Cell Adhesion and Reproduction: Tokyo Conference on Reproductive Physiology Iii, Tokyo, August 30, 1997 (Hormone Research)

Preface

H. Kanzaki A. Miyake H. Suginami M. Taga O. Tsutsumi Y. Yoshimura

The theme of the 9th Annual Meeting of the Tokyo Conference of Reproductive Physiology is ‘Cell Adhesion and Reproduction’. For the present Tokyo meeting, we, the organizing committee, nominated outstanding Japanese researchers in this field of science and all of them consented to attend and give a lecture. We welcome them and greatly appreciate their attendance. We are sure that with their participation, the present meeting will be completed in success, as were the previous meetings. The Tokyo Conference of Reproductive Physiology was first organized in 1989 under the sponsorship of Sandoz, now Novartis. The main purpose of the Conference has been to provide updated information and hot discussion to Japanese researchers dealing with reproductive physiology through which they could, hopefully, develop new directions or ferment new ideas in their own research. In the past we discussed ‘New Aspects of Pathophysiology and Treatment of Polycystic Ovary Syndrome’, ‘New Aspects of Prolactin in Human Reproductive Physiology’, ‘New Aspects of the Physiology and Pathology of the Luteal Phase’, ‘Aging of Reproductive Organs’, ‘Local

Regulators in the Ovary: Paracrine and Autocrine Control’, ‘Endocrine Regulation of Early Embryonic Development and Implantation’, ‘Sex Differentiation and Ovarian Function’, and ‘Apoptosis and Reproduction’ in the years 1989 through 1996, respectively. The contents and discussion of each annual meeting have appeared as a supplement to Hormone Research. Indeed, the Tokyo Conference has had remarkable impacts on the participants who have succeeded in expanding their research fields and most of them have obtained higher positions. This year the organizing committee came to the conclusion that the conference has accomplished the original aim and should be finalized. Opening the curtain is always exciting, but closing it is not. It is also true that the end of an era is also the beginning of a new one, i.e., apoptosis. Here, we declare the closing of the Tokyo Conference with the hope that this closing will be a new opening. Finally, we would like to thank and acknowledge all of the speakers and participants of the Conference, the sponsorship by Novartis, and people dealing with publication of the Conference.

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Organizing Board (in alphabetical order) Hideharu Kanzaki

Hiroshi Suginami

Osamu Tsutsumi

Department of Obstetrics and Gynecology Kansai Medical University Osaka (Japan)

Department of Obstetrics and Gynecology Kyoto National Hospital Kyoto (Japan)

Department of Obstetrics and Gynecology Tokyo University Tokyo (Japan)

Akira Miyake

Michiyoshi Taga

Yasunori Yoshimura

Department of Obstetrics and Gynecology Osaka University Osaka (Japan)

Department of Obstetrics and Gynecology Yokohama City University Yokohama (Japan)

Department of Obstetrics and Gynecology Keio University Tokyo (Japan)

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Horm Res 1998;50(suppl 2):2–6

Cell Adhesion and Reproduction

Michiyoshi Taga a Hiroshi Suginami b a

b

Department of Obstetrics and Gynecology, Yokohama City University School of Medicine, Yokohama, and Department of Obstetrics and Gynecology, Kyoto National Hospital, Kyoto, Japan

An Overview

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Key Words Cell adhesion Adhesion molecule Reproduction

Abstract Cell adhesion is essential for the regulation of many cellular functions. The adhesion molecule plays a critical role as a fundamental substance in various processes of reproduction such as trophoblast-endometrial interaction. Our understanding of the physiology of adhesion molecules will be helpful for clinical application of these molecules in the treatment and assessment of disorders of many processes of reproduction. We will briefly review integrins, cadherins, and laminin, which were the principal subjects of discussion in this conference. OOOOOOOOOOOOOOOOOOOOOO

Introduction One fundamental principle of cellular biology consists of ‘what a cell touches has a major role in determining what a cell does’ [1]. Cell adhesion plays a principal role in the regulation of many cellular functions, because the first contact between cells exerts a crucial effect on the entire course of their subsequent relationship. Adhesion not only binds cells or the extracellular matrix (ECM) to their proper location, as it literally means, but also actively mediates bidirectional signals both into and out of the cell. The importance of cell adhesion has been emphasized in such diverse processes as immune response, wound healing, inflammation and metastasis of malignant cells. Similarly, cell adhesion plays a main role in various processes in reproduction as well. For example, trophoblast-endometrial interaction in the implantation process, one of the most typical phenomena in reproduction, requires the finest regulation of cell adhesion and communication between two different cells. Figure 1 summarizes the junctional and nonjunctional adhesive mechanisms used by animal cells in binding to one an-

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other and to the ECM [2]. A junctional interaction is defined as one that can be seen as a specialized region of contact by electron microscopy. The integrins and cadherins are involved in both junctional and nonjunctional contacts, whereas the immunoglobulin superfamily and selectins are involved in nonjunctional contact. For a better understanding of the interactions of cells with the ECM or with other cells, it is important to identify cellsurface molecules because they are biochemical substances that determine the function of cell adhesion. Transmembrane proteins linking the matrix to the cell’s cortical cytoskeleton are divided into several families based on each structure (table 1). We will briefly review integrins, cadherins, and laminin, which were the principal subjects of discussion in this conference.

Integrins Integrins, a large family of homologous transmembrane linker proteins, are the principal receptors by which cells bind most ECM proteins, including collagen, fibro-

Michiyoshi Taga, MD Department of Obstetrics and Gynecology Yokohama City University School of Medicine 3-9 Fukuura, Kanazawa-ku, Yokohama 236 (Japan) Tel. +81 45 787 2691, Fax +81 45 701 3536

Fig. 1. A summary of the junctional and nonjunctional adhesive mechanisms used by animal cells in binding to one another and to the ECM [from 2].

Table 1. Cell adhesion molecule families [from 2] Some family members

Ca2+ or Mg2+ Homophilic or dependence heterophilic

Cytoskeleton associations

Cell junction associations

E, N, P cadherins

yes

homophilic

adhesion belts

desmosomal cadherins

yes

homophilic

Ig family members

N-CAM, L1

no

no

Selectins (blood cells + endothelial cells only) Integrins on blood cells

P-selectin

yes

homophilic or heterophilic heterophilic

actin filaments (via catenins) intermediate filaments (via desmoplakins, plakoglobin and other proteins) unknown unknown

no

LFA-l (aLb2), Mac-l (aMb2)

yes

heterophilic

actin filaments

no

many types

yes

heterophilic

focal contacts

a6b4 syndecans

yes no

heterophilic heterophilic

actin filaments (via talin, vinculin, and other proteins) intermediate filaments actin filaments

Cell-cell adhesion Cadherins

Cell-matrix adhesion Integrins

Transmembrane proteoglycans

Cell Adhesion and Reproduction

Horm Res 1998;50(suppl 2):2–6

desmosomes

hemidesmosomes no

3

Fig. 2. The receptors undergo conformational changes between at least two states: inactive (closed symbol) and active (open symbol). Only in the latter state do they bind most of their ligands. Signaling via integrins takes two forms: regulation of the affinity and conformation of the receptor from inside the cell (inside-out signaling), and triggering of intracellular events by ligand occupation of the receptors (outside-in signaling) [from 3].

illustrates the role of integrins as two-way signaling molecules [3]. The integrin receptors undergo conformational change between the inactive (closed symbol) and active state (open symbol), and bind most of their ligands only in the active state. Signaling via integrins takes two forms: regulation of the affinity and conformation of the receptor from inside the cell (inside-out signaling) and triggering of intracellular events by ligand occupation of the receptor (outside-in signaling). Recently, the molecular basis for the mechanism underlying a signal transmission mediated through integrins has been clarified. The clustering of integrins at the sites of contact with the matrix or another cell can activate intracellular signaling cascades, such as activation of mitogen-activated protein (MAP) kinase, increases in intracellular pH and Ca2+, and phosphatidylinositol turnover, thereby inducing changes in gene expression, stimulation of cell proliferation, or apoptosis. After cell adhesion, several proteins which become phosphorylated on tyrosine residues are located in focal adhesion, where actin filaments assemble via talin and ·actinin connecting to the intracellular domain of the integrins. Among these proteins, FAK (focal adhesion kinase) plays a central role.

Cadherins nectin, and laminin. They are heterodimers which are composed of two noncovalently associated transmembrane glycoprotein subunits called · and ß. About 20 integrin heterodimers, made from 9 types of ß subunits and 14 types of · subunits, have been discovered, and the number is still rising. Although integrins are crucially important receptor proteins, they differ from cell-surface receptors for hormones and for other soluble signaling molecules in that they bind their ligand with relatively low affinity and are usually present at about 10- to 100-fold higher concentrations on the cell surface. This arrangement makes sense, as binding simultaneously but weakly to large numbers of matrix molecules allows cells to explore their environment without losing all attachment to it [2]. Therefore, individual integrins can often bind to more than one ligand, whereas individual ligands are recognized by more than one integrin; for example, at least 8 integrins bind fibronectin, and at least 5 bind laminin. Integrins are involved not only in cell adhesion by connecting to bundles of actin filaments but also in the mediation of cytoskeletal interactions at the inner face of the membrane at sites of cell-ECM or cell-cell adhesion. This interaction operates in both directions: regulation from within the cell and also from the EMC. Figure 2

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The cadherins, transmembrane linker proteins, play a major role in morphogenesis by mediating Ca2+-dependent homophilic cell-cell adhesion. Ca2+ dependence of cadherin protein function is significant in its biological function, because in the absence of Ca2+, the cadherins undergo a large conformational change, and, as a result, are rapidly degraded by proteolytic enzymes [2]. The first three cadherins that were discovered were named according to the main tissues in which they were found: E-cadherin is present on many types of epithelial cells; N-cadherin on nerve, muscle, and lens cells; and P-cadherin on cells in the placenta and epidermis. As cells approach one another and touch, cadherins begin to cluster and connect, through their cytoplasmic domains and associated proteins (catenins), with the cytoskeleton [4]. The extracellular portion of cadherins, which interacts with that on neighboring cells, typically consists of 5 tandem repeats of an F110 amino acid homology domain, the cadherin repeat, whereas the cytoplasmic domain interacts with the actin cytoskeletons of the cells they join together by means of at least 3 catenins. Prior to their clustering, cadherins associate with the cytoplasmic proteins, ß-catenin or plakoglobin (PKG), through their cytoplasmic domains [5].

Taga/Suginami

The clustered cadherin-ß-catenin/PKG complex appears to act as a nucleus for the formation of a cytoplasmic ‘plague’ composed of other catenins [4]. At the adherens junctions, the plague associated with cadherin tail domains mediates the ‘end-on’ anchoring of microfilaments via the vinculin-like ·-catenin polypeptide and ·-catenin [6]. Increased levels of cytoplasmic ß-catenin/PKG could act to liberate the hypothetical factor X, which then moves to the nucleus and alters gene expression. Alternatively, cytoplasmic ß-catenin/PKG could alter the activity of factor X by forming a complex with it [4].

Laminin The ECM, composed of a variety of versatile proteins and polysaccharides, builds an intricate network of macromolecules by filling extracellular space and organizing a meshwork in close association with the cell surface. Until recently, the ECM was thought to serve mainly as a relatively inert scaffolding to stabilize the physical structure of tissues. But now it is clear that the ECM plays a far more active and complex role in regulating the behavior of the cells that contact it – influencing their development, migration, proliferation, shape, and function [2]. Laminin is a large flexible complex of multidomain glycoproteins composed of three very long polypeptides (·, ß, and Á) that are disulfide bonded into an asymmetric cross-like structure. Five types of · chains, three types of ß chains, and two types of Á chains have been cloned, and 11 isoforms have been found [7]. Each isoform has a distinctive tissue distribution according to the site where it is produced, such as epithelial, endothelial, muscle, and Schwann’s cells. Laminin has diverse functions, including not only cell adhesion but also cell movement and proliferation, and especially plays an important role in development, because laminin is one of the first ECM proteins synthesized in a developing embryo and because early in development basal laminae consist mainly of a laminin network [2]. Recently, laminin has been reported to be involved in the pathogenesis of muscular dystrophy in the human and mouse [8] and a point mutation in the ·2 chain was identified in this disease [9, 10]. A homozygous nonsense mutation in the ·3 chain gene of laminin 5 (LAMA3) was reported in lethal (Herlitz) junctional epidermolysis bullosa [11].

Cell Adhesion and Reproduction

Cell Adhesion in Reproduction The adhesion molecule plays a critical role as a fundamental substance in each stage of reproduction, such as pulsatile secretion of gonadotropin-releasing hormone (GnRH), gonadotropin secretion, follicular development, ovulation, fertilization, preimplantation embryo development, implantation, placentation, and maintenance of pregnancy. Cell migration is important in hypothalamic GnRH secretion, because GnRH-producing neurons originate in the epithelium of the medial olfactory pit and migrate from the nose into the hypothalamus along nerve fibers rich in neural cell adhesion molecules (N-CAM) during fetal development. In Kallmann’s syndrome (hypogonadotropic hypogonadism with anosmia), which is characterized by defects in the olfactory system and hypothalamic GnRH secretion, these symptoms could be explained by a cell migration defect specifically affecting these nasal epithelium-derived neurons. The KALIG-1 gene shares homology with molecules involved in cell adhesion and axonal pathfinding, suggesting that a molecular defect in this gene causes the neuronal migration defect which underlies Kallmann’s syndrome. It has been suggested that the protein product of the KAL gene could be of the N-CAM type [12]. Adhesion molecules function as cell-cell adhesion proteins, promoting sperm binding via an integrin to the egg plasma membrane [13]. Transmembrane heterodimeric ligand on guinea pig sperm is responsible for both recognition of and fusion with the egg plasma membrane [13]. The involvement of cell adhesion molecules has been reported from the earliest stage of embryo development to placentation and the maintenance of pregnancy. With regard to preimplantation and peri-implantation development, modulation of cell-cell interactions plays a predominant role during initial lineage decisions and in promoting epithelial-mesenchymal transitions throughout this period [14]. The preimplantation mouse embryo produces a surprisingly broad repertoire of ECM receptors and ligands [1], and several integrins are detected from the outset of embryonic gene transcription (late two-cell stage) [15]. Laminin is one of the first ECM proteins synthesized in a developing embryo. E-cadherin helps to cause compaction, an important morphological change that occurs at the eight-cell stage of mouse embryo development. During compaction, the loosely attached blastomeres become tightly packed together and are joined by intercellular junctions. By analyzing the distribution of 9 different · and ß integrin subunits in human endometrial tissue at different stages of the menstrual cycle, Lessey et al. [16] reported that some

Horm Res 1998;50(suppl 2):2–6

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integrins normally undergo spatial and temporal changes in expression within the cycling endometrium and that a disruption of this pattern may be associated with decreased uterine receptivity and infertility. Therefore, the adhesion molecule may be a good marker of endometrial function, by which infertility and abortion may be explained. In the implantation process, the exchange of the signals from embryo to endometrium, as well as from endometrium to embryo, is achieved by adhesion molecules. Adhesion mechanisms might be differentially expressed at the fetal-maternal interface in the human and mouse, contributing to significant structural differences in the cytoarchitecture of the placental bed among different species [14]. The expression of several adhesion molecules undergoes dramatic alteration during normal cytotrophoblast differentiation along the invasive pathway in vivo [14]. In trophoblast differentiation and invasion during placentation, not only the role of cell-ECM interaction, but also

the regulation of cell-cell interactions is critical in the acquisition of an invasive phenotype [17]. The maintenance of pregnancy is dependent on adhesion molecules which control trophoblast invasion and their communication. In preeclamptic patients, abnormal expression of adhesion molecules by invasive cytotrophoblasts has been reported [18]. Our understanding of the physiology of adhesion molecules will be helpful for clinical application of these molecules in the treatment and assessment of disorders of many processes of reproduction. For example, the addition of some adhesion molecules into the culture medium may improve the quality of the embryo and the subsequent pregnancy rate in in vitro fertilization and embryo transfer. Furthermore, the analysis of adhesion molecules on embryos could provide us with much information concerning their quality. The research for new adhesion molecules could offer further applications for therapeutic intervention in reproductive disorders.

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References 1 Vinatier D: Integrins and reproduction. Eur J Obstet Gynecol Reprod Biol 1995; 59: 71–81. 2 Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD (eds): Molecular Biology of the Cell, ed 3. New York, Garland Publishing, 1994. 3 Hynes RO: Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 1992;69: 11–15. 4 Klymkowsky MW, Parr B: The body language of cells: The intimate connection between cell adhesion and behavior. Cell 1995;83:5–8. 5 Hinck L, Nathke IS, Papkoff J, Nelson WJ: Dynamics of cadherin/catenin complex formation: Novel protein interactions and pathways of complex assembly. J Cell Biol 1994;125: 1327–1340. 6 Knudsen KA, Peralta Soler A, Johnson KR, Wheelock MJ: Interaction of ·-actinin with the cadherin/catenin cell-cell adhesion complex via ·-catenin. J Cell Biol 1995;130:67–77. 7 Miner JH, Patton BL, Lentz SI, Gilbert DJ, Snider WD, Jenkins NA, Copeland NG, Sanes JR: The laminin · chains: Expression, developmental transitions, and chromosomal locations of ·1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel ·3 isoform. J Cell Biol 1997;137:685–701.

