs pathologically. However, after endarterectomy and during by-pass surgery it is usual that a region of the endothelium is denuded and rapidly proliferates to recover the vessel integrity. The process of reendothelialization has been extensively studied using rat models (Riedy and Schwartz, 1981;Riedy et al., 1983). The entire process is complex, involving the initial release of the cell from the basement membrane proteins, cell division, and the subsequent migration of the cell. Migration has been extensively examined in simple in vitro models. Recently, it was shown that preincubation with HDL could enhance the rate of endothelial cell migration (Murugesan et al., 1994), suggesting yet another point of intervention in which HDL can potentially be of clinical benefit. In these studies, the role of proliferation, G protein-linked interactions, and activation through phospholipase A2 on endothelial migration was investigated. Prevention of cell proliferation using hydoxyurea did not affect the ability of HDL to stimulate migration. Similarly, blocking G protein-mediated interactions with pertussis toxin did not inhibit HDL-mediated migration although it did block migration induced by bFGF. A phospholipase A2 inhibitor, aristolochic acid, was able to block FGF-mediated migration without affecting HDL-mediated migration. Since the tln max of this in vitro effect was shown to be 2540 p g / d HDL, well below physiological level, it is difficult to interpret the in vivo significance of this observation.

111. Effects of HDL on Leukocyte Activation

A. Leukocyte Adhesion Following feeding of a high-cholesterol diet to experimental animals the earliest observable cellular event in the vasculature is the adhesion of mononuclear leukocytes to the endothelium. The molecules which mediate this event have been extensively characterized (Bevilacqua et al., 1987, 1989;Luscinskas et al., 1989;Springer, 1990; Abbassi et aZ., 1993). Following the demonstration that HDL could inhibit the binding of monocytes to an

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in vitro coculture model (Navab et aZ., 1991) of SMCs and ECs, or to endothelialmonolayers (Maier et aL, 1994),we showed that HDL can inhibit the cytokine-induced expression of VCAM-1, ICAM-1, and E-selectin on HUVEC (Cockerill et aZ., 1995; Fig. 5). This effect is both dose and time dependent. The addition of HDL to endothelial cells 30 min after activation by cytokines had no effect on the expression of adhesion molecules. In further studies, reconstituted discoidal particles in which apo A-I, apo AIMlano,or apo A-I1 were the sole apolipoproteins were also shown to inhibit the cytokine-induced expression of VCAM-1, whereas the purified apolipoproteins in the absence of lipids had no effect (Calebresi et aL, 1997). Recently, we demonstrated that preincubation of the cells with HDL had no effect on cytokine-induced translocation of NF-KB or its cytoplasmic inhibitory protein, IKBc~, levels which are normally rapidly degraded and resynthesized following cytokine activation (Fig. 6) (G. Cockerill et aL, submitted for publication). However, HDL was able to mediate differential

A

B 10

la P" i 4

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NIL TNF

TNF HDL

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28s 18 8

FIG. 5 HDL inhibits cytokine-induced adhesion molecule expressionin vitro. Confluent monolayers of endothelial cells were incubated for 16 h with a range of concentrations of HDL. Following the addition of TNF-a (1 ng/ml) for an additional 4 h the cell surface expression of E-selectinwas measured using flow cytometq (A). The values represent the mean fluorescence intensity in response to cytokine (hatched bars) versus unstimulated (solid bars). The results represent data from one experiment and are representative of four experiments. Differences between means were evaluated using an unpaired t test. **p < 0.001. (B) Steady-state mRNA levels of E-selectin in response to HDL preincubations (1 m g / d for 16 h) (TNF, HDL) in comparison to cytokine alone (TNF) or no treatment (NIL). (Bottom) The ethidium bromidestained gel showing equivalent loading of the samples.

0 n

en

A

IOmin

3hrs

FIG. 6 Effects of HDL are independent of the NF-KBfamily of transcription factors per se. (A) Electrophoretic mobility shift assays were used to measure the effect of HDL on NF-KB DNA binding and nuclear translocation. Confluent endothelial cells were either untreated or incubated in the presence of HDL (1 mg/ml apo A-I) for 16 h followed by a I-h incubation with TNF-a! (1 ng/ml). Nuclear extract (5 pg) from each group was incubated with a double-stranded 32P-labeledE-selectinNF-KBdouble-stranded oligonucleotideprobe. Autoradiographs of the size-fractionatedcomplexes show an absence of NF-KB complex in untreated cells (unstimulated)which is present in cytokine-stimulatedcells (TNF) and not altered by preincubation of the cells with HDL (HDL, TNF). The specificNF-KBcomplex could be competed out in the presence of excess unlabeled E-selectin NF-KB(TNF, lOOX cold probe). The band migrating below the NF-KBcomplex appears to represent a nonspecific constitutive factor. (B) Western blot analysis of I&a following a 16-h incubation of confluent endothelial cells in the presence or absence of HDL (1 m g / d apo A-I) after which both groups were stimulated with TNF-a! (1ng/ml). Cell extracts of 2 X 106cells per lane were isolated at 10min and 3 h after cytokine stimulationand Western blotted using an anti-IKBaantibody (Mad-3,Santa Cruz, CA). Protein bands were developed using ECL (Amersham, UK) and show no significant difference between those cells treated with HDL and those cells with no HDL.

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gene regulation since in transient transfection of endothelial cells with the GM-CSF promoter and the E-selectin promoter, both of which have a significant requirement for NF-KB, HDL was able to inhibit the cytokineinduced expression through E-selectin while having no such effect through the GM-CSF promoter (G. Cockerill et d.,1998). The precise reason for this differential effect, although clearly not related to NF-KBdirectly, has yet to be determined. We have also shown that HDL can inhibit the adhesion of neutrophils to endothelial monolayers (Fig. 7). Since leukocyte adhesion is one of the earliest events in the formation of atherosclerotic lesion development, we suggest that this antiinflammatory effect of HDL

T

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4.(

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FIG.7 HDL inhibits neutrophil adhesion to endothelial cells in vifro. Confluent endothelial cells were incubated with HDL (1 mg/ml apo A-I) or PBS control for 16 h before being stimulated with TNF-a(1 ng/ml) for 4 h prior to the addition of chromium-labeledneutrophils. Neutrophils were allowed to attach to the cells for 30 min at 4°C. Following vigorous washing the remaining cells were lysed with NaOH and the relative extent of adhesion was evaluated by measuring the gamma counts remaining in each well. This graph shows data from one experiment which is representative of three similar experiments.

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is another mechanism whereby the lipoprotein can mediate its protective effect.

B. HDL and Leukocyte Degranulation The recruitment and activation of leukocytes are known to play an important role in inflammation and the accumulation of leukocytes into the developing plaque has been well documented (Stemme and Hansson, 1994). Research concerning the effect of HDL on transmigration will be discussed in Section IV. Research reports concerning the effect of HDL on degranulation are limited. Analysis of the direct effect of lipoproteins on neutrophil degranulation has shown that although lipid-free apo A-I is able to inhibit neutrophil degranulation, the native heterogeneous HDL cannot (Blackbum et aZ., 1991). Whether this is a mechanism which operates in vivo depends on whether lipid-free apo A-I is prevalent in vivo. Others have shown that degranulation of neutrophils and mast cells can modulate HDL, leading to oxidation and proteolysis of the lipoprotein (Cogny et aZ., 1994; Linstedt et al., 1996). The question of whether the level of HDL can affect mobilization of leukocytes, which occurs during inflammation, remains unanswered; however, we have shown that HDL has a differential effect on the ability of endothelial cells to synthesize the hemopoeitic growth factors GM-CSF and G-CSF, which are important in the mobilization of leukocytes, (Fig. 8). In addition, GM-CSF has been shown to be a factor underlying macrophage proliferation and accumulation in atherosclerotic plaques (Wang et al., 1994). Thus, modulating the endothelial synthesis of these growth factors may prevent mobilization and/or proliferation of leukocytes in response to inflammatory stimuli.

IV. Effects of HDL in Cell Transmigration

Following capture from the axial stream and firm adhesion to the endothelium, leukocytes will transmigrate through the endothelial monolayer (Dejana et al., 1996; Buckley et al., 1996; Murohara et al., 1996; Wada er al., 1993). Although the precise molecules involved in leukocyte transmigration may vary according to the specific site, there is general agreement that the process requires both chemotactic and haptotactic mediators. HDL has been shown to inhibit the expression of the monocyte chemotractant monocyte chemotactic factor-1, and in so doing reduces the extent of monocyte transmigration in endothelial-smooth muscle cell cocultures (Navab et al., 1991). In simpler in vitro models of transmigration, using the growth of

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60 -r

T 50-

40-

30-

20

-

10-

1 Nil

TNF

HDL+ TNF

HDL+ TNF+ anti418

118

IL8+ anti-118

FIG. 8 Differential regulation of EC cytokines by HDL. Confluent endothelial cells were treated, as indicated, with HDL (1 mg/ml apo A-I) alone or in combination with TNF-a (1 ng/ml) for 24 h, after which time supernatants were harvested and G-CSF and GM-CSF measured using an ELISA (R&D Systems) accordingto the manufacturer's recommendations. This graph shows means and standard deviationsof relative cytokine levels within one experiment.

endothelial cells on Transwell,we have shown that HDL can inhibit TNF-ainduced neutrophil transmigration by 50% (Fig. 9). The addition of blocking anti-IL-8 antibodies,while blocking IL-&inducedtransmigration by approximately 50%, cannot further inhibit the transmigration induced by TNF-a in the presence of HDL (Fig. 8). Using ELISA assays we have shown that HDL can also inhibit the TNF-a-induced expression of the CXC chemokine, IL-8,by endothelial cells, suggesting a possible mechanism for inhibition of transmigration (Fig. 10).

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[7 Nil HDL

T

I

Nil

TNF lOOU/ml

TNF lOU/ml

FIG. 9 Inhibition of neutrophd transmigration by HDL in v i m . Confluent endothelial cells grown on 8-pm transwells were divided into three groups. One group was incubated in HDL for 16 h, after which this group was subdivided and half of the cultures were treated with blocking anti-IL8 antibody for 15 min before both sets of cultures were treated with TNF-(Y (1ng/ml). Half the control group was treated with TNF-a and the remaining were untreated. Half the remaining group were treated with anti-IL-8 antibodies prior to the addition of IL-8 to the whole group. Following a 4-h stimulationof cytokine,chromium-labeledneutrophils were added and allowed to transmigrate for 4 h at 37°C. The extent of transmigration was evaluated by gamma counting the lysed labeled cells which had migrated into the lower chamber. This graph shows data from one experiment which is representative of four similar experiments.

V. HDL, Thrombosis, and Platelet Function

Injury to the vascular endothelium results in both rapid adhesion and aggregation of platelets at the site of injury and activation of site-specific and highly regulated coagulation cascade (Hekman and Loskutoff, 1987). Hence, the consequence of a dysfunctinal endothelium may include changes

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31

NIL

HDL

I

I

TNF

TNFlHDL

**

B

'1

l-7

NIL

HDL

T

I -

TNF

TNFlHDL

FIG. 10 HDL inhibits endothelialcell IG8 synthesis.Following a 16-h stimulation of confluent endothelial cells in the presence or absence of HDL (1 m g / d apo A-I) cultures were washed and TNF-a was added (1 ng/ml). Supernatants were collected at 4 h following stimulation and the level of IG8 was measured using an ELISA kit (R&D Systems, UK) according to the manufacturer's recommendations. These data are representative of three similar experiments.

in the ability of the cell to participate adequately in both coagulation and fibrinolysis. The final step in the coagulation cascade is the conversion of prothrombin to thrombin in the presence of factors Xa, Va, and Ca2'. This process occurs on the surface of membranes containing anionic phospholipids. Both native HDL and purified apo A-I were able to block the generation of procoagulant activity induced by A23187 and Ca2+(Epand er aZ., 1994).

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Data from this study suggested that HDL inhibited the transbilayer diffusion of anionic lipids required for the formation of the procoagulant complex leading to activation of prothrombin. Sulpice and coworkers (1994) showed that PI-4,5 biphosphate plays an important role in this process. Endothelial cells may have a role in both these processes because they can synthesize both fibrinolytic [(e.g., tissue plasminogen activator (tPA)] and procoagulant [(e.g., plasminogen activator inhibitor-l(PA1-1) and von Willebrand factor)] components. Tissue plasminogen activator has been a useful thrombolytic agent in patients with acute myocardial infarction (MI), whereas high levels of PAI-I have been associated with arterial and thrombotic disease incidence (Loscalzo, 1989; Dawson and Henney, 1992), although the association with PAI-1 is conflicting (Hellsten et al., 1992; Oseroff et al., 1989; Blann et al., 1995). Recently, insertional mutations in the tPA gene has been shown to associate with a greater risk of MI (Van der Bom et al., 1996). Endothelial coagulant phenotype is a balance between activation and inactivation modulated by a range of molecules (Ryan et al., 1994). There is evidence that lipoproteins can affect the production and/or secretion of these molecules (Levin et al., 1994). In response to cytokine activation at least 60% of the stimulated tissue factor (TF) is secreted ablurninally by endothelial cells. It is also likely that of the TF secreted onto the luminal surface, a large portion is sequestered by binding to the extracellular matrix components. Hence, the precoagulant effect of TF is regulated by exposure. Interaction with activated leukocytes can cause increased exposure of sequestered TF. Using human umbilical vein endothelial cells, it was shown that while VLDL strongly induced endothelial expression of PAI-1 and TF, the effect of LDL and HDL was modest (Kaneko et al., 1994). However, in further studies, when LDL and VLDL were conditioned by cocultivation with monocytes, both of these conditioned lipoproteins caused a significant increase in the level of expression of endothelial cells PAI-1 and TF (Kaneko et al., 1994). Although monocytes are normally antithrombotic and secrete thrombomodulin and tissue factor pathway inhibitor-1 (TFPI-l), the effect reported in this study could potentially be due to oxidation of the lipoprotein or because of induction of cytokine expression by the monocytes (Kaneko et al., 1994). TFPI-1 is a protease inhibitor containing tandem Kunitz-type inhibitory domains which inhibit coagulation factors Xa and VIIa, thereby blocking the initial step in the extrinsic coagulation pathway. Although the majority of TFPI1is associated with endothelial cells, three other isoforms have been demonstrated-one associated with VLDLLDL, one associated with .HDL, and one which is found as a free protein (Kato, 1996). HDL has been shown to inhibit both thrombin and ADP-stimulated platelet aggregation (Aviram et al., 1983; Nofer et al., 1996). Nofer and colleagues demonstrated that in response to HDL the platelet Na+/H+

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antiporter was activated and that the effect was dependent on GpIIb/IIIa since in platelets from Glanzmann’s thrombocytopenia type I, which lack this integrin, no induction was observed. In these experiments H Dk failed to induce an increase in cytosolic calcium levels. Chen and Mehta (1994) have shown that the inhibitory effect of HDL on platelet function is mediated by the regulation of NO synthase activity in platelets. However, although the ability of HDL to inhibit platelet aggregation has been linked to a reduction of NO production in platelets, the effect of HDL on Ca2+ signaling pathways and PKC-mediated pathways may play a role in this process (Zhao, 1996). In addition to inhibiting cytotoxic damage caused by oxidized LDL in ECs, HDL is also effective at inhibiting the cytotoxic effect in platelets (Zhao et aL, 1994, 1995). This may also partly explain the inhibitory effect of HDL on platelet aggregation. Earlier studies have shown that HDL can inhibit complement-mediated cell lysis (Rossenfeld et aL, 1983; Packman et aZ., 1985). The demonstration of an increased binding of HDL, apo A-I, and apo A-I1 to endothelial cells exposed to activated complement (Hamilton and Sim 1991) led Hamilton and coworkers to further investigate this effect. It was shown that apo A-I bound polymeric C9, not the monomeric form, and that it could block the Zncatalyzed polymerization of C9 in a concentration-dependent manner (Hamilton et al., 1993), suggesting a further protective mechanism. Inhibition of the C5b-C9 deposition would reduce injury to cells of the vascular wall and limit activation of procoagulant responses in both endothelium and platelets. HDL has also been reported to act as a carrier of small amounts of CD59 (protectin), a membrane-bound glycophosphatidylinosito1 lipid-anchored glycoprotein that protects cells against the cytolytic complement membrane attack complex (Vakeva et aL, 1994).

VI. Effetct of HDL on Vasoacthm Molecules Control of vascular tone is achieved by modulation of a balance of vasoconstrictive and vasodilatory factors. Of those which will be discussed in this section, both NO and prostacyclin (PG12)are powerful inhibitors of platelet aggregation,although PGI, is more potent in this respect than NO (Radomski et al., 1987a).

A. Endothelin-I Endothelin-1 (ET-1) is a powerful vasocontrictor and mitogen for SMCs which has been implicated in both acute and chronic vascular diseases

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including atherosclerosis (Yamagisawa et al., 1988;Yamagisawa and Masakai, 1989; Dubin et al., 1989). Reports on the effects of lipoproteins on endothelin secretion are conflicting. Hu and coworkers (1994) showed that native HDL was able to induce a 2.4-fold elevation in the level of ET-1 synthesized from bovine aortic endothelial cells. Using extremely low levels of HDL (0.5-50 pg/ml), these researchers showed that HDL had no effect on mRNA levels, transcriptional regulation, or transcriptional degradation of ET-1. The level of induction observed in these cells was sensitive to PKC inhibition, suggestingthat the posttranslational effect that HDL mediated was PKC dependent. Second, using renal proximal tubular cells, Ong and colleagues (1994) showed that HDL can induce ET-1 synthesis. The 4-fold elevation obtained was not dependent on the class of HDL used; H D L was capable of promoting a similar response to HDL3. Unlike the effect on ECs, the response of renal tubular cells was PKC independent and not affected by the addition of staurosporin or long-term treatment with PMA (Ong et al., 1994). The elevation of ET-1 is not exclusive to HDL because LDL and VLDL have also been shown to induce this peptide in human ECs (Horio et al., 1993). Native LDL was shown in some cases not to induce the secretion of ET from both porcine and human ECs (Boulanger et al., 1992; Jougasaki et al., 1992). On the contrary, studies by Horio have shown native LDL can induce ET-1 secretion. However, while Boulanger and others showed that oxidized LDL did induce ET-1 secretion (Boulanger et al., 1992; Horio et al., 1993);Jougasaki and coworkers (1992) report that oxidized LDL inhibits ET-1 secretion. 6. Nitric Oxide Despite the demonstration that under certain conditions high levels of NO may induce tissue injury (Beckman et al., 1990), the inhibitory effects of endothelial-derived NO on platelet activation (Radomski et al., 1987a,b), vascular tone (Furchgott and Zawadski, 1980), leukocyte adhesion (Bath et al., 1991),and vascular SMC proliferation and dedifferentiation (Cornwell et al., 1994; Lincoln et aZ., 1994) suggest that NO is an atheroprotective molecule (Cooke and Tsao, 1994). Using isolated strips of rabbit thoracic aortae it was shown that HDL was able to reverse the inhibitory effect of oxidized LDL on endothelial-dependentarterial relaxation in response to acetylcholine (Matsuda et al., 1993). In this study it was shown that HDLs reduce the incorporation of [14C]LPC palmitate of oxidized LDL into BAEC by transfer of the LPC into HDL. Since LPC has been shown to mediate elevation of a number of proinflammatory genes, including NO synthase, the ability of HDL to prevent the action of LPC upon endothelial cells may present a further protective mechanism (Kume et al., 1992, Kume