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8 Xu H, Christmas P, Wu X-R, Wewer UM, Engvall E: Defective muscle basement membrane and lack of M-laminin in the dystrophic dy/dy mouse. Proc Natl Acad Sci USA 1994;91: 5572–5576. 9 Xu H, Wu X-R, Wewer UM, Engvall E: Murine muscular dystrophy caused by a mutation in the laminin ·2 (Lama 2) gene. Nat Genet 1994;8:297–302. 10 Helblig-Leclerc A, Zhang X, Topaloglu H, Cruaud C, Tesson F, Weissenbach J, Tomé FMS, Schwartz K, Fardeau M, Tryggvason K, Guicheney P: Mutations in the laminin ·2chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy. Nat Genet 1995;11:216–218. 11 Kivirikko S, McGrath JA, Baudoin C, Aberdam D, Ciatti S, Dunnill MGS, McMillan JR, Eady RAJ, Ortonne J-P, Meneguzzi G, Uitto J, Christiano AM: A homozygous nonsense mutation in the ·3 chain gene of laminin 5 (LAMA3) in lethal (Herlitz) junctional epidermolysis bullosa. Hum Mol Genet 1995;4:959–962. 12 Franco B, Guioli S, Pragliola A, Incerti B, Bardoni B, Tonlorenzi R, Carrozzo R, Maestrini E, et al: A gene deleted in Kallmann’s syndrome shares homology with neural cell adhesion and axonal path-finding molecules. Nature 1991; 353:529–536. 13 Blobel CP, Wolfsberg TG, Turck CW, Myles DG, Primakoff P, White JM: A potential fusion peptide and an integrin ligand domain in a protein active in sperm-egg fusion. Nature 1992;356:248–252.

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14 Damsky C, Sutherland A, Fisher S: Extracellular matrix 5: Adhesive interactions in early mammalian embryogenesis, implantation, and placentation. FASEB J 1993;7:1320–1329. 15 Behrendsten O, Alexander CM, Werb Z: Metalloproteinases mediate extracellular matrix degradation by cells from mouse blastocyst outgrowths. Development 1992;114:447–456. 16 Lessey BA, Damjanovich L, Coutifaris C, Castelbaum A, Albelda SM, Buck CA: Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest 1992;90:188– 195. 17 Behrens J, Vakaet L, Friis R, Winterhager E, Van Roy F, Mareel M, Birchmeier W: Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/ß-catenin complex in cells transformed with a temperature-sensitive vSRC gene. J Cell Biol 1993;120:757–766. 18 Zhou Y, Damsky CH, Chiu K, Roberts JM, Fisher SJ: Preeclampsia is associated with abnormal expression of adhesion molecules by invasive cytotrophablasts. J Clin Invest 1993; 91:950–960.

Taga/Suginami

Horm Res 1998;50(suppl 2):7–14

Hiroto Mizushima a Naohiko Koshikawa a Kayano Moriyama a Hiroyuki Takamura a Yoji Nagashima b Fumiki Hirahara c Kaoru Miyazaki a a

b c

Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, and Departments of Pathology and Obstetrics and Gynecology, Yokohama City University School of Medicine, Yokohama, Japan

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Key Words Laminin-5 Cell adhesion Laminin Á2 chain Distributions Immunohistochemistry

Wide Distribution of Laminin-5 Á2 Chain in Basement Membranes of Various Human Tissues

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Abstract Laminin 5 (LN5), a heterotrimer of laminin ·3, ß3, and Á2 chains, is a laminin isoform which strongly promotes adhesion, migration, and scattering of cells through binding to integrins ·3ß1, ·6ß1 and ·6ß4. To get an insight into the physiological functions of LN5, we prepared a mouse monoclonal antibody to human laminin Á2 chain and used it for immunohistochemical analysis of laminin Á2 chain in normal human tissues. The basement membranes of various epithelial tissues, such as the skin, lung, small intestine, stomach, kidney and prostate, were immunostained with the anti-laminin Á2 chain monoclonal antibody. In addition, the basement membrane of the surface germinal epithelium in the ovary was also positive for laminin Á2 chain. These results suggest general roles of LN5 in the anchorage of various types of epithelial cells to the underlying basement membrane and in the expression of their cellular functions. Moreover, deposition of laminin Á2 chain around small arteries and veins was observed in the thymus and spleen. This lymphatic organ-specific expression of vascular LN5 might provide a novel function of LN5 in immune responses. OOOOOOOOOOOOOOOOOOOOOO

Introduction Laminins are extracellular matrix proteins which are composed of ·, ß, and Á heterotrimeric chains. Different combinations of five · chains, three ß chains, and two Á chains form at least 11 laminin isoforms (laminin-1 to laminin-11) [1, 2]. Laminin-5 (LN5), which consists of laminin ·3, ß3, and Á2 chains, is a component of anchoring filaments underlying the hemidesmosome structure of epidermal keratinocytes [3]. Mutations or deletion in the LN5 genes (LAMA3, LAMB3, and LAMC2) is associated with epidermolysis bullosa, a lethal skin blistering disease [4–7]. LN5 extracted from the amnion exists as a monomer or complexes with other laminins (laminin-6 or laminin-7) linked by disulfide bonds [8].

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It has been proposed that LN5 stabilizes the epidermal/dermal junction of skin through binding with integrins ·3ß1, ·6ß4, and type VII collagen [9]. LN5 strongly promotes adhesion, migration, and scattering of various types of cultured cells compared with other known extracellular matrix proteins [10–12]. These activities are mainly mediated by integrin ·3ß1 [11–15]. We recently identified a G2 subdomain in the carboxyl-terminal globular domain of laminin ·3 chain as an integrin ·3ß1 binding site [16]. In addition, it was found that G4 and G5 subdomains have cell adhesion activity through interaction with heparin-like molecules on the cell surface [16]. Laminin Á2 chain is a unique subunit of LN5. In addition to its truncated structure [17], laminin Á2 chain has

Kaoru Miyazaki, PhD Division of Cell Biology, Kihara Institute for Biological Research Yokohama City University 642-12 Maioka-cho, Totsuka-ku, Yokohama 244 (Japan) Tel. +81 45 820 1905, Fax +81 45 820 1901, E-Mail [email protected]

some features, such as low affinity for nidogen compared with laminin Á1 chain [18] and binding with fibulin-2 via its short arm [19]. It has been reported that cleavage of laminin Á2 chain at a specific site within coiled-coil structure by matrix metalloproteinase-2 stimulates migration of cells on the LN5 substrate [20]. These results suggest that laminin Á2 chain has specific functions for the activity of LN5. Expression of LN5 is modulated by retinoic acid, growth factors, and tumor promoter in vitro [21–24]. In vivo up-regulation of LN5 has been detected at the sites of colon adenocarcinoma invasion and wound healing of skin [23, 25, 26]. On the other hand, decreased expression of LN5 has been reported in transformed keratinocytes in culture [23], prostate carcinomas [27], and invasive pancreatic carcinomas [28]. It has been generally accepted that LN5 has an important role in the maintenance of epidermal-dermal junction of skin. There are a considerable number of papers showing the distribution of laminin ·3 or ß3 chain by in situ hybridization or immunohistochemistry [13, 29, 30]. However, there are only a few studies dealing with the tissue distribution of laminin Á2 chain [17, 31, 32]. In this study, the distribution of laminin Á2 chain in normal adult human tissues was examined using a monoclonal antibody directed against laminin Á2 chain.

Materials and Methods Laminin Á2 Chain Monoclonal Antibody cDNA encoding III domain (amino acid residues 382–608) of laminin Á2 chain was obtained by RT-PCR, and subcloned into pGEX-2TK vector (Pharmacia, Uppsala, Sweden) after confirming the sequence. Primers used in the RT-PCR are 5)-GCGGATCCTGTATATGTCCTGTTG-3) and 5)GCGAATTCAGCTGAATGCTCCATG-3), where underlining denotes restriction enzyme sites for subcloning. The III domain was expressed as a glutathione-S-transferase fusion protein as described previously [16]. The GST fusion III domain of laminin Á2 chain was immunized into Balb/c mice. Monoclonal antibodies were prepared by the standard method using mouse myeloma cell line P3U1.

Fig. 1. Detection of laminin Á2 chain by immunoblotting with monoclonal antibody D4B5. Purified LN5 was run on 6% SDSPAGE under reducing conditions, and was probed with an anti-laminin Á2 chain monoclonal antibody (D4B5). Lane 1, Coomassie brilliant blue staining. Laminin ·3 (·3), ß3 (ß3), and Á2 (Á2) chains are indicated. Lane 2, immunoblotting. Arrowheads indicate laminin Á2 chain proteins at 150 and 105 kD. Molecular weight markers are shown in the left with kilodaltons.

Table 1. Tissue distribution of laminin Á2 chain Tissues (localization of signal)

Laminin Á2

Skin (BM of stratified epithelium) Esophagus (BM of stratified epithelium) Lung (BMs of bronchial surface and alveolar epithelia) Stomach (BM of surface epithelium) Small intestine (BM of surface epithelium) Kidney (BMs of collecting tubules) Spleen (blood vessels) Thymus (blood vessels) Prostate (BMs of glandular alveoli) Ovary (BM of surface epithelium) Thyroid Testis Skeletal muscle Cardiac muscle

++ ++ ++ + + ++ + + + + – – – –

BM = Basement membrane. Immunoblot Analysis Samples were run on SDS-PAGE, transferred to nitrocellulose membranes. After blocking with 5% (w/v) nonfat milk in PBS, the membranes were probed with antibodies, and protein bands were visualized by the alkaline phosphatase method [24]. Tissue Specimens and Sample Preparation All human adult and fetal tissues (28 weeks’ gestation) [33] were obtained by autopsy, and immediately fixed in 10% formalin. The paraffin-embedded sections were mounted on aminoacyl silanecoated glass slides and used for immunohistochemical analysis.

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Immunohistochemistry Immunohistochemical staining of laminin Á2 chain of LN5 was carried out with monoclonal antibody D4B5. Briefly, 4-Ìm thick paraffin sections were dewaxed, rehydrated and immersed in 0.3% hydrogen peroxide-containing methanol for inactivation of intrinsic peroxidase. All sections were treated with Protease XXIV (Sigma, St. Louis, Mo., USA) for 15 min at room temperature. The sections were

Mizushima/Koshikawa/Moriyama/ Takamura/Nagashima/Hirahara/Miyazaki

A

B

C

D Fig. 2. Light microphotographs of immunohistochemistry for laminin Á2 chain in human skin (A), esophagus (B), lung (C) and embryonic lung (D). Paraffin sections of the tissues were subjected to immunohistochemistry with anti-laminin Á2 antibody D4B5. Arrowheads show positive staining of epithelial basement membranes for laminin Á2 chain. Experimental conditions are described in the text. Bar = 30 Ìm.

then incubated with the antibody D4E5 at 37 ° C for 1 h. The labelled antigen was detected by HistoFine kit (Nichirei Pharmaceutical, Tokyo, Japan), and visualized by the 3,3-diaminobenzidine (DAB) reaction. Other experimental conditions were described previously [34].

A mouse monoclonal antibody to human laminin Á2 chain, D4B5, was prepared as described in the Materials and Methods. To confirm the specifity of the antibody, we first carried out Western blotting of native LN5 purified from the culture medium of human gastric carcinoma STKM-1 cells. As shown in figure 1, laminin Á2 proteins at 150 and 105 kD were detected by the anti-Á2 mono-

clonal antibody D4B5 (fig. 1). The 150-kD precursor form of laminin Á2 chain has been shown to be processed to the 105-kD mature form [35]. Normal human tissues were subjected to immunohistochemical analysis with the anti-Á2 monoclonal antibody D4B5. The results of immunohistochemical analysis are summarized in table 1. As expected, intense immunoreactivity for the Á2 chain of LN5 was detected in the basement membranes (BMs) of the stratified squamous epithelia of the skin and esophagus (fig. 2A, B). In addition, strong staining for laminin Á2 chain was detected in the BM of alveolar epithelial cells in a lung tissue (fig. 2C). Bronchiolar epithelium was also positive for laminin Á2 chain (data not shown). Interestingly, an immature lung tissue from a human embryo showed many BM-like ring

Expression of Laminin Á2 Chain in Tissues

Horm Res 1998;50(suppl 2):7–14

Results

9

A

Fig. 3. Light microphotographs of immunohistochemistry for laminin Á2 chain in human ovary (A invaginated surface; B inclusion cyst epithelium), small intestine (C) and kidney (D). Paraffin sections of the tissues were subjected to immunohistochemistry with anti-laminin Á2 antibody D4B5. Arrowheads show positive staining of epithelial basement membranes for laminin Á2 chain. Experimental conditions are described in the text. Bar = 30 Ìm. The magnification of B is half of the others.

structures immunostained by the anti-Á2 monoclonal antibody, although alveolar structures were not yet evident histologically (fig. 2D). This suggests that LN5 may play an important role in the development of the alveolar structure of the lung. In the ovary, the BMs of the surface epithelium (fig. 3A) and the inclusion cyst epithelium (fig. 3B) clearly showed the positive staining for laminin Á2 chain. Laminin Á2 chain was detected in the BMs of mucosal epithelia of small intestine (fig. 3C), stomach (data not shown), collecting tubules of the kidney (fig. 3D), and the glandular

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B

epithelium of the prostate (fig. 4A). Proximal and distal tubules of the kidney and mucosal epithelium of the oviduct were also positive for laminin Á2 chain (data not shown). Unexpectedly, laminin Á2 chain was also detected in the thymus and spleen. In an embryonic thymus tissue, laminin Á2 chain was detected in membranes surrounding small arteries and veins (fig. 4B). Distribution of the immunoreactivity around blood vessels was also observed in some of trabecular arteries, veins and sinusoids vessels in an adult spleen tissue (fig. 4C).

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C

D

We previously found that human laminin ·3, ß3, and Á2 chains are expressed at high levels in the adult lung, placenta, and fetal kidney, and moderately in the prostate,

thymus, testis, and ovary [24]. Recent studies with immunohistochemistry or in situ hybridization have shown that LN5 exists not only in the BM of the skin but also in those of other epithelial tissues. However, the limited number of studies have reported the tissue distribution of laminin Á2 chain. Sugiyama et al. [31] showed the gene expression of laminin Á2 chain by epithelial cells in the oral cavity of fetal mice and the presence of the protein product in the BMs of fetal mouse kidney and pancreas. Airenne et al. [32] reported the expression of laminin Á2 chain by epithelial cells in fetal and neonatal human tissues of the

Expression of Laminin Á2 Chain in Tissues

Horm Res 1998;50(suppl 2):7–14

The thyroid, testis, placenta, skeletal muscle and cardiac muscle did not show significant staining for laminin Á2 chain (data not shown). All sections incubated with preimmune serum demonstrated no immunostaining.

Discussion

11

A

B

Fig. 4. Light microphotographs of immunohistochemistry for laminin Á2 chain in human prostate (A), thymus (B) and spleen (C). Paraffin sections of the tissues were subjected to immunohistochemistry with anti-laminin Á2 antibody D4B5. Solid arrowheads show positive staining of epithelial basement membranes for laminin Á2 chain. Open arrowheads show positive staining of small arteries and veins for laminin Á2 chain. Experimental conditions are described in the text. Bar = 30 Ìm.

lung, kidney, skin, and liver, using in situ hybridization. Hao et al. [27] showed the presence of laminin Á2 chain in the basal lamina surrounding human prostate glands by immunohistochemistry. In addition, the expression of laminin Á2 chain by various types of cancer cells have been reported [24]. In human carcinomas, laminin Á2 chain is expressed at high levels in invading carcinoma cells [25, 26]. Together with these results, our previous finding, that LN5 has potent cell migration-stimulating activity, stronlgy suggests that LN5 may promote tumor cell invasion in vivo [10–12]. The present study demonstrated wide distribution of laminin Á2 protein in the BMs underlying the stratified squamous epithelia of the skin and esophagus and those surrounding the mucosal epithelia in the lung, stomach, small intestine, kidney, prostate, and ovary. This suggests the general role of LN5 in the expression of normal epithelial cell functions. To our knowledge, the localization

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C

of LN5 or laminin Á2 chain in the ovary has not been reported previously. Laminin Á2 chain was clearly detected in the BM surrounding surface epithelial cells in the ovary. Because mRNAs for laminin ·3, ß3, and Á2 chains are expressed in the ovary [24], it is expected that the laminin Á2 chain is deposited in the BMs of the ovary as a subunit of the LN5 heterotrimer. It has been reported that ovarian surface epithelial cells, like keratinocytes, express ·2, ·3, ·6, ß1, and ß4 integrins [36]. The basal cells of stratified squamous epithelia divide at relatively high rates. In general, mucosal epithelial cells are also dynamic: they frequently divide and migrate. LN5 is supposed to be a suitable substrate for these types of epithelial cells. The present study also showed that laminin Á2 protein is deposited in the membranes surrounding trabecular arteries and veins in the spleen. The presence of laminin Á2 protein in blood vessels was also seen in fetal thymus. It has been reported that laminin ·3 chain is detected in

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the endothelia of the skin, lymph node, tonsil, thymus, and lung [37]. We previously demonstrated that LN5 promotes initial attachment of cultured human umbilical endothelial cells through binding with integrin ·3ß1 [12]. Endothelium of large vessels in lymphatic organs, thymus and spleen, is positive for integrin ß4 in mice [38]. These results indicate that LN5 functions as an adhesive ligand for endothelial cells in some tissues. LN5 underlying endothelial cells in the lymphatic organs may play another role. Wayner et al. [37] reported that T lymphocytes expressing integrin ·3ß1 are accumulated in the vicinity

of LN5-containing epithelial BMs at cutaneous inflammation sites [37]. Integrin ·3ß1-positive lymphocytes might penetrate a monolayer of endothelial cells by adhering to the endothelial cell-derived LN5.