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and Gimbrone, 1994). Recently it was demonstrated that LPC could induce the mRNA, protein, and activity of NOS I11 in HUVEC (Zembowicz et al., 1995). It may well be that EC NOS I11 may be elevated through the action of LPC, but that the reduced vasodilatory response of the whole tissue is a result of a balance of effects of other vasoactive agents. Galle and coworkers (1994), in similar studies using rabbit renal arteries, examined the effects of pressure as well as HDL on the effect of oxidized LDL on endothelial-dependent vasodilatation. While they also observed the inhibition of vasodilatation in response to oxidized LDL, they noted that the effect was dependent on lipid infiltration of the vessel wall. Lipid infiltration under high pressure was ameliorated by the presence of HDL. HDL was shown to exert a beneficial effect on abnormal vascular reactivity, a fundamental disturbance associated with coronary atherosclerosis. Patients with focal stenoses were given acetylcholine infusions to evaluate their endothelial-dependent vasodilatation. Patients with elevated HDL showed a blunted response to acetylcholine in comparison to those with low levels of HDL (Zeiher et al., 1994). The effects of NO on vascular reactivity are well documented. There is evidence that NO can affect metabolism. In addition to affecting the regulation of carbohydrate metabolism (Gross and Wolin, 1995), the inhibition of NO synthase by feeding rats nitroargainine resulted in an increase in triglycerides and cholesterol accompanied by a reduction in hepatic fatty acid oxidase (Khedera et aL, 1996).

C. Prostacyclin Many studies have shown that HDL can stimulate production of PGIz (Fleischer et al., 1982; Beitz et al., 1994; Vane and Botting, 1995; Myers et al., 1996; Tamagaki et al., 1995). Since the extent of induction of PG12 correlated with the arachidonate content of HDL, the ability of HDL to supply substrate for the eicosonoid synthesis was postulated as a mechanism of action (Fleischer et al., 1982; Pomerantz et al., 1984). Others have demonstrated that while LDL treatment of endothelial cells results in an increased prostaglandin synthesis, HDL will alter the synthesis of eicosonoids toward elevation of prostacyclin (Jambou et al., 1993), suggesting that the lipoprotein balance of plasma could affect the balance of eicosonoids synthesized by the vascular endothelium. In addition to elevating the level of PG12,it has been shown that the apo A-I component of HDL (and not lipid-free apolipoprotein A-I) can stabilize the eicosonoid, prolonging its active halflife in vivo (Yui et al., 1988; Aoyama et al., 1990). We have shown that HDL, not surprisingly, can increase the level of cyclooxygenase-2 (cox-2;

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prostacyclin synthase) in endothelid cells (Fig. 11). Interestingly, HDL synergizes with cytokines to elevate Cox-2 production, suggesting that in the case of an inflammatory challenge HDL is able to induce prostacyclin, influencing both vascular tone and platelet aggregation. The possibility that low levels of PG12 might be a suitable marker for risk of ischemic heart disease (Peto et aZ., 1991) has been explored. As with most biological processes, maintenance of vascular tone is a balance between vasodilatory and vasoconstrictive agents. In order to understand more fully the role of lipoproteins in this function it will be important to analyze their effect on both sides of the equation. To add to the complexity, some of the vasoactive agents have isoforms or intermediates with differential importance and/or control mechanisms within specific cells. Our understanding of the effects of HDL on vasoactive agents is very limited and worthy of further investigation.

VII. Acutephase Response and HDL A. CReactive Protein That atherogenesis is a chronic inflammatory disease is generally accepted. Although the clinically significant lesions are focal, it is interesting to determine whether inflammation occurring in distal sites could influence the development of these lesions and, if so, to what degree. Recent studies showed a significant relationship between the subclinicallevels of C-reactive protein (CRP) and the risk of MI (Vallance et aL, 1997), suggesting that even normal levels of this acute-phase protein could be a useful surrogate marker for risk and may have some influence on the development of disease. Furthermore, in studies comparing the level of CRF' to coronary event, the association was independent of the severity of the event, suggesting that CRP elevation was not just a passive phenomenon related to the degree of preexisting atheroma (Woodhouse et aL, 1994; Haverkate et al., 1997).

6 . Serum Amyloid A

Much of the preceding discussion has highlighted antiinflammatory actions of HDL. If this usually abundant endogenous lipoprotein HDL is such a good antiinflammatory agent, how does the body mount a normal response to insult through infection? Why are we not a sickly lot suffering from an inability to mount a normal host response to infection? To understand

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FIG. 11 Synergismbetween HDL and cytokines elevates Cox-2levels and induces prostacyclin synthesis in EC. (A) Western blot analysis of whole cell lysates (2 X 106 cells per lane) prepared from confluent EC preincubated with HDL (1 mg/ml apo A-I) alone for 8 h or preincubated with HDL for 4 h and then treated with either TNF-a (1 ng/ml) or IL-1p (1 ng/ml) and HDL for an additional 4 h. The lanes have been probed with antibody against Cox-2 (Biogenics Ltd., UK). Results are representative of three experiments. (B) Following preincubation of confluent EC with HDL (1 mg/ml apo A-I) for 16 h, IL-lP (1 ng/ml) was added for a period of 16 h, after which the supernatants were assayed for levels of 6-0x0 PGFla. Levels in supernatants from cells treated with cytokine in the presence of HDL (IL-1, HDL) and the absence of HDL (IL-1)were compared to cells incubated with HDL alone (HDL) and cells left untreated (NIL). The data are representative of three separate experiments and show means and SD. Differences between means were evaluated using a Student’s unpaired t test: *p < 0.001; **p < 0.005.

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this we must first analyze what happens during an infection and how this affects HDL. Serum amyloid A (SAA), a multigene family of HDL apolipoproteins, is a major acute-phase reactant (Kushner, 1982). Plasma levels of SAA can be elevated several hundred- to 1000-fold as part of a response to various injuries including trauma and infection (Kushner, 1982; Hoffman and Benditt, 1982). Acute-phase responses have been shown to be elicited not only during acute infections but also during active phases of inflammation in patients with rheumatoid arthritis and during MI and depression (Maes et al., 1997). Although acute-phase proteins are primarily synthesized in the liver, both endothelial cells and macrophages have been shown to synthesize SAA, and SAA has been shown to be abundant in cells of the mature plaque (Meek et al., 1994; Urieli-Shoval et al., 1994). In addition to an inverse relationship between levels of acute-phase proteins, CRP, SAA, and levels of HDL (Walter et al., 1989; Kumon et al., 1993), the apolipoprotein composition of HDL is altered. SAA has been found in association with plasma HDL (Benditt and Ericksen, 1977) and can displace apo A-I from HDL (Cabana et al., 1996). There is also evidence that HDL containing SAA is more rapidly cleared than normal native HDL. What effect does incorporation of acute-phase proteins have on the antiatherosclerotic effects of HDL? We have postulated that incorporation of acute-phase proteins into HDL is a mechanism for regulating the anti-inflammatory effects of HDL, thereby allowing an acute-phase response to occur. Thus, HDL isolated from patients with acute vasculitis, and containing a high level of SAA within the HDL, are no longer able to inhibit the cytokine-induced adhesion molecule expression described in Section I11 (Fig. 12). Similarly, it has been shown that acute-phase HDL, containing reduced amounts of PAF acetyl hydrolase and paraoxonase, is no longer able to inhibit oxidation of LDL and is in fact, proinflammatory (Van Lenten et al., 1995).

VIII. HDL Receptors and Cell Signal Transduction A. HDL Receptors/Binding Proteins

Although several cell surface-bindingproteins/receptors have been defined for HDL from a range of cell types, the precise role of these proteins has been difficult to determine because of lack of specificity and an inability to link structure with a discrete cellular function (Koller et al., 1989;Hokland et al., 1992; Hidaka and Fidge, 1992; McKnight et al., 1992; Schmitz et al., 1985a; Vries et al., 1995; Acton et al., 1996). Using cholesterol-poor peritoneal macrophages, Schmitz and colleagues described a surface protein with

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FIG. 12 Acute-phase HDL loses antiinflammatory effects. Confluent cultures of EC were treated with either normal native HDL (nHDL) or HDL isolated from two patients suffering from chronic vasculitis (AP-HDL) (1 m g / d total protein, 16 h). Following a 4-h stimulation with TNF-a (1 n g d ) cell surface levels of VCAM-1 were measured using flow cytometry. Histogram shows the level of VCAM-1 expression as mean fluorescence intensity in response to treatments, as indicated. Data show means and SD.

a KO of 9 X lo7 M,a B , of 320 ng HDL/mg of cell protein, and a BID of 95 ng/mg cell protein. The kinetics of apo E-free '%labeled HDL binding indicated approximately 160,000 receptors per cell. Following cholesterol loading of the same cells, saturation was increased to 510 ng/mg cell protein (Schmitz et d.,1985b). Further work revealed that the binding of HDL to murine macrophages could be abolished by pretreatment with an anti-apo A-I antibody, whereas an anti-apo A-I1 antibody had only a minor effect. Saturation binding of HDL to macrophages occurred at 40 pg/ml and the kinetics showed two components; a linear (with respect to ['251]HDL) component interpreted as nonspecific binding and a nonlinear (limiting) component which was equated with total HDL binding. The difference between the two components is believed to represent specific receptormediated HDL binding (Schmitz et d.,1985a). Lectin affinity chromatography and denaturing gel electrophoresis were used to identify two acidic 4.7) apo A-I-binding proteins from rat liver (Hidaka and Fidge, (pZ 1992). The two proteins, designated HI31 (120 kDa) and HB2 (100 m a ) , were shown to be extensively glycosylated, but apo A-I binding was diminished following treatment with neuraminidase and glycosidases. In similar studies using bovine aortic endothelial cells (BAEC) three HDL-binding

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proteins were described ( M , = 105,110, and 130 kDa). Two of these, pll0 and p130, appear to be specific to BAEC (Hokland et al., 1992). Treatment of these proteins with N-glycanases decreased the apparent mass by approximately 30 kDa and also abolished HDL recognition (Garver el al., 1994). The authors suggest that the 110-kDaprotein was a member of the cadherin superfamily on the basis of amino acid sequence analysis. In 1996, Acton and coworkers described the SR-B1 HDL receptor (Acton et al., 1996; Wang et al. 1996; Landschultz et al., 1996). This receptor appears to have a fairly wide binding selectivity and is expressed in liver and steroidogenic tissues. The SR-B1receptor appears to be important in cholesterol delivery to tissues. In this respect the need for apo A-I was shown by Wang and colleagues when apo A-I-deficient mice were found to be depleted in adrenal corticoids and cholesterol stores. (Wang et al., 1996).Furthermore, such mice were able to upregulate their expression of SR-B1. The expression of SR-B1 in adrenal cortical cells has been shown to be upregulated by adrenocorticotrophic hormone and suppressed by glucocorticoids, providing further evidence for a role of HDL in cholesterol delivery to sites of steroid hormone synthesis. Others have shown an increase in lipoproteinbinding proteins in response to cholesterol loading (McKnight et al., 1992). Demonstration of high-affinity binding sites for HDL in cerebral endothelial cells could also suggest a role for HDL in cholesterol metabolism in the central nervous system (Vries et al., 1995).

8. Protein Kinase CDependent Signal Transduction Currently, the evidence for signaling by HDL points toward a G proteinlinked mechanism. The effect of HDL on initiation of signaling pathways in cells loaded with tritiated cholesterol precursors has led to the demonstration of a PKC-dependent reaction (Hokland et al., 1992; Dusserre et al., 1993).Investigation of the manipulation of cyclic adenosinemonophosphate (CAMP)concentration on cholesterol efflux from BAEC and human skin fibroblasts (Hokland et al., 1993) demonstrated a positive relationship between cAMP concentration and efflux. In contrast to the PKC-dependence, HDL binding was not an essential prerequisite for the CAMP-dependent pathway since tetranitromethane-modified HDL, which does not engange the HDL receptor, produced a definite, albeit blunted, effect on CAMP levels. Similarly, Wu and colleagues have shown complementary activation of both PKC- and CAMP-dependent mechanisms in their studies with human placental tissue (Wu et al., 1988; Wu and Handweger, 1992). Using human placental lactogen (hPL) secretion as the response, 100 pg/ml of HDL elicited a 3- to 5-fold increase in cAMP and an equivalent rise in the release of hPL. This effect was shown to be both dose (100-1000 pg/ml)

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and time (up to 2 h) dependent. In later work the authors were able to show a 2- or 3-fold increase in the hPL secretion in response to HDL, purified apo A-I, and PMA. Additionally, 32Pincorporationinto an 80-kDa protein known to be a PKC substrate elevated the protein by approximately 2.5-fold. The implication that diacylglycerol (DAG) is released from inositide membrane phospholipids is substantiated by the work of Mollers and coworkers (1995). Activation of PI-PLC in human fibroblastswas observed in response to challenge by H D b or LDL. Both lipoproteins (at 100 pg/ ml) caused translocation of PKC activity from the cytosol to the membrane. Activity corresponding to only three major (a,E , and 0 and one minor (8) PKC isoforms were observed and only PKC-a and PKC-Ewere translocated. Interestingly, in fibroblast cultures from patients with Tangier’s disease (familial hypoapolipoproteinemia) there appears to be an impaired PI-PLC activation in response to lipoproteins (Drobnik et al., 1995). In a brief report, Uint and Mendez (1994) described the effects of PKC modulation on apo A-I and HDL-mediated cholesterol efflux from cholesterol-laden fibroblastsand SMCs in culture. The authors noted differencesin the ability of native HDL (no effect) and isolated apo A-I (50%decrease) to stimulate cholesterol efflux when PKC had been down-regulated by long-term treatment of the cells with PMA. Furthermore, apo A-I lost the ability to inhibit Acetyl-CoA cholesterol acyltransferase (ACAT) in PKC downregulated cells. Acute treatment of cells with PMA causing an activation of PKC promoted cholesterol esterification by ACAT but efflux was not changed. They concluded that there are two pools of cholesterol, one available for esterification but not efflux and the other available for efflux but not esterification. Extensive studies using platelets have also shown that HDL3 stimulates phosphatidylcholine breakdown via a G protein-coupled, PKCdependent mechanism (Nazih et al., 1990,1992,1994).

C. lnositol Phosphate Turnover Fibroblast challenge by both HDL and LDL resulted in a very rapid (30 s) but modest (1.6-fold over basal) generation of IP1, a negligible effect on IP2, and an approximately 20% rise in IP3 (Walter et al., 1995). DAG and IP3 generation in response to HDL was relatively slow and sustained, whereas the response to LDL was clearly biphasic (30 s and 5 min). As expected, the concentration of intracellular calcium rose quickly in a dosedependent fashion in response to both LDL and HDL. However, the rise was transient and pretreatment with thapsigargin indicated that the calcium peak was due to the release from intracellular stores within the endoplasmic reticulum rather than as a result of influx of extracellular calcium. CAMP production peaked at about 10 min in these experiments. Additional evi-

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dence of the association of HDL stimulation and IP turnover has come from the work of Bochkov and colleagues (1992) and Resink et aZ. (1993a). Bochkov reported the action of LDL and HDL (100 pg/ml) on aortic vascular SMCs and microarterioles from omentum. In these studies, the actions of the lipoproteins were compared to those of angiotensin I1 (2 nM) and PDGF (2 ng/ml), both of which are known to mediate their effects via PLC mobilization of Ca2+.The two lipoproteins produced a qualitatively similar response to both the growth factor and the vasoactive peptide in terms of phosphoinositide (PI) turnover. Eight IP isoforms were subsequently isolated by high-performance liquid chromatography. However, only five of these could be identified: l-IP, 4-IP,1,4-IP2,1,4,5-IP3,and 1,3,4,5-IP4.The response was biphasic and peak IP mobilization occurred at 30 s (4-IP, 1,4-IP2,and 1,3,4,5-IP4)and 5 min (1-IP).

D. HDL and Calcium Flux Both LDL and HDL at 50 p g / d stimulated a rise in intracellular Ca2+ which was shown to result from cytosolic stores and not via influx from the culture medium (Negre-Salvaryre et aZ., 1992; Schaefer et aZ., 1993). Figure 13 shows the acute response of human coronary artery ECs to stimulation with HDL. Experiments using bovine aortic ECs showed that the initial “acute” rise in Ca2+is significantly blunted in the absence of extracellular calcium, whereas the more sustained effect is unaffected, suggesting that the biphasic response requires both intracellular and extracellular calcium (Su et aZ., 1996). Resink and coworkers showed that the IP turnover in smooth muscle cells from spontaneoushypertensiverats (SHRs) and Wistar Kyoto normotensive (WKY) rats was both dose dependent (0-100 pg/ml) and time dependent (0-5 min) with respect to LDL and HDL3 (Resink et aZ., 1993b). The release of IP, IP2, and IP3 was more pronounced in the SHRs than in the WKY rats in response to both LDL and HDL. The decline in IP3 concentration following HDL challenge showed biphasic kinetics. DAG generation showed a clear biphasic response to both HDL and LDL, with an initial peak at 30 s and a second at 2 min. In SHR cells, PI turnover plateaued at 20 pg/ml, suggesting saturation, but there were less clear results in the WKY cells or for LDL in either cell line. Compared with the WKY cells, SHR cells showed an increased 45Ca2+ uptake and a decrease in intracellular pH in lipoproteins in both stimulated (50 pg/ml) and unstimulated states. This finding was supported by a similar study by Orlov and colleagues (1993). The fact that there was apparent synergism between HDL and LDL for PI turnover implies two parallel activation pathways rather than nonspecific interaction between either

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FIG. 13 HDL induces a a*+ flux in human coronary artery cells. Intracellular calcium concentration changes after addition of HDL to human coronary artery endothelid cells bathed in Krebs-Hepes. HDL (0.5 mg/ml) was added at time 0 s and images were acquired at 4-s intervals. Images were processed using the ionvision software (Improvision,Coventry, UK). Calcium changes are color coded (color bar) such that white indicates high calcium.