Acknowledgments We thank Drs. H. Yasumitsu and K. Udagawa for helpful discussions. This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan, and a grant from Uehara Memorial Foundation, Japan.

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References 1 Burgeson RE, Chiquet M, Deutzmann R, Ekblom P, Engel J, Kleinman H, Martin GR, Meneguzzi G, Paulsson M, Sanes J, Timpl R, Tryggvason K, Yamada Y, Yurchenco PD: A new nomenclature for the laminins. Matrix Biol 1994;14:209–211. 2 Miner JH, Patton BL, Lentz SI, Gilbert DJ, Snider WD, Jenkins NA, Copeland NG, Sanes JR: The laminin · chains: Expression, developmental transitions, and chromosomal locations of ·1–5, identification of heterotrimeric laminins 8–11, and cloning of a novel ·3 isoform. J Cell Biol 1997;137:685–701. 3 Rousselle P, Lunstrum GP, Keene DR, Burgeson RE: Kalinin: An epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments. J Cell Biol 1991;114:567–576. 4 Aberdam D, Galliano M-F, Vailly J, Pulkkinen L, Bonifas J, Christiano AM, Tryggvason K, Uitto J, Epstein EH Jr, Ortonne J-P, Meneguzzi G: Herlitz’s junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the Á2 subunit of nicein/kalinin (LAMININ-5). Nat Genet 1994;6:299–304. 5 Pulkkinen L, Christiano AM, Gerecke D, Burgeson RE, Pittelkow MR, Uitto J: A homozygous nonsense mutation in the ß3 chain gene of laminin 5 (LAMB3) in Herlitz junctional epidermolysis bullosa. Genomics 1994;24:357– 360. 6 Kivirikko S, McGrath JA, Baudoin C, Aberdam D, Ciatti S, Dunnill MGS, McMillan JR, Eady RAJ, Ortonne J-P, Meneguzzi G, Uitto J, Christiano AM: A homozygous nonsense mutation in the ·3 chain gene of laminin 5 (LAMA3) in lethal (Herlitz) junctional epidermolysis bullosa. Hum Mol Genet 1995;4:959–962. 7 Vidal F, Baudoin C, Miquel C, Galliano M-F, Christiano AM, Uitto J, Ortonne J-P, Meneguzzi G: Cloning of the laminin ·3 chain gene (LAMA3) and identification of a homozygous deletion in a patient with Herlitz junctional epidermolysis bullosa. Genomics 1995;30: 273–280.

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8 Champliaud MF, Lunstrum GP, Rousselle P, Nishiyama T, Keene DR, Burgeson RE: Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment. J Cell Biol 1996; 132:1189–1198. 9 Rousselle P, Keene DR, Ruggiero F, Champliaud M-F, van der Rest M, Burgeson RE: Laminin 5 binds the NC-1 domain of type VII collagen. J Cell Biol 1997;138:719–728. 10 Miyazaki K, Kikkawa Y, Nakamura A, Yasumitsu H, Umeda M: A large cell-adhesive scatter factor secreted by human gastric carcinoma cells. Proc Natl Acad Sci USA 1993;90:11767– 11771. 11 Kikkawa Y, Umeda M, Miyazaki K: Marked stimulation of cell adhesion and motility by ladsin, a laminin-like scatter factor. J Biochem (Tokyo) 1994;116:862–869. 12 Kikkawa Y, Akaogi K, Mizushima H, Yamanaka N, Umeda M, Miyazaki K: Stimulation of endothelial cell migration in culture by ladsin, a laminin-5-like cell adhesion protein. In Vitro Cell Dev Biol 1996;32:46–52. 13 Carter WG, Ryan MC, Gahr PJ: Epiligrin, a new cell adhesion ligand for integrin ·3ß1 in epithelial basement membranes. Cell 1991;65: 599–610. 14 Verrando P, Lissitzky J-C, Sarret Y, Winberg JO, Gedde-Dahl T Jr, Schmitt D, BrucknerTuderman L: Keratinocytes from junctional epidermolysis bullosa do adhere and migrate on the basement membrane protein nicein through ·3ß1 integrin. Lab Invest 1994;71: 567–574. 15 Zhang K, Kramer RH: Laminin 5 deposition promotes keratinocyte motility. Exp Cell Res 1996;227:309–322. 16 Mizushima H, Takamura H, Miyagi Y, Kikkawa Y, Yamanaka N, Yasumitsu H, Misugi K, Miyazaki K: Identification of integrin-dependent and -independent cell adhesion domains in COOH-terminal globular region of laminin5 ·3 chain. Cell Growth Differ 1997;8:979– 987.

17 Kallunki P, Sainio K, Eddy R, Byers M, Kallunki T, Sariola H, Beck K, Hirvonen H, Shows TB, Tryggvason K: A truncated laminin chain homologous to the B2 chain: Structure, spatial expression, and chromosomal assignment. J Cell Biol 1992;119:679–693. 18 Myer U, Pochl E, Gerecke DR, Wagman DW, Burgeson RE, Timpl R: Low nidogen affinity of laminin-5 can be attributed to two serine residues in EGF-like motif Á2III4. FEBS Lett 1995;365:129–132. 19 Utani A, Nomizu M, Yamada Y: Fibulin-2 binds to the short arms of laminin-5 and laminin-1 via conserved amino acid sequences. J Biol Chem 1997;272:2814–2820. 20 Giannelli G, Falk-Marziller J, Schiraldi O, Stetler-Stevenson WG, Quaranta V: Induction of cell migration by matrix metalloproteinase-2 cleavage of laminin-5. Science 1997;277:225– 228. 21 Verrando P, Pisani A, Ortonne J-P: The new basement membrane antigen recognized by the monoclonal antibody GB3 is a large size glycoprotein: Modulation of its expression by retinoic acid. Biochim Biophys Acta 1988;942:45– 56. 22 Korang K, Christiano AM, Uitto J, Mauviel A: Differential cytokine modulation of the genes LAMA3, LAMB3, and LAMC2, encoding the constitutive polypeptides, ·3, ß3, and Á2 of human laminin 5 in dermal keratinocytes. FEBS Lett 1995;368:556–558. 23 Ryan MC, Tizards R, Van Devanter DR, Carter WG: Cloning of the LamA3 gene encoding the ·3 chain of the adhesive ligand epiligrin: Expression in wound repair. J Biol Chem 1994; 269:22779–22787. 24 Mizushima H, Miyagi Y, Kikkawa Y, Yamanaka N, Yasumitsu H, Misugi K, Miyazaki K: Differential expression of laminin-5/ladsin subunits in human tissues and cancer cell lines and their induction by tumor promoter and growth factors. J Biochem (Tokyo) 1996;120: 1196–1202.

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25 Pyke C, Rømer J, Kallunki P, Lund LR, Ralfkiaer E, Dano K, Tryggvason K: The Á2 chain of kalinin/laminin 5 is preferentially expressed in invading malignant cells in human cancers. Am J Pathol 1994;145:782–791. 26 Pyke C, Salo S, Ralfkiaer E, Romer J, Dano K, Tryggvason K: Laminin-5 is a marker of invading cancer cells in some human carcinomas and is coexpressed with the receptor for urokinase plasminogen activator in budding cancer cells in colon adenocarcinomas. Cancer Res 1995; 55:4132–4139. 27 Hao J, Yang Y, McDaniel KM, Dalkin BL, Cress AE, Nagle RB: Differential expression of laminin 5 (·3ß3Á2) by human malignant and normal prostate. Am J Pathol 1996;149:1341– 1349. 28 Soini Y, Maatta M, Salo S, Tryggvason K, Autio-Harmainen H: Expression of the laminin Á2 chain in pancreatic adenocarcinoma. J Pathol 1996;180:290–294. 29 Galliano M-F, Aberdam D, Aguzzi A, Ortonne J-P, Meneguzzi G: Cloning and complete primary structure of the mouse laminin ·3 chain: Distinct expression pattern of the laminin ·3A and ·3B chain isoforms. J Biol Chem 1995; 270:21820–21826.

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30 Tani T, Karttunen T, Kiviluoto T, Kivilaakso E, Burgeson RE, Sipponen P, Virtanen I: ·6ß4 integrin and newly deposited laminin-1 and laminin-5 form the adhesion mechanism of gastric carcinoma. Am J Pathol 1996;149:781– 793. 31 Sugiyama S, Utani A, Yamada S, Kozak CA, Yamada Y: Cloning and expression of the mouse laminin Á2 (B2t) chain, a subunit of epithelial cell laminin. Eur J Biochem 1995;228: 120–128. 32 Airenne T, Haakana H, Sainio K, Kallunki T, Kallunki P, Sariola H, Tyggvason K: Structure of the human laminin Á2 chain gene (LAMC2): Alternative splicing with different tissue distribution of two transcripts. Genomics 1996;32: 54–64. 33 Nagashima Y, Miyagi Y, Udagawa K, Taki A, Misugi K, Sakai N, Kondo K, Kaneko S, Yao M, Shuin T: Von Hippel-Lindau tumor suppressor gene. Localization of expression by in situ hybridization. J Pathol 1996;180:271– 274.

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34 Akaogi K, Okabe Y, Sato J, Nagashima Y, Yasumitsu H, Sugahara K, Miyazaki K: Specific accumulation of tumor-derived adhesion factor in tumor blood vessels and in capillary tubelike structures of cultured vascular endothelial cells. Proc Natl Acad Sci USA 1996;93:8384– 8389. 35 Marinkovich MP, Lunstrum GP, Burgeson RE: The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor. J Biol Chem 1992;267: 17900–17906. 36 Skubitz APN, Bast RC Jr, Wayner EA, Letoureau PC, Wilke MS: Expression of ·6 and ß4 integrins in serous ovarian carcinoma correlates with expression of the basement membrane protein laminin. Am J Pathol 1996;148: 1445–1461. 37 Wayner EA, Gil SG, Murphy GF, Wilke MS, Carter WG: Epiligrin, a component of epithelial basement membranes, is an adhesive ligand for ·3ß1 positive T lymphocytes. J Cell Biol 1993;121:1141–1152. 38 Kennel SJ, Godfrey V, Ch’ang LY, Lankford TK, Foote LJ, Makkinje A: The ß4 subunit of the integrin family is displayed a restricted subset of endothelium in mice. J Cell Sci 1992;101: 145–150.

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Kenji Sobue Ken’ichiro Hayashi Wataru Nishida Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, Osaka University Medical School, Osaka, Japan

Molecular Mechanism of Phenotypic Modulation of Smooth Muscle Cells

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Key Words Smooth muscle Phenotypic modulation Caldesmon Gene expression Splicing Contractile protein ·-Smooth muscle actin SM22 Myosin heavy chain ·1 Integrin Atherosclerosis

Abstract Phenotypic modulation of smooth muscle cells is closely associated with vasculogenesis, enterogenesis and some diseases such as atherosclerosis, hypertension and leiomyogenic tumorigenicity. During phenotypic modulation, smooth muscle cells change their morphology, cell function and biochemical characteristics. Recent studies have focused on the regulation mechanism of smooth muscle cell-specific genes at the levels of transcription and/or alternative splicing in a phenotype-dependent manner. Typical examples of such genes include caldesmon, ·-tropomyosin, myosin heavy chain, SM22, calponin and ·1 integrin. Cell adhesion molecules and growth factors/cytokines also play a critical role for controlling phenotype of smooth muscle cells via signal transduction pathways such as phosphoinositide 3-kinase and mitogen-activated protein kinases. OOOOOOOOOOOOOOOOOOOOOO

1. Introduction The smooth muscle cells (SMCs) are involved in control of blood pressure, enteric peristalsis and bronchial, uterus and bladder contraction. The origin of the precursor cells that give rise to SMCs remains unclear. At least three different lineages (cardiac neural crest, nodosa placode, and lateral mesoderm) are candidates to produce SMCs. In the case of vasculogenesis [1, 2], vascular precursor cells, angioblasts, initially differentiate from mesoderm to endothelial cells, which then coalesce to form a single layer of endothelial tubes. During development, these initial vessels are involved in the recruitment of SMC precursor cells onto the endothelial tubes, with subsequent differentiation of SMCs and morphogenesis of

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the blood vessel. However, the precise molecular mechanism regarding recruitment of SMC precursor cells from mesenchymal cells are still unknown. Phenotypic modulation of SMCs is also crucially involved in the onset of vascular diseases such as atherosclerosis and hypertension. It is, therefore, important for current vascular biology to reveal the molecular mechanism of phenotypic modulation of SMCs. Investigations in this field are limited because primary culture systems or clonal cell lines of SMC have not been established. Here, we have summarized the current knowledge regarding the regulation mechanism of SMC-specific genes in association with phenotypic modulation.

Dr. Kenji Sobue Department of Neurochemistry and Neuropharmacology Biomedical Research Center, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 (Japan) Tel. +81 6 879 3680, Fax +81 6 879 3689, E-Mail [email protected]

Fig. 1. Ca2+-dependent dual regulation of smooth muscle and nonmuscle actomyosin systems. Under a lower Ca2+ concentration (Ca2+ ! 10 –6 M ), the 20-kDa myosin light chain is dephosphorylated by myosin light chain phosphatase, and caldesmon on the actin/tropomyosin complex inhibits actin-myosin interaction (relaxation). As an increasing Ca2+ concentration (Ca2+ 1 10 –6 M ), myosin light kinase is activated by binding to Ca2+/calmodulin complex and phosphorylates the myosin light chain. Simultaneously, the inhibitory effect of caldesmon is also released by binding of Ca2+/calmodulin complex to caldesmon, inducing actin-myosin interaction (contraction). CaM = Calmodulin; CaD = caldesmon; A = actin filament; TM = tropomyosin; MLCK = myosin light chain kinase; p-M = phosphorylated myosin; MLCP = myosin light chain phosphatase; M = dephosphorylated myosin.

2. Structural Organization and Function of SMC The structural characteristics of SMC are as follows: (1) Myofibrils (contractile apparatus which are mainly composed of actin and myosin) in SMC are organized in three-dimensional direction. (2) Two prominent electrondense structures are localized in the cytoplasm and at the cell-cell contact: dense body and dense membrane (dense plaque), respectively. The dense body is located at the terminal site of myofibrils and plays a Z-line-like role in striated muscles. The dense membrane is the heterophilic cell adhesion structure mediated by the extracellular matrices. Intermediate filaments link the dense body and the dense membrane. These SMC-specific structures lead to a three-dimensional contraction [3]. In the muscle layer, SMCs are filled with the extracellular matrix components mainly composed of collagen type IV, laminin, and elastin [4, 5]. It has been reported that the ·1ß1 integrin and membrane skeletal proteins such as vinculin, ·-actinin, and talin are colocalized in the dense membrane [6, 7]. The main function of SMC is contraction. A molecular basis of SMC contraction is actin-myosin interaction like striated muscle contraction. In fact, 50% of total proteins in SMCs are occupied by contractile proteins. However, the Ca2+-dependent regulation of SMC contraction is different from that of striated muscle contraction. In SMCs, actin-myosin interaction is regulated by a dual mechanism: The myosin-linked and actin-linked regulations (fig. 1). The myosin-linked regulation is based on phos-

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phorylation of the 20-kDa regulatory light chain of myosin by Ca2+/calmodulin-dependent myosin light chain kinase and dephosphorylation by myosin phosphatase [8]. This regulation might be involved in the degree of actinmyosin interaction, presumably resulting in control of contractile force. The actin-linked regulation is the functional regulation of actin filament by caldesmon and tropomyosin in a Ca2+/calmodulin-dependent manner, and plays a role as an on/off switch of contraction [9]. Since calmodulin, caldesmon, tropomyosin, myosin light chain kinase and myosin light chain phosphatase are ubiquitously distributed in nonmuscle cells in addition to SMCs, the dual mechanism summarized here is a Ca2+-dependent regulation in common contractile events of cells, except for striated muscles.