HDL or LDL and a single receptor protein. Immunoblot revealed a Gi protein implicated in mediation of both LDL and HDL responses.

IX. Concluding Remarks It is clear from the wealth of evidence presented in this review that HDL can mediate its protective effect at many levels, operating on many cells in the vasculature. For many of these effects HDL appears to be the ying to LDLs yang, operating in a manner counter to that of LDL. However, in some aspects of the cellular response to lipoproteins the effects of HDL and LDL are similar, varying only by degrees. Although HDL is antiinflammatory in many of its responses it is likely that its antiinflammatory responses are perturbed when an acute-phase response is required. The

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mechanism of this perturbation has been demonstrated to involve modulation of proteins and apolipoproteins within the HDL particle, resulting in HDL changing from an antiinflammatory to a proinflammatory lipoprotein. In order to understand more fully the mechanisms for these pleotropic actions we need to know more about cell signal transduction in response to HDL. The cloning of novel cell-specific HDL receptors, facilitated by the range of functions we now know HDL to have, will be an important advance in our understanding of the protective mechanisms of HDL. Acknowledgment The authors thank Dr. Sean Allen for providing the data for endothelial cell calcium flux in response to HDL.

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Cooke, J. P., and Tsao, P. S. (1994). Is NO an endogenous antiatherogenic molecule? Arterioscler. Thromb. 14, 653-655. Cornwell, T. L., Arnold, E., Boerth, N. J., and Lincoln, T. M. (1994). Incubation of smooth muscle cell growth by nitric oxide and activation of CAMP-dependent protein kinase by cGMP. Am. J. Physiol. C 267,1404-1413. Darbon, J.-M., Tournier, J.-F., Tauber, J.-P., and Bayard, F. (1988). Possible role of protein phosphorylation in the mitogenic effect of high density lipoproteins on cultured vascular endothelial cells. J. Biol. Chem. 261, 8002-8008. Davies, M. J. (1995). Acute coronary thrombosis-The role of plaque disruption and its initiation and prevention. Eur. Heart J. 16, 3-7. Davies, M. J., Woolf, N., Rowles, P. M., and Pepper, J. (1988). Morphology of the endothelium over atherosclerotic plaques in human coronary areteries. Br. Heart J. 60,459-464. Dawson, S., and Henney, A. (1992). The status of PAI-1 as a risk factor for arterial and thrombotic disease: A review. Atherosclerosis 95,105-117. Dejana, E., Zanetti, A., and Del Maschino, A. (19%). Adhesive proteins at endothelial cellto-cell junctions and leukocyte extravasation. Haemostasis 26,210-219. Des Mouliere, A., Badid, C., Cochalon-Piallat, M. L., and Gabbiani, G. (1997). Apoptosis during wound healing, fibrocontractive disease and vascular wall injury. Znt. J. Biochem. Cell Biol. 29, 19-30. Drobnik, W., Mollers, C., Resink, T., and Schmh, G. (1995). Activation of phosphatidylinositol-spedc phospholipase C in response to HDL, and LDL is markedly reduced in cultured fibroblasts from Tangier patients. Arterioscler. Thromb. Vasc. Biol. l5, 1369-1377. Dubin, D., Pratt, R. E., Cooke, J. P., and Dzau, V. J. (1989). Endothelin, a potent vasoconstrictor, is a vascular smooth muscle mitogen. J. Vasc. Med. Biol. l, 50-154. Dusserre, E., Pulcini, T., Bourdillon, M. C., and Berthezene, F. (1993). High-density lipoprotein 3 stimulates phosphatidylcholine breakdown and sterol translocation in rat aortic smooth muscle cells by a phospholipase Uprotein kinase C-dependent process. Biochem. Med. Metab. Biol. 52, 45-52. Epand, R. M., Stafford, A., Leon,B., Lock, P. E., Tytler, E. M., Segrest, J. P., and Anantharamaiah, G. M. (1994). HDL and apolipoprotein A-I protect erythrocytes against the generation of procoagulant activity. Arterioscler. Thromb. 14,1775-1783. Evensen, S . A. (1979). Injury to cultured endothelial cells: The role of lipoproteins and thromboactive agents. Huemostasis 8,203-210. Favre, G., Tazi, K. A., LeGaillard, F., Bennis, F., Hachem, H., and Soula, G. (1993). High density lipoprotein.3 binding sites are related to DNA biosynthesis in adenocarcinoma cell line A549. J. Lipid Res. 34,1093-1106. Fischer-Dzoga,K., and Wissler,R. W. (1976). Stimulation of proliferation in stationary primary cultures of monkey aortic smooth muscle cells. Part 2. Effect of varying concentrations of hyperlipemicserum and low density lipoproteins of varying dietary fat origins. Atherosclerosis 24,515-525. Fischer-Dzoga, K., Chen, R., and Wissler, R. W. (1974). Effects of s e w lipoproteins on the morphology, growth, and metabolism of arterial smooth muscle cells. Adv. Exp. Med. Biol. 43,299-311. Fischer-Dzoga, K., Fraser, R., and Wissler, R. W. (1976). Stimulation of proliferation in stationary primary cultures of monkey and rabbit aortic smooth muscle cells. I. Effects of lipoprotein fractions of hyperlipemic serum and lymph. Exp. Mol. Pathol. 27,346-359. Fleischer, L. N., Tall, A. R., Witt, L. D., Muller, R. W., and Cannon, P. J. (1982). Stimulation of arterial endothelial cell prostacyclin synthesis by high density lipoproteins. J. Biol. Chem. 257,6653-6655. Fukuo, K., Nakahashi, T., Nomura, S., Hata, S., Suhara, T., Shimizu, M., Tamatani, M., Morimoto, S., Kitamura, Y., and Ogihara, T. (1997). Possible participation of Fas-mediated apoptosis in the mechanism of atherosclerosis. Gerontology 43,35-42.

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Van Lenten, B. J., Hama, S. Y., de Beer, F. C., Stafforini, D. M., McIntyre, T. M., Prescott, S. M., h D u , B. N., Fogelman, A. M., and Navab, M. (1995). Antiinflammatory HDL becomes pro-inflammatory during acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wall cell co-culture. J. Clin. Invest. !M,i 2758-2767. Velican, C., and Velican, D. (1980). The precursors of coronary atherosclerotic plaques in subjects up to the age of 40 years old. Atherosclerosis 37,33-46. Vries, H. E., Breedveld, B., Kuiper, J., De Boer, A. G., Van Berkel, T. J. C., and Breimer, D. D. (1995). High-density lipoprotein and cerebral endothelial cells in vitro: Interactions and transport. Biochem. Phannacol. SO, 271-273. Wada, W. H., Moni, Y., Kaneko, T., Wakita, Y., Nakase, T., Minimikawa, K., Ohura, M., Tamaki, S., Tamgawa, M., kageyanas, S., et al. (1993). Elevated plasma levels of vascular endothelial cell markers on patients with hypercholesterolemia.Am. J. Hematol. 44,112-116. Walter, M., Reinecke, H., Nofer, J.-P., Seedorf, U., and Assman, G. (1995). HDI, stimulates multiple signaling pathways in human skin fibroblasts. Arterioscler. Thromb. Vmc. Biol. 15, 1975-1986. Walter, M. T., Stevenson, F. K., Goswami, R., Smith, J. L., and Cowley, M. I. (1989). Comparison of serum and synovialfluid concentration of beta-2-microglobulinand C reactive proteins in relation to clinical disease active macrophage synovial inllammation in rheumatoid arthritis. Ann. Rheum. Dis.48, 905-911. Wang, J., Wang, S., Lu, Y., Weng, Y., and Gown, A. M. (1994). GM-CSF and M-CSF expression is associated with macrophage proliferation in progressing and regressing rabbit atheromatous lesions. Exp. Mol. PathoL 61,109-118. Wang, N., Weng, W., Breslow, J. L., and Tall,A. R. (1996). Scavenger receptor B1 (SR-Bl) is upregulated in adrenal gland in apolipoprotein A-I and hepatic lipase knock-out mice as a response to depletion of cholesterol stores. In vivo evidence that SR-B1 is a functional HDL receptor under feedback control. J. Biol. Chem. 2% 21001-21004. Wissler, R. W. (1984). Principles of the pathogenesis of atherosclerosis. In “Heart Disease” (E. Braunwald, ed). Saunders, Philadelphia. Woodhouse, P. R., Meade, T. W., and Khaw, K.-Y. (1994). Plasminogen activator inhibitor1, the acute phase response and vitamin C. Atherosclerosis l33,71-76. Wu, Y. Q., and Handweger, S. (1992). HDL stimulates molecular weight 80kDa protein phosphorylation in human trophoblast cells. Evidence for a protein b a s e C dependent pathway in human placental lactogen release. Endocrinology Wl, 2935-2940. Wu, Y. Q., Jorgensen, V., and Handweger, S. (1988). HDL stimulates placental lactogen release and adenosine 3’5’ monophosphate (CAMP)production in human trophoblast cells. Evidence for CAMPas a second messenger in human placental lactogen release. Endocrinology l31,1879-1884. Yamigisawa,M., and Masaki,T. (1989).Molecular biology and biochemistry of the endothelins. Trends Pharmacol. Sci 10,374-378. Yamagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, Y., Mitsui, Y., Yazaki, K., Goto, K., and Masaki, T. (1988). A novel vasoconstrictor peptide produced by vascular endothelial cells. Nature 332,411-415. Yui, Y., Aoyama, T., Morishita, H., Takahashi, M., Takatsu, Y., and Kawai, C. (1988). Serum prostacyclin (PGI?) stabilizing factor is identical to apolipoprotein A-I (apo A-I): A novel function of apo A-I. J. Clin Invest. 82,803-807. Zeiher, A. M., Schachinger,V., Hohnloser, S. H., Saurbier, B., and Just, H. (1994). Coronary atherosclerotic wall thickening and vascular reactivity in humans. Circulation89,2525-2532. Zembowicz, A., T a d , J., and Wu, K. K. (1995). Transcriptional induction of endothelial nitric oxide synthase type I11 by lysophosphatidylcholine. J. BioL Chem 270,17006-17010. Zhao, B. (1996). Role of lipoproteins in platelet activation. Blood Coag. Fibrin. 7,270-273. Zhao, B., Dierichs, R., Lui, B., and Holling-Rauss, M. (1994). Functional morphological alterations of human blood platelets induced by oxidised low density lipoproteins. Thromb. Res. 74,293-301.

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A Absorption, retinoids, 79-80 Acidic epididymal glycoprotein aging effect, 184 epididymal expression, 166-167 Acute-phase response, and HDL, 279-281 ADAM7, epididymal expression, 162 Adhesion leukocyte, to endothelium, 267-271 platelet in Bernard-Soulier syndrome, 22-23, 27-28 to subendothelium, 20-23 Aging and epididymal gene expression alterations, 181-184 free radical theory, 215 Allografts cultured, 55 keratinocyte, 63-64 Amyloid A, serum, HDL containing, 279, 281 Androgen-binding protein role in epididymis, 173-174 in testicular fluid, 153 Androgen receptors epithelial, expression, 144-145 expression in adult epididymis, 170-172 Androgen-response element, in gene promoter regions, 170-171 Androgens early action, growth factor role, 146 expression in adult epididymis, 170-172 and production, 143-144

Animal studies, see also Mouse vitamin A deficiency, 75-76 Antioxidants endogenous activity, 218 a-lipoic acid as, 213 and ROS homeostasis in animal cells, 211-213 Apical ectodennal ridge, retinoic acid in, 106-107 Apolipoprotein A-I binding proteins, 282-283 transgenic insertion, 260 Apoptosis LDL-induced, 266-267 result of oxidative stress, 216 thiol status role, 229-230 A-raJ epididymal expression, 179-180 Arteries, subendothelium, type VI collagen, 5 Associated microfibril protein, extraction, 15 Atherogenesis, cellular mechanisms, 260-262

B Bacteria, transcriptional activity, redox regulation, 233-235 Basement membrane, microfibrils linked to, 16 Basic fibroblast growth factor epididymal expression, 180 role in developing genital tract, 146

299

300

INDEX

Bead periodicity, fibrillin-containing microfibrils, 11 Bernard-Soulier syndrome, platelet adhesion in, 22-23,27-28 Biosynthesis, phytoalexins, and ROS production, 236-237 Blood, platelet reactivity with type VI collagen, 25 BMP, see Bone morphogenetic protein Bone morphogenetic protein, gene expression, 138

C Calcium homeostasis alteration, 216 ROS role, 225 low-Ca cultures of keratinocytes, 46-47,54 Calcium-binding protein, TSP-1, 17-18 Calcium flux, and HDL,285-286 Calmodulin, activator of Ca2+-ATPase, 231-232 Cancer, photodynamic therapy, 216 p-Carotene, dietary sources, 79 Catalase, decomposition of Hz02, 212 Cauda epididymis, transformed, 142-143 CD52 epididymal expression, 167, 169 mRNAs, androgen responsiveness, 171 CD52, mRNA stability, 181 Cell adhesion molecules, cytokine-induced expression, HDL effect, 268 Cell attachment, type VI collagen as substrate, 7-8 Cell death, hypersensitive, 237 Cells animal, ROS homeostasis, 211-214 nonphagocytic, ROS generation, NADPH oxidase role, 209 oxygen reduction steps, 204-206 phagocytic, NADPH oxidase, 206-209 plant, oxidative burst, 210-211 responses to ROS direct effects, 220 Cell surface-binding proteins, and HDL receptors, 281-283 Cellular retinoic acid-binding protein, see CRABP Chloroplasts, ROS generation, 210-211

Cholesterol, epididymal, 164 Chondrogenesis, in skull,retinoic acid receptor role, 105-106 Clinical application, transplantation of allogeneic dermis, 58 Coagulation, cascade, HDL effect, 274-275 Collagen binding to von Willebrand factor, 21 type VI affinity with von Willebrand factor, 30 dimer aggregation, 6-7 microfibrillar structure, 4-5 thrombogenicity, 23-25 Collagenase, microfibrils resistant to, 20 Colony formation, efficiency of trypsinized keratinocytes, 52 Coronary artery disease, HDL protective effect, 258-260 CRABP epididymal expression, 158-159 functions, 89, 95-96 tissue distribution, 92 embryonic, 93-94 Craniofacial development, retinoic acid role, 99-106 CRBP epididymal expression, 158-159 retinol bound to, 87 role in exocytosis, 92 tissue distribution, 89 CRBP 11, enterocytes containing, 80 C-reactive protein, as surrogate marker, 279 CRES, epididymal expression, 158 CRISP-1, epididymal expression, 166-167 c-ros, targeted mutation, 138-139 c-ros, epididymal expression, 178-179 Cross-linking, transglutaminase-catalyzed, MAGP-1, 13 Cryopreservation, keratinocytes, 61-62 y-F-Crystallin gene, RA response element, 88 Cyclic AMP,elevated levels, effect on ROS production, 226 Cyclooxygenase-2,HDL effect, 278-279 Cystatin-related epididymal and spermatogenic gene, see CRES Cytochrome b5=, NADPH oxidase membrane, 207-208 Cytokines, synergism with HDL,280

301

INDEX

Cytoplabmicretinol-binding protein, see CRBP Cytotoxicity LDL for endothelial cells, 266 ROS, 214-220

D Degranulation, leukocyte, HDL effect, 271 Dermal-epidermal junction, de novo formation, 60 Development, cellular, redox responses during, 220-222 Diacylglycerol, generation in response to HDL, 284-285 Differentiation keratinocytes, 51 markers, 59-60 vitamin A role, 76-77 Diffuse intimal thickening, 1 Disintegrin, see ADAM7 Dispase, in keratinocyte transplantation, 55-57 Dispersed cultures, keratinocytes, 44-45 DNA epidermal cells, recipient-originated, 55 ROS-induced damage, 215

E EAP I protein, androgen-dependent, 162 EGF-like repeats fibrillin-1, 9-11 fibulin-2, 14 Egr-1, expression, lipoprotein effect, 265 Elastin, microfibrils associated with, 3-4 proteins from, 8-19 thrombogenicity, 25-28 Elastogenesis, and microfibrils, 19 Embryo and pregnancy, vitamin A role, 73-75 retinoid receptors, 98-99 tissues, retinoic acid synthesis and catabolism, 85-96 Embryogenesis, retinoic acid signaling pathway in, 77-85 Endodermal cells, synthesis of retinolbinding protein, 83-85

Endoplasmic reticulum, glutathione redox state in, 231 Endothelial cells, HDL in virro effects, 262-267 Endothelin-1, HDL effect, 276-277 Enterocytes, retinol esterification in, 80 Enzymes defense, and ROS homeostasis in animal cells, 211-213 NADPH-oxidase-like, 209 Epidermal growth factor effect on keratinocyte migration, 50 in keratinocyte dispersed cultures, 44-45 role in developing genital tract, 146 Epidermal growth factor receptor, in Wolffian duct development, 139 Epidermis reconstructed, keratin expression in, 59 reorganized, long-term maintenance, 57 Epididymal duct, gene expression throughout, 164-166 Epididymis adult gene expression, 153-169 aging-related changes, 181-184 temperature effect, 180-181 nonnuclear protooncogenes, 178-180 steroid hormone receptors, 170-174 transcription factors, 176-178 vitamin A role, 174 distal part, gene expression control, 185 duct epithelium, regionalized gene expression in, 134-135 gene expression pattern, ontogenesis, 135-153 juvenile and peripubertal, 148-153 role in mammalian reproduction, 133-134 Epithelium androgen receptor expression, 144-145 epididymal duct, regionalized gene expression in, 134-135,184-185 retinoic acid synthesis in, 95 Estrogen receptors early expression in epididymis, 147 in fluid reabsorption, 172-173 Estrogens, expression in adult epididymis, 172-173 Exocytosis, CRJ3P role, 92 Explant cultures, keratinocytes, 43

302

INDEX

Extracellular matrix role in keratinocyte migration, 49-50 von Willebrand factor binding to, 24-25 Extraction GP128,16 microfibrils, 5-6 type VI collagen, 7 Eye, developing, effect of maternal vitamin A deficiency, 88

F Facial development, retinoic acid role, 104-105 FBNI, mutations, 12 FBN2, mutations, 12 Fenton reaction, 219 Fibers, oxytalan, in elastin-free tissues, 3 Fibrillin-1, EGF-like repeats, 9-11 Fibrillin-2, domain structure, 11-12 Fibrillin-like protein, gene location, 15 Fibrillins, in elastin-associated microfibrils, 3-4 Fibroblasts contamination of keratinocyte cultures, 43-44 proliferation, OH’-mediated, 221 ROS generation, 209 Fibronectin, localization to microfibrils, 18-19 Fibulin-2, amino acid structure, 13-14 Filaggrin, differentiation marker, 59-60 Flavocytochrome, NADPH oxidase membrane, 208 Fluid reabsorption, estrogen receptor role, 172-173 Fos, DNA-binding activity, redox regulation, 232-233 Free radical theory, aging, 215