3. Phenotypic Modulation of SMCs Phenotypic modulation of vascular SMCs is one of the early events of atherosclerosis [10]. As an initial step, vascular SMCs undergo a transition in phenotype from a contractile (differentiated) to a synthetic (dedifferentiated) state. During this process, the cells obtain alternative properties and undergo proliferation and migration, whereby the proliferated cells migrate into the intima, causing intimal thickening. Finally, progression of atherosclerotic lesions in intima is thus characterized by the accumulation of alternating layers of dedifferentiated SMCs and lipid-laden macrophages. Before describing the

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molecular mechanism of phenotypic modulation of SMCs, it is necessary to characterize both differentiated and dedifferentiated phenotypes. The functional, morphological and biochemical characteristics of SMC in different phenotypes are summarized in table 1. As outlined in section 2, the dense membrane, the dense body, and myofibrils are well developed in differentiated SMCs which are able to contract in response to SMC-specific ligands. In place of losing these characteristic structures, dedifferentiated SMCs show extensive Golgi complex and rough endoplasmic reticulum [11] and activated protein synthesis. Therefore, the dense membrane and the dense body are morphological markers for phenotype of SMCs. Unlike differentiated SMCs, dedifferentiated SMCs are not able to contract, but extensively profilerate and migrate. Changes in expression and/or isoform interconversion of SMC-specific proteins in association with phenotypic modulation of SMCs are well studied. These changes are listed in table 1. They are regulated at the level of gene including SMC phenotype-dependent transcription and alternative splicing. The expression of caldesmon, calponin, SM22, ß-tropomyosin, and ·1 integrin are regulated by transcription. These proteins are upregulated in differentiated SMCs, but are downregulated in dedifferentiated SMCs. Isoform changes of caldesmon, ·-tropomyosin, vinculin/metavinculin, and smooth muscle myosin heavy chain are controlled by alternative splicing. These biochemical parameters are splendid aids to quantify phenotype of SMCs, and are favorable targets to elucidate the molecular mechanism of phenotypic determination. In section 6, we will describe the regulation mechanism of SMC-specific genes in detail.

4. Effects of Cell Adhesion Molecules and Growth Factors/Cytokines on Phenotype of SMCs Under usual culture conditions, SMCs prepared by either enzyme dispersion or explant rapidly display their phenotypic change from a contractile to synthetic state [11]. Until recently, there have been no reports regarding primary culture systems of SMCs maintaining a differentiated phenotype and established cell lines controlling a phenotypic change. Because of these reasons, it has been difficult to analyze a differentiated phenotype of SMCs. As described in section 2, collagen type IV, laminin and elastin are main components of the extracellular matrix in SMC layer. s-Laminin, an isoform of laminin ß2 chain, is also expressed in well-developed vascular SMC layer, but not in visceral SMC layer [12, 13]. High expression of ·1

Phenotypic Modulation of Smooth Muscle Cells

Table 1. Expression changes of SMC-specific marker proteins Marker proteins

Differentiated SMC

Dedifferentiated SMC

Caldesmon ·-Tropomyosin ß-Tropomyosin Myosin heavy chain

h-CaD ↑ ·-TM-SM ↑ SMemb ↓ SM1 ↑ SM2 ↑

l-CaD ↓ ·-TM-F1 and ·-TM-F2 ↓ ↑ ↓ ↓

↑ ↓ ↑ ↑ ↑ ↑

↓ ↑ ↓ ↓ ↓ ↓

·-Smooth muscle actin Vascular SMC Visceral SMC Metavinculin SM22· Calponin ·1 Integrin

↑ = Upregulation; ↓ = downregulation.

and ·3 integrins, receptors for collagen and laminin, are detected in tissues containing SMCs [13, 14]. In particular, the ·1 integrin expression is closely associated with phenotypic modulation of SMCs; its expression is upregulated in developing SMCs, but is downregulated during dedifferentiation [14, 15]. By contrast, the expression of ·V and ·5 integrins increases in dedifferentiated SMCs [16]. These findings suggest that the extracellular matrix might be one of the determinants for SMC phenotype. Thyburg and co-workers [17] reported using primary cultured vascular SMCs that laminin and collagen type IV induced a delay of the progression of dedifferentiation, whereas fibronectin stimulates it. To establish a primary culture system, Hayashi et al. [18] have investigated the effects of extracellular matrix components on phenotype of SMCs under serum-free culture conditions. The results are summarized in figure 2. Among several extracellular matrix components examined, laminin had the potency to delay the progression of dedifferentiation, while collagens type I and type IV and fibronectin did not possess such ability. Laminin was, however, not able to maintain a differentiated phenotype of SMCs for a long culture, suggesting the requirement of additional factors. Serum and most growth factor cytokines such as PDGFs, bFGF, angiotensin II, Arg-vasopressin, EGF, and TGFßs induced dedifferentiation of SMCs even when they were cultured on laminin. Under these conditions, dedifferentiation and proliferation of SMCs did not always link because serum induced proliferation of SMCs, but PDGFs did not. Sur-

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Fig. 2. Effects of cell adhesion molecules and growth factors/cytokines on the phenotype of cultured SMCs. Maintenance of a differentiated phenotype of SMCs depends on cell adhesion mediated by laminin and stimulation by IGFs or insulin. By contrast, dedifferentiation of SMCs is induced by other cell adhesion molecules such as fibronectin or vitronectin and a variety of growth factors and cytokines such as serum, angiotensin II, PDGF, Arg-vasopressin, TGFß, bFGF, or EGF.

Fig. 3. Distinct signal transduction pathways controlling SMC phenotypes. Signaling pathway generated from IGF-I/IGF-I receptor to PI3 kinase plays a vital role in maintaining a differentiated phenotype of SMCs. This signaling pathway is not able to activate MAPKs. On the other hand, MAPK (ERK) and a stress-activated MAPK (p38 MAPK) are activated during dedifferentiation of SMCs induced by PDGF or serum.

prisingly, insulin-like growth factor I (IGF-I), II (IGF-II), and insulin were able to maintain a differentiated phenotype of SMCs for a long culture [18]. Among them, IGF-I showed the most potent activity and the effect of IGF-I on SMCs links to the expression of IGF-I receptor in a phenotype-dependent manner; high levels of IGF-I receptor express in differentiated SMCs, whereas its expression decreases in serum-induced dedifferentiated cells [18]. These results suggest that IGF-I/IGF-I receptor and laminin are essential for maintaining a differentiated phenotype of SMCs.

SMCs (fig. 3). Downstream targets of PI3 kinase have not been unknown. Hayashi et al. [18] have obtained partial evidence that IGF-I activates Akt1 (protein kinase B), but rapamycin, a specific inhibitor of p70 ribosomal S6 kinase, does not inhibit the activity of IGF-I on SMCs. Unlike IGF-I, PDGF-BB and serum, which are potent factors for promoting dedifferentiation of SMCs, enhanced the activity of MAPKs. Specific MAPK kinase (MEK) inhibitor, PD98059, partially inhibitied the downregulation of calponin and caldesmon expressions and isoform change of caldesmon under PDGF-BB- or serum-stimulated culture conditions. In accordance with these findings, Hu et al. [19] and Pyles et al. [20] have demonstrated that the ERK activity is enhanced in balloon-injured vessels. One of the stress-activated MAPKs, p38 MAPK, was also activated by PDGF-BB or serum stimulation. p38 MAPK-specific inhibitor, SB203580 [21], was not able to solely protect PDGF-BB- or seruminduced dedifferentiation. However, both PD98059 and SB203580 completely inhibited PDGF-BB- or seruminduced dedifferentiation of SMCs as measured by SMCspecific proteins such as caldesmon and calponin and ligand-induced contractility [18]. Another type of stressactivated MAPKs, JNK, is not involved in dedifferentiation. Taken together, coordinate activation of MAPK and p38 MAPK would trigger to induce dedifferentiation of SMCs (fig. 3).

5. Signal Transduction Controlling Phenotypic Modulation of SMCs As described above, IGF-I/IGF-I receptor- and laminin-generated intracellular signal transductions play a critical role for maintaining a differentiated phenotype of SMCs. Under these culture conditions, IGF-I activated phosphoinositide 3-kinase (PI3 kinase). By contrast, PI3 kinase inhibitors, wortmannin and LY294002, induced dedifferentiation even when SMCs were cultured on laminin under IGF-I-stimulated conditions. IGF-I stimulation was not able to activate mitogen-activated protein kinases (MAPKs) [18]. These findings suggest that signaling pathway from IGF-I/IGF-I receptor to PI3 kinase is essential for maintenance of differentiated phenotype of

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6. SMC Phenotype-Dependent Gene Expression Transcription ·-SM actin is a major actin isoform expressed in vascular SMCs [22, 23]. Expression of this actin isoform in rat and human aortic SMCs is developmentally upregulated; steady-state levels of ·-SM actin mRNA increase in developing aortae, reaching about 90% of total actin mRNA in adult tissue [24]. By contrast, in primarily cultured aortic SMCs, the expression of this actin isoform decreases with serum-induced dedifferentiation [25]. Because of these properties, ·-SM actin has been used as a favorable molecular marker for vascular SMC phenotpye. However, recent studies demonstrate that this actin is ectopically expressed in nonmuscle stromal cells [26] and under certain pathological conditions such as massively proliferated mesangial cells [27] and myofibroblasts [28]. It has been recently found that the expression of ·-SM actin in visceral SMCs is quite opposite to that in vascular SMCs regarding a phenotype [29]. This finding suggests that the transcriptional regulation of ·-SM actin gene in visceral SMCs is completely different from that in vascular SMCs. Promoter analyses revealed that the ·-SM actin expression was paradoxically regulated by a combination of multiple cis-elements/transacting factors interactions. In visceral SMCs, a novel negative cis-element was predominantly involved in the suppression of ·-SM actin expression [29]. Kimura et al. [30] have recently identified one of the negative trans-acting factors as c-myc gene single-strand binding protein-1. Among these regulatory machineries, one positive enhancer is CArG box/serum response factor (SRF) interaction in both vascular and visceral SMCs. In vascular and visceral SMCs together, other SMCspecific genes including caldesmon [31], calponin [32, 33], SM22 [34, 35], ß-tropomyosin [36], and ·1 integrin [13, 14, 37] are upregulated during differentiation, but are downregulated during dedifferentiation. Interestingly, the CArG box or CArG box-like motif are present in the promoter regions of such SMC-specific genes in addition to myosin heavy chain [38] and vinculin [39] (fig. 4). The caldesmon promoter contains multiple E boxes, CArG, CCAAT, TATA boxes and Sp1 binding site. Promoter analysis revealed that the SMC-specific transcription of the caldesmon gene depended on a single cis-element, CArG box [31]. In the case of ·1 integrin, its high level expression in differentiated SMCs was also dependent on the CArG box, but not on Ap1, AP2 and Sp1 binding sites [37], which are located in the promoter regions of a family of integrin genes such as ·2 [40], ·4 [41–44], CD11 a (·L) [45], and CD11c (·X) [46]. Gel shift

Phenotypic Modulation of Smooth Muscle Cells

Fig. 4. The CArG box is a common cis-element regulating SMCspecific transcription. Schematic structures of the promoter regions of SMC-specific genes are listed. The CArG box is the only ciselement conserved in the promoter regions of these genes. The sequences of respective CArG box are shown. The SMC-specific transcription of each marker gene is dependent on the CArG box.

assay using anti-SRF antibody indicated the involvement of SRF in specific DNA-nuclear protein complex formation with the two CArG boxes of the caldesmon and ·1 integrin genes, suggesting that the SRF is a core transacting factor for such complex. Among CArG box-containing SMC-specific genes listed in figure 3, CArG box/SRF interaction has been identified in myosin heavy chain [47], SM22· [48], and ß-tropomyosin [49] genes. These results suggest that the CArG box and the SRF would function as SMC-specific transcriptional machinery. The SRF was originally discovered as a transcription factor of c-fos responsive to the serum [50]. It is widely distributed in a variety of cells. In particular, high levels of SRF expression are observed in striated and smooth muscles [51]. Therefore, the SRF is not the sole transcription factor for SMC-specific gene expression. There may be another factor which confers the SMC specificity on CArG box/SRF interaction. An attractive candidate for the SMC-specific regulator is a family of homeodomain proteins. These proteins containing a highly conserved sequence of homeodomain are encoded by homeobox genes which specify segmental identity and positional

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Fig. 5. Tissue-specific transcriptions with homeodomain proteins and SRF. In the heart, Nkx-2.5 activates transcription of cardiac ·-actin gene in accordance with SRF. In the visceral SMCs, we have found that bagpipe and barx activate transcription of SM22· and ß-tropomyosin genes, respectively. Note that SRF is expressed in all tissues and plays an important role as a core activator. Homeodomain proteins regulate cardiac and SMC-specific transcription in their tissue-specific manners. Vascular SMC-specific homeodomain protein has not been identified.

information along the antero-posterior axis [52]. Gruenberg et al. [53] reported that a homeodomain protein, Phox1, significantly enhanced the binding of SRF with c-fos SRE probe. However, stable ternary complexes composed of Phox1, SRF, and SRE probe were not observed in vitro. The affinity of SRF with SRE probe was quite weak in the case of SRF alone, but it became stable in the presence of an initiator protein, TFII-I [54]. Similar activation of SRF with a homeodomain protein have been reported in ·-cardiac muscle actin gene. Nkx-2.5, a mouse homologue of Drosophila tinman, which is a heart-specific homeobox gene and a regulator of cardiogenesis, activates the promotor activity of ·-cardiac muscle actin gene in collaboration with SRF [55]. Taking into account with this evidence, SMC-specific genes containing CArG box(es) may also need homeodomain protein(s) for the coactivation of SRF. Nishida et al. [in preparation] have screened a chicken gizzard smooth muscle cDNA library with a degenerative oligo-probe targeted for the most conserved region of third helix in the homeodomain. Several homeobox genes have been cloned, and it has been revealed that some of them activate the transcription of SM22· or ß-tropomyosin genes via CArG box/SRF interaction in vitro. They have also cloned a chicken homologue of Drosophila bagpipe. Bagpipe and another homeobox gene tinman are involved in the determination of cell fates in the dorsal mesoderm of Drosophila [56]. In bagpipe mutant, visceral mesoderm formation is strongly disrupted; tinman regulates expression of bagpipe and it is required for the formation of visceral musculature, the

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heart, and the body wall muscles [56]. Thus, tinman is an upper regulator of bagpipe, and three muscle lineages possess a common transcription pathway in Drosophila. This fact resembles the phenomenon that an SMC-specific gene, SM22·, is transiently transcribed in the heart and myotome before its expression in mouse embryo smooth muscles [35]. It seems likely that the NK family homeobox genes also form a cascade of genetic interactions like tinman/bagpipe and determine the differentiation of mesoderm in the vertebrates (fig. 5). Nishida et al. [57] have also searched for the transcription factors whose expressions are activated during an early phase of dedifferentiation of SMCs and are not generated in differentiated SMCs. As a result, several clones which are specifically expressed in dedifferentiated SMCs were obtained. One of the clones was Msx-1, a homeobox gene which is transiently expressed in limb buds and craniofacial structures of embryo, and progressively disappears after birth [58, 59]. In cultured SMCs, Msx-1 was not expressed in differentiated cells at all, but it came to be expressed several hours after serum-induced dedifferentiation. Overexpression of Msx-1 was also observed in the balloon-injured rat carotid artery. Therefore, Msx-1 seems to be an important transcriptional regulator in phenotypic modulation of SMCs, and it is a useful and clinical molecular marker for the dedifferentiated smooth muscle-like cells in the chronic proliferating diseases discussed in the following section. Splicing As shown in table 1, SMC phenotype-dependent isoform interconversion of caldesmon and ·-tropomyosin are most notable. The two different Mr isoforms of caldesmon have been identified [60, 61]: h-Caldesmon (high Mr-form) is predominantly expressed in differentiated SMCs, while l-caldesmon (low Mr-form) widely distributes in nonmuscle tissues and cells [62]. Isoform interconversion of cadesmon is a first example of alternative splicing in association with phenotypic modulation of SMCs. The caldesmon isoform converts from the l- to h-form during differentiation and vice cersa during dedifferentiation [62]. ·-Tropomyosin also diversifies eight spliced variants [63]. During dedifferentiation, the SMC-specific ·-tropomyosin (·-TM-SM) converst to fibroblast type 1 and 2 ·-tropomyosin isoforms (·-TM-F1 and ·-TM-F2) by a change in the exon selection from exon 2a to exon 2b [36]. Interestingly, the caldesmon and ·-tropomyosin genes are coordinately regulated by transcription and alternative splicing in a SMC phenotype-dependent manner [36]. In SMCs, two variants of myosin heavy chain

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with Mrs of 204 kD (SM1) and 200 kD (SM2) have been reported. They are encoded in a single gene and produced by alterantive splicing [64]. The SM1 and SM2 diverge in their C-terminus. Vascular SMCs express two nonmuscle isoforms, NMHC-A and NMHC-B (SM emb), under pathological conditions [65]. During development, embryonic/fetal nonmuscle-type myosin heavy chains replace with adult-type variants [66]. These isoform changes are similar to those of caldesmon and ·-tropomyosin. Therefore, h-caldesmon, ·-tropomyosin, and SM22 are thought to be molecular markers for a late stage of SMC differentiation. Vinculin/meta-vinculin interconversion is also SMC phenotype-dependent. The meta-vinculin expression is developmentally upregulated, but is downregulated during dedifferentiation [67]. The vinculin/meta-vinculin interconversion is regulated by a selection of exon 19 within a single gene [68]. Meta-vinculin is, however, expressed in other muscles of mesodermal origin indicating that it is not a specific molecular marker for SMCs and a splicing machinery of the vinculin gene seems to be different from that of caldesmon and ·-tropomyosin genes (fig. 6). The genomic structures of caldesmon gene have revealed that exon 3 is unique. The common domain of h- and l-caldesmons is encoded in exon 3a, whereas hcaldesmon-specific central repeating domain is entirely encoded in exon 3b. Therefore, the expression of caldesmon isoforms is determined by an SMC phenotypedependent selection of alternative 5)-splice sites within exon 3; predominant selection of proximal 5)-splice site generates h-caldesmon and that of distal 5)-splice site generates l-caldesmon. It has been recently reported that repeating purine-rich motifs in exon 3b act as an exon enhancer element, causing predominant selection of distal 5)-splice site in myofibroblasts and nonmuscle cells [69]. This report is curious because these cells, when examined, do not endogenously express h-caldesmon. Hayashi et al. [in preparation] have investigated the splicing mechanism of the chicken caldesmon gene in cultured SMCs using a series of caldesmon mini-gene contructs composed of exon 3 and downstream intron. They have obtained a different result, that the intron sequence between exons 3b and 4 might be involved in alternative selection of distal and proximal 5)-splice sites within the exon 3; the selection of proximal 5) splice site was inhibited in the transcripts from such mini-gene constructs carrying deleted or mutated intron sequence. In the ·-tropomyosin gene, the exon selection among a couple of mutually exclusive exon pairs (exons 2a and 2b, and exons 6a and 6b) is regulated in a tissue-specific man-

Phenotypic Modulation of Smooth Muscle Cells

Fig. 6. SMC phenotype-dependent splicing in the caldesmon, ·-tropomyosin, and vinculin genes. Alternative splicings of the caldesmon (CaD) gene and the ·-tropomyosin (·-TM) gene are coordinately regulated in an SMC phenotype-dependent manner. In differentiated SMCs, exon 3ab in the CaD gene and exon 2a in the ·-TM gene are spliced in respective mRNAs, generating SMC-specific isoforms h-CaD and ·-TM-SM. By contrast, in dedifferentiated SMCs, exon 3a and exon 2b are selectively spliced in the respective mRNAs, causing the expression of l-CaD and ·-TM-F1 and ·-TM-F2, respectively. In the vinculin gene, exon 19 encoding meta-vinculin-specific sequence is specifically selected in differentiated SMCs. However, this splicing is not specific to SMCs because meta-vinculin is also expressed in skeletal and cardiac muscles.

ner. The selection of a pair of internal exons (exons 6a and 6b) is closely associated with the differentiation process of skeletal muscle cells; exons 6a and 6b are used in myoblasts and myotubes, respectively [70, 71]. Exon 2b rather than exon 2a (termed exons 3 and 2, respectivley) is spliced in the ·-tropomyosin mRNAs in all types of cell except for SMCs [72, 73]; exon 2a is specifically selected in SMCs (fig. 6). The molecular mechanism of mutually exclusive splicing between exons 2a and 2b has been intensively studied. The functional strength of exon selection depends on an affinity of splicing factor U2AF to the pyrimidine tract for exon 2b or that for exon 2a [74]. The branch point/pyrimidine tract in the downstream intron of exon 2b is stronger than those of exon 2a [75]. Thus,

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7. Conclusion

Fig. 7. Smooth muscle or smooth muscle-like cells play the leading part in the myofibroblastosis. The smooth muscle-like cells, such as mesangial cells in the kidney, Ito cells in the liver, alveolar myofibroblasts in the lung, and pericytes in the capillary vessel contain a well-developed actomyosin system and show contractility. In the case of pathological conditions, the smooth muscle-like cells change their phenotype, lose contractility, activate protein synthesis, and proliferate like vascular SMCs in the atherosclerotic lesion. In this way, the smooth muscle-like cells play the leading part in nephritis, lung fibrosis, and hepatic fibrosis. We would like to propose a new concept, myofibroblastosis, to include all of the chronic proliferating diseases.

exon 2b is predominantly selected in other types of cell except for SMCs. Alternatively, the SMC-specific selection of exon 2a may be due to inhibition of exon 2b selection. In fact, two conserved elements in each of the introns flanking exon 2b play a role for such SMC-specific inhibition. It is still unclear whether common factor(s) are involved in such exon selection in the ·-tropomyosin and caldesmon genes. Two smooth muscle variants of myosin heavy chain (SM1 and SM2) and two vinculin isoform (vinculin and metavinculin) are generated from respective single genes by alterantive splicing [62, 68]. These splicing regulations have not been characterized.

There are several kinds of smooth muscle-like cells, such as mesangial cells in the kidney, Ito cells (lipocytes or liver satellite cells) in the liver, alveolar myofibroblasts in the lung, and pericytes in the capillary vessel. All of them contain a well-developed actomyosin system and show contractility. Their origins remain unclear, but they seem to be derived from the mesoderm. It seems likely that the pericytes share the same origin with vascular SMCs [76]. As a consequence of phenotypic modulation, these smooth muscle-like cells lose contractility, activate protein synthesis, proliferate like vascular SMCs in the atherosclerotic lesion, and finally cause nephritis [27], lung fibrosis [77], and hepatic fibrosis [78], respectively. Interestingly, ·-SM actin is overexpressed in the proliferated mesangial cells [79] and Ito cells [80]. Imai et al. [81] have found that caldesmon is overexpressed in ·SM actin-positive mesanigal cells of the IgA nephritis patients. They have also reported that caldesmon regresses promptly during the improvement of the nephritis with therapy, and its reduction is faster than that of ·SM actin. As described here, caldesmon, ·-SM actin and other SMC-secific proteins are molecular markers for phenotypic modulation of SMCs and smooth muscle-like cells, and there may be common mechanism(s) which regulate the chronic proliferating diseases, such as nephritis, lung fibrosis, and liver fibrosis in addition atherosclerosis. We have shown that these diseases are commonly based on the dedifferentiated smooth muscle-like cells, so-called myofibroblasts, and we would like to propose a new concept, myofibroblastosis, to include all of the chronic proliferating diseases (fig. 7). The underlying mechanism of myofibroblastosis is unclear, and there is no definitive plan for a therapeutic strategy and prevention. We believe that analyses of phenotypic modulation of SMCs will lead us to the understanding of, and a cure for, myofibroblastosis.

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58 Robert B, Sassoon D, Jacq B, Gehring W, Buckingham M: Hox-7, a mouse homeobox gene with a novel pattern of expression during embryogenesis. EMBO J 1989;8:91–100. 59 Davidson D: The function and evolution of Msx genes: Pointers and paradoxes. Trends Genet 1995;11:405–411. 60 Owada MK, Hakura A, Iida K, Yahara I, Sobue K, Kakiuchi S: Occurrence of caldesmon (a calmodulin-binding protein) in cultured cells: Comparison of normal and transformed cells. Proc Natl Acad Sci USA 1984;81:3133– 3137. 61 Sobue K, Tanaka T, Kanda K, Ashino N, Kakiuchi S: Purification and characterization of caldesmon-77: A calmodulin-binding protein that interacts with actin filaments from bovine adrenal medulla. Proc Natl Acad Sci USA 1985;82:8025–5029. 62 Ueki N, Sobue K, Kanda K, Hada T, Higashino K: Expression of high and low molecular weight caldesmons during phenotypic modulation of smooth muscle cells. Proc Natl Acad Sci USA 1987;84:9049–9053. 63 Lees-Miller JP, Helfman DM: The molecular basis for tropomyosin isoform diversity. Bioessays 1991;13:429–437. 64 Nagai R, Kuro-o M, Babij P, Periasamy M: Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis. J Biol Chem 1989; 264:9734–9737. 65 Kuro-o M, Nagai R, Nakahara K, Katoh H, Tsai RC, Tsuchimochi H, Yazaki Y, Ohkubo A, Takaku F: cDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in arteriosclerosis. J Biol Chem 1991; 266:3768–3773. 66 Kuro-o M, Nagai R, Tsuchimochi H, Katoh H, Yazaki Y, Ohkubo A, Takaku F: Developmentally regulated expression of vascular smooth muscle myosin heavy chain isoforms. J Biol Chem 1989;264:18272–18275. 67 Glukhova MA, Kabakov AE, Frid MG, Ornatsky OI, Belkin AM, Mukhin DN, Orekhov AN, Koteliansky VE, Smirnov VN: Modulation of human aorta smooth muscle cell phenotype: A study of muscle-specific variants of vinculin, caldesmon, and actin expression. Proc Natl Acad Sci USA 1988;85:9542–9546. 68 Moiseyeva EP, Weller PA, Zhidkova NI, Corben EB, Patel B, Jasinska I, Koteliansky VE, Critchley DR: Organization of the human gene encoding the cytoskeletal protein vinculin and the sequence of the vinculin promoter. J Biol Chem 1993;268:4318–4325. 69 Humphrey MB, Bryan J, Cooper TA, Berget SM: A 32-nucleotide exon-splicing enhancer regulates usage of competing 5) splice sites in a differential internal exon. Mol Cell Biol 1995; 15:3979–3988.

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70 Helfman DM, Cheley S, Kuismanen E, Finn LA, Yamawaki-Kataoka Y: Nonmuscle and muscle tropomyosin insoforms are expressed from a single gene by alternative RNA splicing and polyadenylation. Mol Cell Biol 1986;6: 3582–3595. 71 Libri D, Lemonnier M, Meinnel T, Fiszman MY: A single gene codes for the beta subunits of smooth and skeletal muscle tropomyosin in the chicken. J Biol Chem 1989;264:2935– 2944. 72 Wieczorek DF, Smith CW, Nadal-Ginard B: The rat alpha-tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth, and nonmuscle isoforms by alternative splicing. Mol Cell Biol 1988;8:679– 694. 73 Lemonnier M, Balvay L, Mouly V, Libri D, Fiszman MY: The chicken gene encoding the alpha isoform of tropomyosin of fast-twitch muscle fibers: Organization, expression and identification of the major proteins synthesized. Gene 1991;107:229–240. 74 Zamore PD, Patton JG, Green MR: Cloning and domain structure of the mammalian splicing factor U2AF. Nature 1992;355:609–614. 75 Mullen MP, Smith CW, Patton JG, NadalGinard B: Alpha-tropomyosin mutually exclusive exon selection: Competition between branchpoint/polypyrimidine tracts determines default exon choice. Genes Dev 1991;5:642– 655. 76 Herman IM, D’Amore PA: Microvascular pericytes contain muscle and nonmuscle actins. J Cell Biol 1985;101:43–52. 77 Adler KB, Craighead JE, Vallyathan NV, Evans JN: Actin-containing cells in human pulmonary fibrosis. Am J Pathol 1981;102:427– 437. 78 Schmitt-Graff A, Kruger S, Bochard F, Gabbiani G, Denk H: Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers. Am J Pathol 1991;138:1233–1242. 79 Alpers CE, Hudkins KL, Gown AM, Johnson RJ: Enhanced expression of ‘muscle-specific’ actin in glomerulonephritis. Kidney Int 1992; 41:1134–1142. 80 Ramadori G, Veit T, Schwogler S, Dienes HP, Knittel T, Rieder H, Meyer zum Buschenfelde KH: Expression of the gene of the alphasmooth muscle-actin isoform in rat liver and in rat fat-storing (ITO) cells. Virchows Arch B 1990;59:349–357. 81 Ando Y, Moriyama T, Miyazaki M, Akagi Y, Kawada N, Isaka Y, Izumi M, Yokoyama K, Yamauchi A, Horio M, Ando A, Udea N, Sobue K, Imai E, Hori M: Enhanced glomerular expression of caldesmon in IgA nephropathy and its suppression by glucocorticoid-heparin therapy. Nephrol Dial Transplant (in press).

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Hiroshi Fujiwara a Nobuhiko Kataoka a Tetsuro Honda a Masamichi Ueda b Shigetoshi Yamada a Kimihiko Nakamura e Hiroshi Suginami d Takahide Mori a Michiyuki Maeda c a

b c

d e

Department of Gynecology and Obstetrics, Faculty of Medicine, Virus Research Institute, Chest Disease Research Institute, Kyoto University, Kyoto National Hospital, Kyoto, and Department of Obstetrics and Gynecology, Kobe City General Hospital, Kobe, Japan

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Key Words Corpus luteum formation Extracellular matrix Follicular growth Integrin Ovary

Physiological Roles of Integrin ·6ß1 in Ovarian Functions

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Abstract We previously reported that human ovarian cells express integrin ß1 families. The physiological role(s) of integrins was investigated using in vitro human granulosa cell culture and in vivo mouse ovulation model. In human luteinizing granulosa cell culture obtained from the patients undergoing in vitro fertilization treatment, laminin, which is a ligand for integrin ·6ß1, suppressed the production of progesterone by granulosa cells. On the other hand, the anti-·6 monoclonal antibody (mAb) GoH3, which partially inhibits the interaction between integrin ·6ß1 and laminin, enhanced production of progesterone by 2-fold of the control under the culture with laminin, indicating that integrin ·6ß1 regulates the luteinization of human granulosa cell during the periovulatory phase. In an immature superovulated 13-day-old ICR (CD-1) mice model, intraperitoneal administration of GoH3 induced successful ovulation, whereas no ovulation was observed in the GoH3-nontreated groups, showing that integrin ·6ß1 is related to gonadotropin-induced follicular growth. These findings suggest that the interaction between integrin ·6ß1 and laminin plays an important role in the corpus luteum formation and follicular growth. OOOOOOOOOOOOOOOOOOOOOO

Introduction We have developed a monoclonal antibody (mAb), OG-1, which recognized a cell surface molecule of human granulosa cells [1]. The expression profiles of OG-1 antigen show that human large luteal cells are derived from granulosa cells and that luteal cells in pregnancy can be classified as a further differentiated stage of granulosa and thecal cells. N-terminal amino acid sequencing of the purified OG-1 antigen shows that it is identical to human integrin ·6 [2]. We also developed a mAb POG-2, recog-

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nizing a cell surface molecule of the porcine granulosa cells; POG-2 antigen is a porcine homologous form of OG-1 antigen, integrin ·6 [3]. With these mAbs, expression profiles of integrin ·6 are shown to be different between animal species. In the human ovary, integrin ·6 is expressed on granulosa cells of medium to large follicles, and on luteinizing granulosa cells in the early luteal phase. The expression level was the highest during the periovulatory stage. In contrast, in the porcine ovary, integrin ·6 was expressed on granulosa cells in the small follicles (1–2 mm in diameter) with maximal immunoreactivity,

Hiroshi Fujiwara, MD Department of Gynecology and Obstetrics Faculty of Medicine, Kyoto University Sakyo-ku, Kyoto 606-01 (Japan) Tel. +81 75 751 3269, Fax +81 75 761 3967, E-Mail [email protected]

Fig. 1. Human granulosa cells were cultured on LN-coated or collagen type IVcoated dishes with the treatment of GoH3 or control antibodies. A Granulosa cells cultured on LN-coated dishes. Progesterone production was higher in the group given GoH3 than in the controls, either with hCG or without hCG. B GoH3 had no effect on progesterone production by the granulosa cells cultured on collagen type IV-coated dishes. Bars indicate the mean B SEM.

whereas the integrin ·6 was undetectable in corpora lutea. Thus the stage-specific expression profiles suggest the involvement of integrin ·6 in ovarian physiology [4]. We have demonstrated that human granulosa cells express integrin ß1, but not integrin ß4, indicating that integrin ·6 expressed on granulosa cells forms a heterodimer with ß1 but not with ß4 [2]. The ligand for integrin ·6ß1 is laminin (LN) [5]. We found LN on human granulosa cells, and that LN was bound to the cell surface of some granulosa cells obtained from preovulatory follicles of patients undergoing in vitro fertilization (IVF) by flow cytometry and was detected between luteinizing granulosa cells in the early corpora lutea by immunohistochemistry [6]. Additionally, the expression of integrin ·5 and its ligand, fibronectin, has been shown to be rapidly induced after ovulation on human luteinizing granulosa cells [7]. Firstly, we investigated the physiological function of the interaction between integrin ·6ß1 and LN using in vitro human granulosa cell culture. Granulosa cells located in the inner layers of the follicles, which are not in contact with basal lamina, expressed integrin ·6 on the cell surface, although the highest expression stages of integrin ·6 on granulosa cells during follicular development are different between human and porcine ovaries [2, 3]. In the mouse follicles, integrin ·6 was detected on all layers of granulosa cells in the primordial, primary, and secondary follicles by immunohistochemistry [8]. Thus, we examined the effect of systemic administration of anti-integrin ·6 antibody on ovulation induction in the immature ICR mice by exogenous gonadotropin in vivo.