G Gene expression in adult epididymis, 153-169 caput region, 156-164 distal part, 166-169 regulating factors, 169-184 throughout epididymal duct, 164-166

androgens and androgen receptors, 143-146 Cys4 zinc linger nuclear receptors, 147-148 in early Wolflian duct, 137-143 in juvenile and peripubertal epididymis, 148-153 regionalized, in epididymal duct epithelium, 134-135 Gene therapy, keratinocyte role, 62-63 Genital tract developmental role of Hox genes, 141-143 growth factor role, 146 male, 5a-reductase expression, 161 GGT epididymal expression, 159-160 mRNAs, androgen responsiveness, 171-172 PEA3-binding, 149 Glanzmann’s thrombasthenia, platelet adhesion in, 22-23 y-Glutamyl transpeptidase, see GGT Glutathione apoptotic role, 229-230 depletion, during oxidative stress, 231 role as redox buffer, 213-214 ROS reductant, 212-213 Glycopeptides, HE5/CD52, 167, 169 Glycoprotein CL, type VI collagen characteristics, 7 platelet membrane interaction with microfibrils, 27-28 reacting with subendothelium, 22-23 structure of microfibrils, 3 GP128, degradation product of thrombospondin, 15-16 G proteins, in ROS production, 226-227 GPX5 androgen responsiveness, 171 PEA3-binding, 149 GPX3 protein, genital tract expression, 160 GPXS protein epididymal expression, 160-161 regionalized expression pattern, 151 Growth, cellular, redox responses during, 220-222 Guanidium chloride, in microfibril extraction, 6

303

INDEX

H Half-life, OH', 205 HDL, see High-density lipoprotein HE2, epididymis-specific,162-163 HE3, a and 0 forms, epididymal expression, 163 HE1 gene product, epididymal duct expression, 164 Hemophilia B, treatment by gene therapy, 63 HE4 protein, epididymal expression, 165 HE6 protein, seven transmembranedomain, 163-164 High-density lipoprotein acute-phase response and, 279-281 effects cell transmigration, 271-272 endothelial cells, 262-267 leukocyte activation, 267-271 vasoactive molecules, 276-279 protective effect against heart disease, 258-260 structure, 257-258 and thrombosis and platelet function, 273-276 High-density lipoprotein receptors, and cell signal transduction, 281-286 Hindbrain development, and vitamin A deficiency, 102 effect of excess retinoic acid, 99-102 normal patterning, and retinoic acid, 102-103 retinoid receptors, 103-104 Historical perspective, vitamin A and cell proliferation, 76-77 early studies, 75-76 Holoclones, founded by stem cells, 48 Homeostasis calcium alteration, 216 ROS role, 225 ROS, in animal cells, 211-214 vitamin A, 82 Hoxa-10, genital tract expression, 142-143 Hoxa-11, mutants, 142-143 Hoxal, regulatory role of RAREs, 101-102

Hoxc-8, epididymal expression, 149 HUSI-11, testicular expression, 165-166 HUSI-YSLPI, produced in genital tract, 165 Hydrogen peroxide cellular levels, and toxicity, 219-221 decomposition by catalase and peroxidase, 212 generation, 204-206 in plant cells, 211 induction of DNA damage, 215 role in hypersensitive cell death, 237 Hydroxyl radical, generation, 204-206 Hypothermia, scrotal, physiological, 180-181

I Inositol phosphate turnover, and HDL, 284-285 Inositol 1,4,5-trisphosphate,ROS-induced production, 225 Integrins, ( ~ $ 3downregulation, ~~ 51 Intima collagen, see Collagen, type VI Involucrin, differentiation marker, 59-60

J Jun, DNA-binding activity, redox regulation, 232-233

K Keratinocyte growth factor, role in developing genital tract, 146 Keratinocytes cryopreservation, 61-62 culture methods, 43-47 differentiation, 51 differentiation markers, 59-60 migration, 49-50 progenitor, lacking in full-thickness bums, 42 proliferation, 47-48,52-54 role in gene therapy, 62-63 transplantation technique, 55-58 Keratins, expression in reconstructed epidermis, 59

304

INDEX

Kinetics, NADPH oxidase activation, 207-208 Krox20, expression in hindbrain, 100

L LDL, see Low-density lipoprotein Lens, induction, retinoic acid role, 88 Lesions fatty streak, 260 reepithelihtion, 58 Leukocytes adhesion to endothelium, 267-271 degranulation, HDL effect, 271 Ligaments, nuchal, studies of microfibrils, 5-7 Limb development, retinoic acid role, 106-109 Lipid peroxidation, ROS-induced, 216 Lipids anionic, transbilayer diffusion, 275 hydrophobic, forming central lipid droplet, 258 a-Lipoic acid, antioxidant role, 213 Lipoproteins, cellular response, 286-287 Low-calcium cultures, keratinocytes, 46-47,49 Low-density lipoprotein cytotoxicity for endothelial cells, 266 effects [Ca2+]i,285-286 endothelin-1,277 Lysophosphatidylchole, transfer into HDL, 277-278

M Macromolecules, subendothelial thrombogenic, 19-20 Malformations, congenital, vitamin A effect, 76-77 Mammalian development, vitamin A role, 74-75 MAP kinase, activation, ROS role, 224-225 Marfan’s syndrome, FBNI mutations, 12 Maturation, sperm, epididymis role, 134-135

Mechanical stimuli, perception of, ROS role, 228-229 Mesenchyme androgen receptor presence, 144 CRABP I distribution, 94 WT-I expression, 141 Messenger RNA CD52, androgen responsiveness, 171 CD52, stability, 181 GGT, androgen responsiveness, 171-172 proenkephalin, epididymal levels, 151, 184 SGP-I, localization in efferent duct, 156 Metabolites, ROS as, 235 Metal proteins containing, and redox state, 233 redox-active complexes, 205 Metalloprotease, see ADAM7 Microcarrier cultures, keratinocytes, 46,58 Microfibril-associatedproteins MAGP-1 and MAGP-2,12-13 MAGP-36, 15 Microfibrils elastin-associated, 3-4 proteins from, 8-19 thrombogenicity, 25-28 and elastogenesis, 19 extraction, 5-6 subendothelial, ultrastructure, 2-5 type VI collagen, 6-8 Migration endothelial cells, HDL effect, 267 keratinocytes, 49-50 neural crest cells, to face, 104 type VI collagen as substrate, 7-8 Milk, maternal, source of vitamin A, 79 Mitochondria, plant, ROS generation, 210-211 Models, platelet-microfibril interaction, 28 Monoclonal antibodies, AS02, fibroblastspecific, 44 Morphology, HDL effect on endothelial cells, 262 Mouse CRABP null mutant, 95-96 RARy null mutant, 98-99 Mutations FBNI and FBN2, 12 targeted c-ros, 138-139 epididymal gene products, 186

305

INDEX

NADPH oxidase, regulation of ROS production, 206-211 Neural crest cell migration to face, 104 excess retinoic acid effect, 99-102 NF-KB cytokine-induced translocation, 268, 270 oxidative activation, 232-233 as target for receptor-driven ROS production, 224 Nitric oxide HDL effect, 277-278 as muscle relaxant, 231 Nitric oxide synthase, endothelial cell, 278 Nutritional studies, vitamin A, 75-76

0 02'-, see Superoxide anion O R , see Hydroxyl radical Ontogenesis, epididymal gene expression pattern, 135-153 Osteogenesis, in skull, retinoic acid receptor role, 105-106 Oxidative burst NADPH oxidase activation-induced,230 in plant cells, 211 as plant defense, 235-236 Oxidative stress glutathione depletion during, 231 resulting in apoptosis, 216 Oxygen partial pressure, and ROS, 228-229 Oxygen reduction, steps, 204-206 OxyR, oxidized and reduced forms, 234 oxyR, transcription, 233-234 Oxytalan fibers, in elastin-free tissues, 3

P p110, HDL recognition, 283 Patterning genital tract, Hox gene role, 141-143 hindbrain, retinoic acid role, 102-103 in limb skeleton, 106-109 Pax-2 epididymal expression, 177 in mesoderm differentiation, 141

PEA3, role in epididymal gene expression, 149,176 Pem epididymal transcripts, 177-178 ontogenetic expression pattern, 149 Peptides HE2 and HE3, epididymal expression, 162-163 neuroendocrine, epididymal expression, 157 Periodicity, bead, fibrillin-containing microfibrils, 11 Peroxidases, decomposition of H202,212 Phagocytic cells, NADPH oxidase, 206-209 Phosphoinositide turnover, 285-286 Phosphorylation, protein, HDL effect, 265 Photodynamic therapy, cancer, 216 Phytoalexins, biosynthesis, and ROS production, 236-237 Placenta, transplacental transfer of retinoids, 83-85 Plants oxidative burst as defense, 235-236 proteins, rapid immobilization, 236 ROS in, 235-237 Plaque, atherosclerotic, apoptotic cells in, 266 Plasma, lipoprotein balance, 278 Plasma membrane, phagocytes, NADPH oxidase complex assembly at, 207-208 Plasminogen activator inhibitor-1, endothelial expression, 275 Platelet adhesion, to subendothelium, 20-23 Platelets function, and HDL, 273-276 interaction with microfibrils model, 28 thrombospondin role, 25-26 von Willebrand factor role, 26-27 Polyomavirus enhancer activator-3, see PEA3 Postnatal changes, epididymal gene expression levels, 148-151 Pregnancy and embryo, vitamin A role, 73-75 stage retinol requirement specific to, 85 and vitamin A deficiency, 76-77 Proenkephalin, mRNA, epididymal levels, . . 151,-184