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Human Luteinizing Granulosa Cell Culture on LN Human granulosa cells were isolated from the patients who had undergone IVF treatment by Ficoll-Hypaque method as described previously [9]. Dishes were coated with the whole mouse LN fragments as described with minor modification [10]. The granulosa cells were suspended in DMEM/F-12 (1:1 v:v) medium containing 5% fetal calf serum (FCS) and 10 mM HEPES, inoculated on 96-well noncoated polystyrene, or mouse LN-coated dishes, and cultured in the absence or presence of hCG (1 IU/ml). After 24 h culture, there was no significant difference in cell morphology and the number of viable granulosa cells between the groups cultured on LN-coated or noncoated dishes. In the absence or presence of hCG, progesterone production cultured on LN-coated dishes was about 0.7-fold less than those on noncoated dishes (p ! 0.05) [6]. Granulosa cells were incubated with the antihuman and mouse integrin ·6 mAb, GoH3, which partially blocks the interaction between integrin ·6 and LN [6, 10], or with the control mAb at a final concentration of 5 mg/ml at 4 ° C for 30 min. The cells were inoculated on 96-well mouse LN- or mouse type IV collagen-coated dishes (Becton Dickinson Labware), in the absence or presence of hCG (1 IU/ml). After 24 h, there was no significant difference in cell morphology and the number of viable cells between the two groups. Progesterone production by granulosa cells cultured with GoH3 was about 2fold higher than that with control mAb when cultured both without and with hCG (fig. 1) [6]. The augmentation of progesterone production was not observed when granu-

Fujiwara/Kataoka/Honda/Ueda/Yamada/ Nakamura/Suginami/Mori/Maeda

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Superovulated Mouse Treated with Anti-Integrin ·6 Antibody

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Immunohistochemical study reveals that LN is rapidly expressed among luteinizing granulosa cells and its expression gradually decreases as granulosa cells become large luteal cells. Flow cytometrical analysis demonstrated that LN is conjugated with the cell surface of the granulosa cells isolated from the patients undergoing IVF treatment [6]. The expression of integrin ·6 is gradually decreased during luteinization, as well [1]. From the results of granulosa cell culture, LN suppresses the luteinization of human granulosa cells via interaction with integrin ·6ß1, indicating that integrin ·6ß1-LN interaction acts as a local inhibitory regulator of luteinization. It is reasonable that the intensity of integrin ·6ß1 expression on human luteinizing granulosa cells decreases from the early luteal to the midluteal phases [7, 8]. Granulosa cell luteinization is a process in which the capacity of progesterone production is augmented, coupled with the morphological changes of cytoplasmic organelles. Human granulosa cells gradually differentiate to large luteal cells within 5 days after ovulation. The serum level of progesterone gradually increases until it reaches a peak 5–6 days after LH surge, which corresponds to the period of embryo implantation. When this luteinization process is accelerated and the lifespan of corpus luteum is shortened, the steroid hormone-induced endometrial environment for embryo implantation is impaired. It is necessary for successful pregnancy to regulate the process of luteinization. In this regard, integrin ·6ß1 is proposed as a local regulator of granulosa cell luteinization during the periovulatory phase (fig. 2).

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Physiological Roles of Integrins in Corpus luteum Formation

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losa cells were cultured on mouse type IV collagen-coated dishes. OG-1 mAb, which reacts with integrin ·6 without inhibitory action, showed no effect on steroid production by granulosa cells.

Fig. 2. Regulatory mechanism of luteinization of granulosa cells by the interaction between integrin ·6ß1 and laminin. Integrin ·6ß1 suppresses the luteinization of human granulosa cells via interaction with laminin, acting as a local inhibitory regulator of luteinization during the corpus formation when the expression intensity of integrin ·6ß1 and laminin on luteinizing granulosa cells gradually decreases.

the other hand, ovulation was induced in the 15- and 16day-old mice [8]. The mice were divided into three groups, without the treatment of antibodies, with the treatment of GoH3 mAb, and with the treatment of control mAb. At 14:00 and 16:00 h on the 13th day, GoH3 mAb or control mAb or PBS without antibodies was intraperitoneally injected into immature mice. At 16:00 h on the 13th day, the development of multiple follicles was induced by an intraperitoneal injection of PMSG (10 IU), and at 16:00 h on the 15th day, hCG (5 IU) was intraperitoneally administrated to induce ovulation as described previously [11]. At 12:00 h on the 16th day, the mice were killed by cervical dislocation and the ovulated ova in the bilateral oviducts were examined. Ovulation rate was significantly higher in the group of GoH3 treatment than in the GoH3 nontreated groups (fig. 3) [8].

Physiological Roles of Integrins in Follicular Growth

Ten-day-old immature female ICR (CD-1 strain) mice were purchased from Charles River Japan Inc. (Kanagawa, Japan) and housed under normal controlled lighting (12 h of light, 12 h of darkness) with their lactating mothers. In 12- and 13-day-old mice, no ovulation was observed by PMSG (10 IU) and hCG (5 IU) stimulation. On

In general, follicular recruitment needs the selection and regulation of entry of the follicles to start follicular growth. If all the follicles react to gonadotropin stimulation and enter the follicular recruitment, the ovary rapidly loses available follicles and cannot maintain estrus or menstrual cycles. Our superovulated mouse experiments

Integrin ·6ß1 in Ovarian Function

Horm Res 1998;50(suppl 2):25–29

27

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Fig. 3. Effect of GoH3 administration on ovulation induction. The 13-day-old mice were superovulated by PMSG (10 IU) and hCG (5 IU) with the treatment of intraperitoneal injection of anti-integrin ·6 mAb (GoH3), control (CTR) mAb or PBS. The ovulation rate per animal was significantly higher in the group of GoH3 treatment than in the GoH3 nontreated groups (p ! 0.01).

indicated that binding of GoH3 mAb to integrin ·6 in the ovary enhanced follicular response against exogenous gonadotropins, suggesting the involvement of integrin ·6 in the responsiveness in immature follicles to gonadotropins [8]. There is evidence showing that rat granulosa cells produce LN by immunoelectron microscopy [12], and that bovine granulosa cells produce LN-B2 chain by Northern blotting [13]. Thus, murine granulosa cells, including not only in a basal layer, but also in inner layers, may produce LN and physiologically interact with LN via integrin ·6ß1 in the follicles [4]. Integrins cooperate with other classes of cell surface receptors to regulate cell growth and function; integrins and platelet-derived growth factor receptors cooperate in the regulation of the Na+/H+ antiporter [14]. Several extracellular matrices have been shown to exert differential effects on apoptosis and steroidogenesis of cultured rat mature granulosa cells, showing that LN suppressed progesterone production by rat mature granulosa cells [15]. Therefore, integrins expressed on granulosa cells provide a new category of regulators, which maintain the stability of the follicles and regulate the follicular recruitment (fig. 4). The critical periods for follicular selection are different among species. This may explain the reason why the high expression stages of integrin ·6 on granulosa cells are different among human, porcine, and murine follicles [1, 3, 8].

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Fig. 4. Proposed physiological roles of integrin ·6ß1 in follicular selection. Integrins expressed on granulosa cells may maintain the stability of the follicles and regulate the follicular recruitment.

Conclusions We demonstrated that integrin ·6 is involved in ovarian function. In the mouse follicles, integrin ·6 regulates the responsiveness of the follicles to gonadotropins. On the other hand, it controls the process of luteinization of human granulosa cells. Thus, integrin provides a new candidate for local regulators which control follicular recruitment and corpus luteum formation. This concept may contribute to further clarification of the regulation mechanism of ovarian function.

Acknowledgment This work was supported in part by Grants-in-Aid for Scientific Research (Nos. 09671673, 09671674, 09671676).

Fujiwara/Kataoka/Honda/Ueda/Yamada/ Nakamura/Suginami/Mori/Maeda

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References 1 Fujiwara H, Maeda M, Ueda M, Fukuoka M, Yasuda K, Imai K, Takakura K, Mori T: A differentiation-related molecule on the cell surface of human granulosa cells. J Clin Endocrinol Metab 1993;76:956–961. 2 Honda T, Fujiwara H, Ueda M, Maeda M, Mori T: Integrin ·6 is a differentiation antigen of human granulosa cells. J Clin Endocrinol Metab 1995;80:2899–2905. 3 Fujiwara H, Ueda M, Takakura K, Mori T, Maeda M: A porcine homolog of human integrin ·6 is a differentiation antigen of granulosa cells. Biol Reprod 1995;53:407–417. 4 Fujiwara H, Maeda M, Honda T, Yamada S, Ueda M, Kanzaki H, Suginami H, Mori T: Granulosa cells express integrin ·6: Possible involvement of integrin ·6 on folliculogenesis. Horm Res 1996;46(suppl 1):24–30. 5 Hynes RO: Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 1992;69: 11–25. 6 Fujiwara H, Honda T, Ueda M, Nakamura K, Yamada S, Maeda M, Mori T: Laminin suppresses progesterone production by human luteinizing granulosa cells via interaction with integrin ·6ß1. J Clin Endocrinol Metab 1997; 82:2122–2128.

Integrin ·6ß1 in Ovarian Function

7 Honda T, Fujiwara H, Yamada S, Fujita K, Nakamura K, Nakayama T, Higuchi T, Ueda M, Maeda M, Mori T: Integrin ·5 is expressed on human luteinizing granulosa cells during corpus luteum formation, and its expression is enhanced by hCG in vitro. Mol Hum Reprod 1997;3:979–984. 8 Nakamura K, Fujiwara H, Higuchi T, Honda T, Nakayama T, Kataoka N, Fujita K, Ueda M, Maeda M, Mori T: Integrin ·6 is involved in follicular growth in mice. Biochem Biophys Res Commun 1997;235:524–528. 9 Fujiwara H, Fukuoka M, Yasuda K, Ueda M, Imai K, Goto Y, Suginami H, Kanzaki H, Maeda M, Mori T: Cytokines stimulate dipeptidyl peptidase-IV expression on human luteinizing granulosa cells. J Clin Endocrinol Metab 1994; 79:1007–1011. 10 Sonnenberg A, Linders CJT, Modderman PW, Damsky CH, Aumailley M, Timpl R: Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that ·6ß1 but not ·6ß4 functions as major laminin receptor for fragment E8. J Cell Biol 1990;110: 2145–2155.

11 Nakamura K, Fujiwara H, Nakayama T, Goto Y, Tachibana T, Suginami H, Ueda M, Maeda M, Mori T: An aminopeptidase inhibitor, bestatin, enhances gonadotropin-stimulated ovulation in mice. Hum Reprod 1996;11:1952– 1957. 12 Leardkamolkarn V, Abrahamson DR: Immunoelectron microscopic localization of laminin in rat ovarian follicles. Anat Rec 1992;233:41– 52. 13 Zhao Y, Luck MR: Gene expression and protein distribution of collagen, fibronectin and laminin in bovine follicles and corpora lutea. J Reprod Fertil 1995;104:115–123. 14 Schwartz MA, Lechene C: Adhesion is required for protein kinase C-dependent activation of the Na+/H+ antiporter by PDGF. Proc Natl Acad Sci USA 1992;89:6138–6141. 15 Aharoni D, Meiri I, Atzmon R, Viodavsky I, Amsterdam A: Differential effect of components of extracellular matrix on differentiation and apoptosis. Curr Biol 1996;7:43–51.

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29

Horm Res 1998;50(suppl 2):30–36

Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu City, Japan

Significance of Sex Steroids in Roles of Cadherin Subfamily and Its Related Proteins in the Uterine Endometrium and Placenta

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Key Words Cadherin Catenin Sex steroids Vessel permeability

Abstract To know the biological functions of the adherens junction in uterine endometrium, mRNA expressions of E-cadherin and ·- and ß-catenin, which mainly comprise the adherens junction, were determined. Furthermore, to understand various functions related to V-cadherin in the placenta, vessel permeability was determined by assessing V-cadherin mRNA expression in HUVEC-C cells, derived from the endothelial cells in human umbilical cord. The levels of E-cadherin and ·- and ß-catenin mRNAs in the endometria of the proliferative phase were significantly less than those of the secretory phase. Treatment with estradiol dipropionate significantly reduced their levels in the endometria of the secretory phase. These suggest that the functions of the adherens junction in endometrial epithelial cell are controlled by sex steroids. On the other hand, estradiol decreased the endothelial cell barrier properties in HUV-EC-C cells, whereas progesterone partly reversed the changes induced by estradiol. While estradiol decreased the level of V-cadherin mRNA in HUV-EC-C cells, progesterone partly reversed the level decreased by estradiol. Therefore, sex steroids play a role in placental development and function related to cadherins on the endothelial cells, probably via placental vessel permeability.

Jiro Fujimoto Hideki Sakaguchi Reiko Hirose Teruhiko Tamaya

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Introduction The adherens junction consists of transmembranous Ecadherin, intracellular actin filament-attachment proteins (·- and ß-catenins, vinculin, actinin), and actin cytoskeleton filament, and regulates the function of cell-to-cell adhesion [1]. E-cadherin exists as a cell-to-cell homophilic adhesion molecule at the adherens junction [2, 3], and has a catenin binding site [4–6], which binds to ß-catenin complexed with ·-catenin as a regulatory part [7, 8]. The

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expression of wild-type E-cadherin family is essential for the normal function of the adherens junction [9], and dysfunction of the adherens junction in cancers of the female genital tract has been demonstrated [10–13]. Furthermore, homophilic cell-to-cell adhesion activated by cadherin might contribute to morphogenesis in reproduction [14]. And, it is known that in reproductive tissues, sex steroids alter the expression of E-cadherin [15, 16]. These phenomena prompted us to study the functional role of cadherins and their related proteins in the human endo-

Jiro Fujimoto, MD, PhD Department of Obstetrics and Gynecology Gifu University School of Medicine 40 Tsukasa-machi, Gifu City 500-8705 (Japan) Tel. +81 58 267 2631, Fax +81 58 265 9006

metrium and placenta. To study the significance of reproductive functions in endometria and placenta, we determined the effect of sex steroids on E-cadherin and ·- and ß-catenin mRNA expressions in endometria during the menstrual cycle and by estrogen administration. We also investigated the effect of sex steroids on cell-to-cell adhesion analyzing cell aggregation and E-cadherin mRNA expression in 3A(tPA-30-1) cells, derived from human term placenta and transformed by SV40 as well as on vessel permeability analyzing endothelial cell barrier properties (ECBP) and V-cadherin mRNA expression in HUVEC-C cells, derived from the endothelial cells in human umbilical cord, as a substitute for the placenta [17].

Patients and Methods Patients Consents for the studies were obtained from all patients and the Research Committee for Human Subjects, Gifu University School of Medicine. Twenty patients (32–45 years of age), with a regular menstrual cycle confirmed by basal body temperature, underwent hysterectomy for uterine huge leiomyoma without hypermenorrhea at the Department of Obstetrics and Gynecology, Gifu University School of Medicine, during November 1993 through December 1994. None of the patients had received any hormonal therapy if not indicated. Five patients with their consent were given the intramuscular injection of 10 mg estradiol dipropionate (Ed) 5 days before hysterectomy in the luteal phase of the menstrual cycle in order to observe the effect of estradiol on the levels of E-cadherin and ·- and ß-catenin mRNA expression. The uterine endometrium was obtained immediately after hysterectomy. A part of each endometrium was snapfrozen in liquid nitrogen to determine the levels of their mRNA expressions, and a neighboring part of the endometrium was submitted for histological endometrial dating [18]. Chemicals 17ß-Estradiol and progesterone were purchased from Sigma Chemical Co. (St. Louis, Mo., USA). All other chemicals and reagents were of the experimental grade. These hormones were solubilized in ethanol, and added to the culture media to obtain a final concentration of ethanol below 0.1%.

lized. Afterwards, the culture was proceeded in the conventional medium without FBS or phenol red. Forty-eight hours later, estradiol or progesterone alone and estradiol plus progesterone were added to culture dishes in the concentrations indicated in the legends for the corresponding figures. Cell Aggregation Activity Bioassay of this activity is designed to determine the function of cell-to-cell adhesion, namely the activity of adhesion molecules [10– 13]. Cells (1.5 ! 105 cells) dispersed by treatment with 0.01% trypsin and 1 mM CaCl2 (conservable of the expression of cadherin on the cell membrane) were seeded in HCMF buffer [1 mM CaCl2, 150 mM NaCl, 5.5 mM KCl, 5.5 mM glucose, 0.35 mM Na2HPO4 W 12H2O, 10 mM N - (2 - hydroxyethyl)piperazine - N) - 2 - ethanesulfonic acid (HEPES), pH 7.4] on a 24-well plate coated with 1% albumin. The plate was rocked at 80 rpm at 37 ° C for 60 min. The number of cell clusters and single cells was counted in 5 high-power fields using a phase-difference microscope. Aggregation index was defined as the ratio of the sum number of single cells and cell clusters to the initial number of dispersed cells [19, 20]. Measurement of Endothelial Cell Barrier Properties [21, 22] The upper face of filters in a Chemotaxicell (Krabo Biochemical, Osaka, Japan) was coated with 10 Ìg/ml human fibronectin (60 Ìl/ filter) for 1 h at room temperature, and rinsed with serum-free F12 K medium. Dispersed cells (105 cells) in 200 Ìl of the conventional medium (90% F12 K medium and 10% FBS with 100 Ìg/ml heparin and 30 Ìg/ml ECGS) were seeded on the upper compartment of the filter. The lower compartment was filled with 600 Ìl of the conventional medium. The culture was continued for 5 days with daily refeeding. Before the experiment, the culture medium in both the upper and lower compartments was replaced with serum-free and phenol red-free medium. Horseradish peroxidase (HRP) of 22.7 ÌM (1.1 Ìl) was added to the upper compartment as a final concentration of 0.126 ÌM. Thirty minutes later, 30 Ìl of culture medium in the lower compartment was transferred to a 2-ml-tube containing 860 Ìl of a reaction buffer (50 mM NaH2PO4 and 5 mM guaiacol). The reaction to measure the concentration of HRP was started by adding 100 Ìl H2O2 and was proceeded for 25 min at room temperature, then the absorbance was measured at 470 nm.