306

INDEX

Proliferation endothelial cells, HDL effect, 262-266 fibroblasts, OH'-mediated, 221 keratinocytes, 47-48,52-54 vitamin A role, 76-77 Proopiomelanocortin, epididymal expression, 157 Prostacyclin, HDL effect, 278-279 Proteinase inhibitors, secretory, putative, 165-166 Protein b a s e C, signal transduction dependent on, 283-284 Proteins from elastin-associated microfibrils, 8-19 plant, rapid immobilization, 236 Pseudohermaphroditism, 145

R RA response element, y-F-crystallin gene, 88 Reactive oxygen species cellular damage, mechanisms, 215-216 effect on bacterial cells, 233-235 homeostasis in animal cells, 211-214 intracellular targets, mechanisms of activation, 229-233 and oxygen partial pressure, 228-229 in plants, 235-237 production, regulation by NADPH oxidase, 206-211 in receptor-mediated intracellular signaling, 222-228 regulation of cell functions, 220-222 as signal molecules, 222-235 steps in oxygen reduction, 204-206 toxicity, 217-220 as useful metabolites, 235 Receptor tyrosine kinase gene expression in early Wolffian duct, 138-139 HER/Neu, 176 role in epididymis, 178-179 Reconstruction, skin, 59-61 Redox responses, during cell growth and development, 220-222 Redox state affecting gene expression, 221 intracellular, control of, 213-214

and metal-containing proteins, 233 protein modification, 229-233 5a-Reductase developmental expression pattern, 145-146 epididymal expression, 161 Regionalization, specific gene expression in epididymis, 134-135, 154 Reproduction, mammalian, epididymis role, 133-134 Resistance, systematic acquired, in plants, 236-237 Retinal dehydrogenase, localization to embryonic lens, 88 Retinoic acid CRABP-binding, 9 5 % excess, effect on early hindbrain and neural crest, 99-102 and limb development, 106-109 localized generation from retinol, 93 role adult epididymis, 174 mammalian development, 74 signaling pathway, 77-78 teratogenic effects, 77 tissue-spdc provision, 85-89 Retinoic acid receptors activation, 97-98 in embryo, 98-99 in hindbrain, 103-104 role developing Wolffian duct, 147-148 skull osteogenesis and chondrogenesis, 105-106 Retinoic acid response elements, 97-98, 101-103 Retinoids, absorption and storage, 79-80 Retinoid X receptors epididymal localization, 174 in hindbrain, 103-104 retinoic acid receptor-binding, 97-98 subtype distribution, 87 Retinol localized generation of retinoic acid from, 93 transfer from maternal blood to embryo, 83-85 Retinol-binding protein encoding genes, 82 synthesis by endodermal cells, 83-85 Retinyl esters, conversion to retinol, 79-80

307

INDEX

Retroviral vector, neo gene-containing, 63 Rhombomeres, gene expression in, retinoic acid effect, 100 ROS, see Reactive oxygen species

S Second messengers, ROS role, 222-224 Secretions, epididymal, sperm contacting, 135 Segmentation genes, Par-2, 177 Serum amyloid A, HDL containing, 279, 281 Serum-free cultures, keratinocytes, 45-46 SGP-I, mRNA localization in efferent duct, 156 SGP-2, epididymal expression, 156-157 Sheets epithelial cryopreserved, 62 in vitro cultured, 41-42 keratinocyte cultured allogeneic, 58 transplantation, 56 SH groups, oxidation, 230-231 Signaling pathway retinoic acid in embryogenesis, 77-85 in limb skeleton, 106-107 sonic hedgehog, and retinoic acid, 107-109 Signal molecules, ROS role, 222-235, 238 Signal transduction cellular, and HDL receptors, 281-286 retinoic acid, 97-99 Skin allograft, 55 reconstruction, 42, 59-61 Skull, osteogenesis and chondrogenesis, 105-106 Smooth muscle cells lipid accumulation, 260-262 proliferation HDL effect, 264-266 02*role, 221 Sonic hedgehog and Bmp expression, 138 signaling pathway, and retinoic acid, 107-109 soxS, transcription, 234-235

Sperm maintenance, in distal epididymis, 185-186 maturation, epididymis role, 134-135 ROS generation, 222 Stem cells, retention in keratinocyte culture, 48-49 Steroid hormone-binding globulin, role in epididymis, 173-174 Storage, retinoids, 79-80 Subendothelium microfibrils, ultrastructure, 2-5 thickening, 1 thrombogenicity, 19-23 thrombospondin localization, 29-30 Subpopulations, keratinocytes, 48 Superoxide anion cellular levels, and toxicity, 219-220 conversion to H20z by superoxide dismutase, 212 effect on enzyme activities, 218 as electron donor, 235 generation, 204-206 release by phagocytes, 208-209 Superoxide dismutase, conversion of 02'to HzOz,212

T Temperature, effect on epididymal gene expression, 180-181 TGF-pl-binding proteins activation by thrombospondin, 29-30 latent, 14-15 Thickening intimal, HDL effect, 264-266 subendothelium, 1 Thiol status, in apoptotic pathway, 229-230 Thioredoxin, in NF-KB DNA-binding activity, 232 Three-dimensional cultures, keratinocytes, 45 Thrombogenicity elastin-associated microfibrils, 25-28 subendothelium, 19-23 type VI collagen, 23-25 Thrombosis, HDL effect, 273-276 Thrombospondin GP128 as degradation product, 15-16 microfibrils containing, 29-30

308

INDEX

Thrombospondin (continued) role in platelet-microfibril interaction, 25-26 TSP-1, on chromosome 15,17-18 Tissue distribution CRABP I in embryo, 93-94 elastin-associated microfibrils, 3-4 Tissue factor pathway inhibitor-1, isoforms, 275 Tissues bioartificial, 42 embryonic, retinoic acid synthesis and catabolism, 85-96 a-Tocopherol, ROS-scavenging, 212 Transcription factors epididymal, 176-178 regulatory role of ROS, 224 segmentation gene, 139-143 Transforming growth factor, see TGF Transmigration, cell, HDL effect, 271-272 Transplantation, see also Xenotransplantation cultured keratinocytes, 54-63 Transport, vitamin A, 82 Trypsinization, in isolation of keratinocytes,

44 Tumor necrosis factor-a, cell transmigration induced by, 272

maternal nutritional sources, 79 and placental function, 85 role adult epididymis, 174 embryogenesis and pregnancy, 73-75 Vitamin A deficiency and hindbrian development, 102 maternal, effect on developing eye, 88 Vitamin A deficiency syndrome, 75,84 Vitamin E, redox reactions with, 214 Vitamins, as ROS scavengers, 212 von Willebrand factor binding to type VI collagen, 23-25 microfibrillar location, 21 role in platelet-microfibril interaction, 26-27

W Wolffian duct 5a-reductase activity, 145-146 c-ros expression, 179 early, gene expression, 137-143 Wound covering, by cultured keratinocytes, 55-56 WT-1, mesenchymal expression, 141

X U Ultrastructure, subendothelial microfibrils, 2-5 Urokinase, plasminogen activator, binding sites on keratinocytes, 50, 57

Xanthine dehydrogenase, release after hepatic ischemia, 218 Xenotransplantation cultured human keratinocytes, 57-58 dermal, 64

Y Vacuolization, cytoplasmic, in epididymis, 183 Vas deferens, gene expression, 166-169 , Vasoactive molecules, HDL effect, 276-279 Virilization, in response to androgen production, 143-144 Vitamin A absorption and storage, 79-80 historical perspective, 75-77 homeostasis and transport, 82

Yolk sac CRJ3P expression, 92, 96 derived retinol-binding protein, 84-85

z Zinc finger, Cys4, nuclear receptor-type superfamily, 147-148 Zone of polarizing activity, in facial processes, 104-105

FIG. 3 Anterior homeotic transformation of the epididymis in Hoxu-lO-’- mutant mice. (A and B) Comparison of gross morphology of caput (cp), corpus (co), cauda (ca) epididymidis, and ductus deferens (dd) of wild-type (A) and mutant (B) 4-week-old males. The mutant shows increased width of corpus (open arrowhead), expansion of the cauda, and continued tortuosity of the ductus deferens exiting the cauda (bracketed). Scale bar = 1 mm. (C and D) Endogenous P-galactosidase activity in the distal epididymis as visualized by X-gal staining in the epididymis of wild-type (C) and homozygous mutant (D) males at 7 months of age. A sharp decrease in activity is seen between the corpus and the cauda region of the wild-type epididymis (C, arrow). The activity was weaker in the mutant (D) but had the same intensity throughout the corpus and cauda, with a distal limit in the ductus deferens (arrow). Scale bar = 1.5 mm [reproduced with permission from Benson, G . V., Lim, H., Paria, B. C., Satokata, I., Dey, S. K., and Maas, R. L. (1996). Mechanisms of reduced fertility in Hoxa-10 mutant mice: Uterine homeosis and loss of maternal Hoxa-10 expression. Development 122, 2687-26961.