Cell Culture 3A(tPA-30-1) cells (ATCC CRL 1583), derived from human term placenta and transformed by SV40, were cultured in 90% ·MEM and 10% fetal bovine serum (FBS). The cells express transformed phenotype at permissive temperature (33 ° C) and nontransformed phenotype at nonpermissive temperature (40 ° C). Therefore, 3A(tPA-30-1) cells were cultured before experiments at 40 ° C. HUV-EC-C cells (ATCC CRL 1730), derived from the endothelial cells in human umbilical cord, were cultured in 90% F12 K medium and 10% FBS with 100 Ìg/ml heparin and 30 Ìg/ml endothelial cell growth supplement (ECGS, Harbor Bio-Products, Norwood, Mass., USA). ECGF is a partially purified preparation from bovine hypothalamus. The product is lyophilized from a phosphate-buffered saline solution (pH 7.4) and is sterile filtered (0.22 Ìm) and lyophi-

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) to Amplify the mRNAs for E- and V-Cadherins and ·- and ß-catenins Total RNA was isolated from the tissues by the acid guanidium thiocyanate-phenol-chloroform extraction method [23]. Total RNA (3 Ìg) was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (MMLV-RTase, 200 units; Gibco BRL, Gaithersburg, Md., USA) in a buffer of 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl2, 0.1 mg/ml bovine serum albumin, 10 mM dithiothreitol, and 0.5 mM deoxynucleotides to generate cDNAs using random hexamer (50 ng; Gibco BRL) at 37 ° C for 60 min. The RT reaction mixture was heated at 94 ° C for 5 min to inactivate MMLV-RTase. To determine the level of E-cadherin and ·- and ß-catenin mRNAs, PCR (denaturation for 1 min at 94 ° C, annealing for 1 min at 55 ° C, and extension for 1 min at 72 ° C) was carried out with reverse transcribed cDNAs and 0.1 ÌM specific primers using the Iwaki thermal sequencer TSR-300 (Iwaki Glass, Tokyo, Japan), with Vent DNA polymerase (New England Biolabs, Beverly, Mass., USA)

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31

E-cadherin

Fig. 1. Effects of sex steroids on the expression of E-cadherin and ·- and ß-catenin mRNAs in uterine endometria. The levels of E-cadherin, ·- and ß-catenin mRNAs were standardized with that of GAPDH mRNA. The mean mRNA levels in the endometria of the early/mid proliferative phase in the menstrual cycle were assigned as arbitrary units/GAPDH mRNA (AU/ GAPDH mRNA). The dark shaded area is the composite pattern [18]. PP = Proliferative phase; SP = secretory phase; Ed = estradiol dipropionate (10 mg) was given intramuscularly 5 days before hysterectomy in the luteal phase. *1 p ! 0.05 versus PP; *2 p ! 0.05 versus SP.

in a buffer of 10 mM KCl, 20 mM Tris-HCl, pH 8.8, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, and 0.15 mM deoxynucleotide phosphates for 25 cycles. PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a housekeeping gene) mRNA was simultaneously performed in the same manner as an internal standard. The oligodeoxynucleotides of specific primers in PCR for reverse transcribed RNA isolated from uterine endometrium were synthesized according to the published information on cDNA for E-cadherin [24], ·-catenin (Genbank M94151), ß-catenin (Genbank Z19054), and GAPDH [25] as follows:

-catenin

-catenin

✽

✽ ✽

✽ ✽

✽

sense primer for E-cadherin mRNA, 5)-CCATCAGCTGCCCAGAAAAT-3); antisense primer for E-cadherin mRNA, 5)-TTGGATGACACAGCGTGAGA-3); sense primer for V-cadherin mRNA, 5)-TGGAACCAGATGCACATTGA-3); antisense primer for V-cadherin mRNA, 5)-AAGCTGGAAGGAGTCTCCAG-3); primers for GAPDH mRNA, the same as above.

sense primer for GAPDH mRNA, 5)-TGAAGGTCGGAGTCAACGGATTTGGT-3); antisense primer for GAPDH mRNA, 5)-CATGTGGGCCATGAGGTCCACCAC-3).

Southern Blot Analysis for Quantities of mRNA Expressions for E- and V-Cadherins and ·- and ß-Catenins PCR products were applied to 1.2% agarose gel, and electrophoresis was performed at 50–100 V. PCR products were capillary-transferred to an Immobilon transfer membrane (Millipore Corp., Bedford, Mass., USA) for 16 h. The membrane was dried at 80 ° C for 30 min, and was UV-irradiated to tightly fix PCR products. PCR products on the membrane were prehybridized in a buffer of 1 M NaCl, 50 mM Tris-HCl, pH 7.6, and 1% sodium dodecyl sulfate at 42 ° C for 1 h, and hybridized in the same solution with the biotinylated oligodeoxynucleotide probes synthesized from the sequences of cadherins, catenins and GAPDH cDNAs between the specific primers at 65 ° C overnight. Specific bands hybridized with the biotinylated probes were detected with Plex Luminescent Kits (Millipore Corp.), and Xray film was exposed on the membrane at room temperature for 10 min. The quantification of Southern blot was carried out with Bio Image (Millipore, Ann Arbor, Mich., USA). The intensity of specific bands was standardized with that of GAPDH mRNA.

The oligodeoxynucleotides of specific primers in PCR for reverse transcribed RNA isolated from 3A(tPA-30-1) and HUV-EC-C cells were synthesized according to the published information on cDNA for E-cadherin [27], V-cadherin [26] and GAPDH [25] as follows:

Statistics Statistical analysis was performed with Student’s t-test and oneway ANOVA; differences were considered significant when p was ! 0.05.

sense primer for E-cadherin mRNA, 5)-AGTGCCAACTGGACCATTCA-3); antisense primer for E-cadherin mRNA, 5)-TCTTTGACCACCGCTCTCCT-3); sense primer for ·-catenin mRNA, 5)-CAGAGGGAGCATGACTTCGG-3); antisense primer for ·-catenin mRNA, 5)-CTACAGCAGCCACCAACTCT-3); sense primer for ß-catenin mRNA, 5)-TTGAAAATCCAGCGTGGACA-3); antisense primer for ß-catenin mRNA, 5)-TCGAGTCATTGCATACTGTC-3);

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Fujimoto/Sakaguchi/Hirose/Tamaya

Fig. 2. Effects of sex steroids on ECBP of HUV-EC-C cells. HUVEC-C cells on the filter in a Chemotaxicell were incubated in the serum-free and phenol red-free conventional medium with 10 –8 M estradiol, 10 –8 M estradiol plus 10 –6 M progesterone, and 10 –6 M progesterone for 1 h. Thirty minutes after addition of HRP to the upper compartment, the quantity of HRP transferred to the lower compartment was determined. Data are the mean B SD of 6 determinations. E2 = 17ß-Estradiol; P = progesterone, * p ! 0.05 versus controls; ** p ! 0.1 versus treated with only E2.

Results The mRNA expression levels were determined within the linear range in the signal intensity curve by RT-PCRSouthern blot analysis [27, 28]. Therefore, semiquantitative alternation of the mRNA levels was thought to be reliable. Effects of Sex Steroids on the Expression of E-Cadherin and ·- and ß-Catenin mRNAs in Endometria The levels of E-cadherin and ·- and ß-catenin mRNAs in endometria of the early/mid proliferative phase and the late proliferative phase were significantly (p ! 0.05) less than those of the secretory phase (SP). The Ed treatment significantly (p ! 0.05) reduced the levels of ·- and ß-catenin mRNAs in endometria of the SP, and tended to reduce the E-cadherin mRNA levels (fig. 1).

Cadherin in Female Reproduction

Fig. 3. Effects of sex steroids on cell aggregation of HUV-EC-C and 3A(tPA-30-1) cells. The cells were incubated in the serum-free and phenol red-free conventional medium with 10 –8 M estradiol, 10 –8 M estradiol plus 10 –6 M progesterone, and 10 –6 M progesterone for 6 h. After the cells were rocked, the aggregation index was determined. Data are the mean B SD of 6 determinations. E2 = 17ß-Estradiol; P = progesterone.

Effects of Sex Steroids on ECBP of HUV-EC-C Cells Estradiol decreased the ECBP of HUV-EC-C cells dose-dependently up to 10 –8 M in 1 h, while progesterone at 10 –6 M partly reversed the estradiol-induced decrease of ECBP in 1 h [28]. Thus the concentration of 10 –8 M estradiol or 10 –6 M progesterone was selected for the following experiments. Estradiol significantly (p ! 0.05) decreased the ECBP in 1 h, and the addition of progesterone tended (p ! 0.1) to reverse the estradiol-induced decrease of ECBP. Progesterone alone did not demonstrate any effect on the ECBP (fig. 2). Effects of Sex Steroids on Cell Aggregation of 3A(tPA-30-1) and HUV-EC-C Cells In the dose-response curve and time course for the effect of sex steroids on the cell aggregation index of 3A(tPA-30-1) and HUV-EC-C cells [28], neither estradiol nor progesterone demonstrated any effect on the activity of cell aggregation (fig. 3).

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of E-cadherin mRNA in 3A(tPA-30-1) cells [28], neither estradiol nor progesterone demonstrated any effect (fig. 4).

Discussion

Fig. 4. Effects of sex steroids on the expression of E-cadherin mRNA in 3A(tPA-30-1) cells and V-cadherin mRNA in HUV-EC-C cells. The cells were incubated in the serum-free and phenol red-free conventional medium with 10 –8 M estradiol, 10 –8 M estradiol plus 10 –6 M progesterone, and 10 –6 M progesterone for 1 h. Then RTPCR-Southern blot analysis was carried out. Data are the mean B SD of 6 determinations. E2 = 17ß-Estradiol; P = progesterone, * p ! 0.05 versus controls; ** p ! 0.1 versus treated with only E2.

Effects of Sex Steroids on the Expression of E-Cadherin mRNA in 3A(tPA-30-1) Cells and V-Cadherin mRNA in HUV-EC-C Cells Estradiol decreased the level of V-cadherin mRNA dose-dependently up to 10 –8 M in 1 h, while progesterone at 10 –6 M reversed the estradiol-induced decrease of Vcadherin mRNA in 1 h [28]. The concentration of 10 –8 M estradiol or 10 –6 M progesterone was selected for the following experiments. Estradiol significantly (p ! 0.05) decreased the level of V-cadherin mRNA in 1 h, and the addition of progesterone tended (p ! 0.1) to reverse the estradiol-induced decrease of V-cadherin mRNA. Progesterone alone did not demonstrate any effect on the V-cadherin mRNA level (fig. 4). In the dose-response curve and time course for the effect of sex steroids on the level

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As a cell-to-cell adhesion molecule, E-cadherin contains cell adhesion recognition (HAV) sequences at the Nterminus for homophilic binding of the adherens junction [4], peptide B (KVSATDADDDVLL) for Ca2+ dependent binding [7], and an exon 16-encoding catenin binding site for catenin dominant cell-to-cell adhesion activity [5]. Ecadherin, a direct extracellular binding site, binds to ßcatenin, which further binds to ·-catenin; ·-catenin comprises a regulatory part in the adherens junction [6, 9]. The role of cadherins for conversion, rearrangement and separation between epithelium and nonepithelium in morphogenesis can be demonstrated from the evidence related to E-cadherin (or P-cadherin) and N-cadherin switch-on and -off mechanisms [29, 30]. In endometrium, the levels of E-cadherin and ·- and ß-catenin mRNA increased after ovulation, but reduced by the estrogen treatment, suggesting that the function of the adherens junction in the endometrium is regulated by sex steroids. Estrogen reduces cell-to-cell adhesive function and progesterone stimulates it. From the clinical aspect, the functions of the adherens junction to regulate the adhesive capacity in endometrial epithelial cells may be activated after ovulation. There are some related phenomena: estrogen stimulates the expression of E-cadherin in rat granulosa cells [15]; progesterone induces the expression of a cadherin-like protein, which leads to oocyte maturation in fertilized Xenopus oocyte [16]. In the well-differentiated endometrial cancer cell line Ishikawa, estrogen suppresses the function of the adherens junction and the expressions of E-cadherin and ·- and ß-catenin mRNAs, while progesterone reverses the estrogen-suppressed events [31]. This endometrial cancer cell line might conserve the regulatory mechanism of the adherens junction under the influence of sex steroids as does the normal endometrium. Therefore, this steroidal action could have some effect on the first step of invasion and metastasis in endometrial cancers. Endometriotic cells, although derived from normal endometrium, appear to possess the potential for detachment and invasion to the intra-abdominal linings of pelvic organs, likely as a behavior of cancer cells [32–34]. There is no alteration in E-cadherin or ·- and ß-catenin mRNA expressions in ovarian endometriosis during the menstrual cycle [35],

Fujimoto/Sakaguchi/Hirose/Tamaya

however, those levels in normal endometria increase after ovulation [27]. These data suggest that progesterone secreted after ovulation might upregulate the adherens junction in the normal endometria, and that progesterone effects might be less demonstrated in endometriotic tissue, probably due to the lesser contents of progesterone receptor [36]. In addition, the level of ·-catenin mRNA in ovarian endometriosis is lower than that in normal endometria regardless of the stage of the menstrual cycle, and the expression of E-cadherin and ß-catenin mRNAs decreases in the endometriotic cells [35]. This evidence corroborates the potential of detachment and invasiveness of endometriotic cells due to a defect in E-cadherin family adhesion molecules, especially during the luteal phase. On the other hand, V-cadherin does not conserve the amino acid sequence ‘HAV’ in the first extracellular domain, specifically related to homophilic cell-to-cell adhesion [26]. Specific expression of V-cadherin is seen in endothelial cells of human umbilical vein and placenta

[37]. V-cadherin, consisting of interendothelial junctions, plays a role in vessel permeability [21]. In the present study, the influence of sex steroids on vessel permeability and cell aggregation activity related to cadherins in 3A(tPA-30-1) as trophoblastic cells and in HUV-EC-C cells as endothelial cells in the placenta. 3A(tPA-30-1) cells expressed E-cadherin mRNA, but not V-cadherin mRNA. Sex steroids did not alter the expression of E-cadherin mRNA or cell aggregation as an index of cell adhesion potential in 3A(tPA-30-1) cells [28]. On the other hand, HUV-EC-C cells expressed V-cadherin mRNA, but not E-cadherin mRNA. In addition, estrogen decreased the expression of V-cadherin mRNA and increased vessel permeability, while progesterone reversed the estrogen-induced events. Therefore, it appears likely that sex steroids play a role in placental development and function related to endothelial V-cadherin, probably via vessel permeability in the placenta.

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References 1 Takeichi M: Cadherin cell adhesion receptor as a morphigenetic regulator. Science 1991;251: 1451–1455. 2 Blacschuk OW, Sullivan R, David S, Pouliot Y: Identification of a cadherin cell adhesion recognition sequence. Dev Biol 1990;139:227–229. 3 Ozawa M, Engle J, Kemler R: Single amino acid substitutions in one Ca2+ binding site of uvomorulin abolish the adhesive function. Cell 1990;63:1033–1038. 4 Ozawa M, Ringwald M, Kemler R: Uvomorulin-catenin complex formation is regulated by a specific domain in the cytoplasmic region of the cell adhesion molecule. Proc Natl Acad Sci USA 1990;87:4246–4250. 5 Nagafuchi A, Takeichi M: Cell binding function of E-cadherin is regulated by the cytoplasmic domain. EMBO J 1988;7:3679–3684. 6 Nagafuchi A, Takeichi M: Transmembrane control of cadherin-mediated cell adhesion: A 94 kDa protein functionally associated with a specific region of the cytoplasmic domain of Ecadherin. Cell Regul 1989;1:37–44. 7 Ozawa M, Kemler R: Molecular organization of the uvomorulin-catenin complex. J Cell Biol 1992;116:989–996. 8 Matsuyoshi N, Hamaguchi M, Taniguchi S, Nagafuchi A, Tsukita Sh, Takeichi M: Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts. J Cell Biol 1992;118:703–714. 9 Nagafuchi A, Takeichi M, Tsukita Sh: The 102 kD cadherin-associated protein: Similarity to vinculin and posttranscriptional regulation of expression. Cell 1991;65:849–857.

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10 Fujimoto J, Ichigo S, Hirose R, Sakaguchi H, Tamaya T: Expression of E-cadherin and ·and ß-catenin mRNAs in uterine cervical cancers. Tumor Biol 1997;18:206–212. 11 Fujimoto J, Ichigo S, Hirose R, Sakaguchi H, Tamaya T: Expression of E-cadherin, and ·and ß-catenin mRNAs in ovarian cancers. Cancer Lett 1997;115:207–212. 12 Fujimoto J, Ichigo S, Hori M, Misao R, Hirose R, Tamaya T: Effect of sex steroids on cell-tocell adhesion molecules in various endometrial cells of the uterus, and their biological implication; in Kuramoto H, Gurpide E (eds): In vitro Biology of Sex Steroid Hormone Action. Tokyo, Churchill Livingstone, 1996, pp 17–30. 13 Fujimoto J, Ichigo S, Hirose R, Sakaguchi H, Tamaya T: Suppression of E-cadherin and ·and ß-catenin mRNA expression in metastatic lesions of gynecological cancers. Eur J Gynaecol Oncol 1997;18:484–487. 14 Takeichi M: The cadherins: Cell-cell adhesion molecules controlling animal morphogenesis. Development 1988;102:639–655. 15 Blasschuk OW, Farookhi R: Estradiol stimulates cadherin expression in rat granulosa cells. Dev Biol 1989;136:564–567. 16 Choi YS, Sehgal R, McCrea P, Gumbinder B: A cadherin-like protein in eggs and cleaving embryos of Xenopus laevis is expressed in oocytes in response to progesterone. J Cell Biol 1990;110:1575–1582. 17 Kaufman P: Functional anatomy of the nonprimate placenta. Placenta 1981;1(suppl):3– 28.

18 Noyes RW, Hertis AT, Rock J: Dating the endometrial biopsy. Fertil Steril 1950;1:3–25. 19 Urushihara H, Ozaki HS, Takeichi M: Immunological detection of cell surface components related with aggregation of Chinese hamster and chick embryonic cells. Dev Biol 1979;70: 206–216. 20 Matsuyoshi N, Hamaguchi M, Taniguchi S, Nagafuchi A, Tsukita S, Takeichi M: Cadherinmediated cell-cell adhesion is perturbed by vsrc tyrosine phosphorylation in metastatic fibroblasts. J Cell Biol 1992;118:703–714. 21 Lampugnani MG, Resnati M, Raiteri M, Pigott R, Pisacane A, Houen G, Ruco LP, Dejana E: A novel endothelial-specific membrane protein is a marker of cell-cell contacts. J Cell Biol 1992;118:1511–1522. 22 Ortiz de Montellano PR, David SK, Ator MA, Tew D: Mechanism-based inactivation of horseradish peroxidase by sodium azide. Formation of meso-azidoprotoporphyin. IX. Biochemistry 1988;27:5470–5476. 23 Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–159. 24 Bussemakers MJG, van Bokhoven A, Kemler MR, Schalken JA: Molecular cloning and characterization of human E-cadherin cDNA. Mol Biol Rep 1933;17:123–128. 25 Arcari P, Martinelli R, Salvatore F: The complete sequences of a full length cDNA for human liver glyceraldehyde-3-phosphate dehydrogenase: Evidence for multiple mRNA species. Nucleic Acids Res 1984;12:9179–9189.

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26 Suzuki S, Sano K, Tanihara H: Diversity of the cadherin family: Evidence for eight new cadherins in nervous tissue. Cell Regul 1991;2: 261–270. 27 Fujimoto J, Ichigo S, Hori M, Tamaya T: Alteration of E-cadherin, ·- and ß-catenin mRNA expression in human uterine endometrium during the menstrual cycle. Gynecol Endocrinol 1996;10:187–191. 28 Fujimoto J, Sakaguchi H, Hirose R, Tamaya T: Sex steroidal regulation of vessel permeability associated with vessel endothelial cadherin (Vcadherin). J Steroid Biochem Med Biol, in press. 29 Hatta K, Takagi S, Fujisawa H, Takeichi M: Spatial and temporal expression pattern of Ncadherin cell adhesion molecule correlated with morphogenetic process of chicken embryos. Dev Biol 1987;120:215–227.

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30 Bronner-Fraser M, Wolf JJ, Murray BA: Effects of antibodies against N-cadherin and NCAM on the cranial neural crest and neural tube. Dev Biol 1992;153:291–301. 31 Fujimoto J, Ichigo S, Hori M, Morishita S, Tamaya T: Progestins and danazol effect on cell-to-cell adhesion, and E-cadherin and ·and ß-catenin mRNA expressions. J Steroid Biochem Mol Biol 1996;57:275–282. 32 Shinoyama Y, Hirohashi S: Expression of Eand P-cadherin in gastric carcinomas. Cancer Res 1991;51:2185–2192. 33 Oda T, Kanai Y, Shimoyama Y, Nagafuchi A, Tsukita Sh, Hirohashi S: Cloning of the human ·-catenin cDNA and its aberrant mRNA in human cancer cell line. Biochem Biophys Res Commun 1993;193:897–904.

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34 Shinoyama Y, Nagafuchi A, Fujita S, Gotoh M, Takeichi M, Tsukita Sh, Hirohashi S: Cadherin dysfunction in a human cancer cell line: Possible involvement of loss of ·-catenin expression in reduced cell-cell adhesiveness. Cancer Res 1992;52:5770–5774. 35 Fujimoto J, Ichigo S, Hori M, Tamaya T: Expression of E-cadherin, and ·- and ß-catenin mRNAs in ovarian endometriosis. Eur J Obstet Gynecol 1996;67:179–183. 36 Tamaya T, Motoyama T, Ohno Y, Ide N, Tsurusaki T, Okada H: Steroid receptor levels and histology of endometriosis and adenomyosis. Fertil Steril 1979;31:398–400. 37 Muller WA, Ratti CM, McDonnell SL, Cohn ZA: A human endothelial cell-restricted, externally disposed plasmalemmal protein enriched in intercelluar junctions. J Exp Med 1989;170: 399–414.

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Studies on the Role of Adhesive Proteins in Maintaining Pregnancy

Toshihiko Asahina Takao Kobayashi Yoshichika Okada Mariko Itoh Miwa Yamashita Yutaka Inamoto Toshihiko Terao Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Japan

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Key Words Early pregnancy Fibrinogen Factor X III Fibronectin Macrophage

Abstract It is well known that maternal fibrinogen (Fg) and factor X III are essential for maintaining early pregnancy. We studied their role by analysis of clinical reports and immunohistochemical investigation. Methods: (1) We analyzed the pregnancy cases of congenital afibrinogenemia and congenital factor X III deficiency. (2) Immunohistochemical staining of Fg, subunit A of factor X III (X IIIA) and fibronectin (Fn) were performed in the human implantation site, placenta, and endometrial cells cultured in serum-free medium. Results: (1) Afibrinogenemia needed to be administrated Fg from 4 weeks’ gestation (4 wG), and factor X III deficiency needed factor X III concentrate from 5 wG, in order to prevent abortion. (2) Implantation tissues: Fg, cellular X IIIA and Fn were present at the decidual stroma around invasive cytotrophoblasts at 5 wG. X IIIA-positive cells coincided with LN-5-positive macrophages. Placenta: Fg, cellular X IIIA and Fn were present in the decidual layer. Endometrial culture cells: Fn was secreted by spindle-like shaped cells. X IIIA was secreted by round-shaped cells. Conclusion: Maternal Fg and factor X III are essential just after 4F5 wG, and in that period they and Fn are present abundantly in decidual stroma around invasive cytotrophoblasts. It is concluded that when cytotrophoblasts invade endometrium maternal Fg, factor X III and Fn are concerned with cytotrophoblasts’ anchoring as adhesive proteins. OOOOOOOOOOOOOOOOOOOOOO

Introduction Several adhesive factors, such as fibronectin (Fn), laminin, integrin families, cadherin and vitronectin, are believed to be essential for maintaining early pregnancy. In human pregnancy, it is usually impossible to prove whether defect or shortage of these factors definitely causes clinical abortion, because congenital deficiencies of these factors have not been reported in the literature. However, in cases of special adhesive proteins, such as fibrinogen (Fg)

ABC

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and blood coagulation factor X III, congenital deficiencies and pregnancy cases are present. As to congenital afibrinogenemia, 2 cases were reported [1, 2] as summarized in table 1. Their pregnancies resulted in spontaneous abortions without any replacement therapy. Inamoto and Terao [3] reported a precise review of pregnancy cases of afibrinogenemia and hypofibrinogenemia, and also showed their own first case of delivery with replacement therapy. As to congenital factor X III deficiency, Fisher et al. [4] demonstrated a case that

Dr. Toshihiko Asahina Handa-cho 3600 Hamamatsu, Shizuoka 431-3124 (Japan) Tel. +81 53 435 2309, Fax +81 53 435 2308 E-Mail [email protected]

Table 1. Two reported cases of congenital afibrinogenemia and pregnancy Case 1 Case 2

Year

Author

Ref.

Patient’s Graage vidity

Abortion

Delivery

1970 1977

Dube Matsuno

1 2

20 28

1 (3 months) 1 (2 months)

– –

had suffered recurrent abortion without any replacement therapy and finally succeeded in cesarean section delivery with replacement therapy. Thereafter, Kobayashi et al. [5] reported a first case of vaginal delivery with replacement therapy in 1990. According to previous reports, in cases with these congenital deficiencies, when the patient herself was a fetus and her mother was healthy, abortion did not occur. In contrast, abortion always occurred at an early stage of gestation without replacement therapy of deficient factor when the patient was the mother, even if her fetus was healthy. It is clear that maternal Fg and factor X III are essential for maintaining early pregnancy. In this report, we analyzed the pregnancy cases of these congenital deficiencies (previous reports and our new cases), made clear when and how much they needed these adhesive proteins for preventing abortion, and investigated immunohistochemical localization of these proteins at the site of implantation in early pregnancy. Thereafter, we discussed the role of these adhesive proteins in maintaining early pregnancy.

Case Reports Case 1 This case was reported by Inamoto and Terao [3] in 1985. The patient was a 34-year-old woman. Her bleeding history started on the 5th day after birth, with a severe umbilical vein hemorrhage after ligation of the umbilical cord. Since the age of 4–5 years she had sometimes suffered from bleeding of the lip. Since 12-years she had repeatedly suffered from arthralgia and fever. At 18-years she was finally diagnosed as congenital afibrinogenemia. In 1973 she married at the age of 24-years. Her first and second pregnancies ended in spontaneous abortions with genital bleeding at 2 months gestation. In 1982 the patient became pregnant. At 5 weeks and 2 days’ gestation (5w2dG), genital bleeding occurred. She was hospitalized and 3 g of Fg was administrated. Bleeding stopped. After hospitalization, 8 g of Fg was administrated per week (8 g/w). Thereafter the administration volume decreased to 4 g/w after 9 wG, and 2 g/w after 15 wG. The patient’s plasma concentration of Fg (plasma Fg) ranged from 5 to 40 mg/dl. At 21w1dG, genital bleeding occurred, and then plasma Fg was 40 mg/dl. Thereafter, administration volume was increased to 12 g/w. Plasma Fg increased to 84 mg/ dl. In the next week, administration volume was decreased again to 6 g/w. No genital bleeding occurred. Plasma Fg ranged from 20 to

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1 1

40 mg/dl. At 37w1dG, elective cesarean section was performed by obstetric indication. The infant’s plasma Fg was 55 mg/dl. In this report, Inamoto and Terao [3] proposed that a minimum plasma Fg was 60 mg/dl to maintain pregnancy. Case 2 This case was reported by Kobayashi et al. [6] in 1996. The patient was a 28-year-old woman. The bleeding history started at birth, with a severe umbilical vein hemorrhage after ligation of the umbilical cord. Since childhood, purpura was repeatedly noticed. At 7 years she was finally diagnosed as congenital afibrinogenemia. She had sometimes suffered from severe bleeding after dental extraction (at 10 years), arthrorrhagia, subarachnoidal hematoma (at 18 years) and ovarian bleeding (at 23 years). After the diagnosis, she was usually treated with administration of Fg during these episodes. Firstly her menstrual period lasted 10–14 days, and was very heavy. She needed administration of Fg during menstruation. Two pregnancies were interrupted by spontaneous abortions due to severe decidual bleeding. In July 1991, the patient became pregnant. She was administrated 6 g of Fg at 4 wG in another hospital. After admission to our hospital, we started prophylactic treatment with 4 g of Fg per week (4 g/w). No genital bleeding occurred. We increased the administration volume gradually from 7 g/w (6 wGF) to 10.5 g/w (15 wGF), in order to keep the patient’s plasma Fg Q60 mg/dl. At 36w6dG, spontaneous onset of labor occurred, and at the time her plasma Fg was 96 mg/dl. At midnight, she complained of severe abdominal pain, and massive genital bleeding occurred. When she was diagnosed as having placental abruption, her plasma Fg had decreased to 33 mg/ dl. 10 g of Fg was immediately administrated, and emergency cesarean section was performed. During the operation, 10 g of Fg was administrated. Plasma Fg was 147 mg/dl (during the operation), and 199 mg/dl (just after the operation). The operation was smoothly performed without any abnormal bleeding. The newborn female infant was 3,120 g at birth and in good condition. The infant’s plasma Fg was 18 mg/dl. We confirmed the sign of placental abruption in one third of the whole placenta. Case 3 This case was reported by Kobayashi et al. [5] in 1990. The patient was a 23-year-old married woman. The bleeding history started at birth, with a severe umbilical vein hemorrhage after ligation of the umbilical cord, and at the time the patient was transfused fresh blood. Since childhood she had repeatedly suffered from severe bleeding after head bruises. Subdural hemorrhage was treated with fresh blood transfusion. At 6 years she was finally diagnosed as congenital factor X III deficiency. Her first pregnancy was interrupted at 2 months’ gestation by artificial abortion. In 1986, the patient became pregnant again. At 6w5dG, genital bleeding occurred. She was administrated 2 vials of

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factor X III concentrate (a vial contains the activity of factor X III in 240 ml of fresh frozen plasma), because Fisher et al. [4] proposed that a minimum plasma activity of factor X III was 10% to maintain pregnancy. She was administrated 2 vials per week (2 V/w). The patient’s plasma activity of factor X III ( plasma X III ) ranged from 7 to 30%. No genital bleeding occurred. At 37w3dG, spontaneous onset of labor occurred. Before the delivery, we administrated 4 vials, according to Fisher’s cesarean section case. She delivered vaginally without any abnormal bleeding. During the delivery the patient’s plasma X III was 36%. The infant’s plasma X III was 32%.

International Co., USA, undiluted), horseradish peroxidase-conjugated goat IgG fraction anti-rabbit IgG (Cooper Biomedical Inc., USA; 1:200 dilution).

Antibodies Two rabbit polyclonal antibodies, one rabbit antisera and one rabbit monoclonal antibody, were employed. Specificities, references, sources and dilution are detailed as follows: rabbit IgG antihuman Fg (Organon Teknika Co., USA; 1:75 dilution), rabbit IgG anti-human Fn (Biomedical Technologies Inc., UK; 1:75 dilution), rabbit antisera to human subunit A of factor X III (X IIIA) (Behringwerke AG, Germany; 1:75 dilution), rabbit antisera to human subunit S of factor X III (X IIIs) (Behringwerke AG; 1:75 dilution), rabbit monoclonal IgG anti-human macrophage; LN-5 (Techniclone

Experimental Procedures (1) Immunohistochemical staining of tissues: Sections were labeled by an indirect immunoperoxidase technique, as follows: Paraffin-embedded tissues were cut to a thickness of 3 Ìm. Sections were dehydrated in the incubator overnight at 37 ° C. Thereafter, paraffin was liquefied in the incubator for 30–60 min at 60 ° C and was removed by xylene, and xylene was removed by ethanol. Sections were soaked in methanol, containing 0.3% hydrogen peroxide for 20 min, in order to remove intrinsic peroxidases. The sections were washed with 0.01 M sodium phosphate-buffered saline, pH 7.6 (PBS) and then incubated with antibodies or antisera diluted with PBS for 30 min. After three PBS washes, sections were incubated with peroxidase-conjugated goat IgG fraction anti-rabbit IgG diluted with PBS for 30 min. After three further PBS washes, the reaction was developed in 0.5 mg/ml 3,3)-diaminobenzidine (Sigma Chemical Co., UK), containing 0.02% hydrogen peroxide, to give a brown reaction product. The reaction was monitored microscopically and after 6–7 min stopped in excess distilled water. Sections were lightly counterstained with Mayer’s hematoxylin, dehydrated, cleared and mounted in synthetic resin. On the other hand, we made a pair of mirror sections. One side was stained with antisera to human X IIIA as above, and the other side was stained with LN-5. In the case of LN-5, after reaction with it, sections were labeled using a streptavidin-peroxidase kit (Dakopatts A/S, Denmark). The sections were sequentially incubated with biotinylated goat IgG fraction anti-rabbit IgG for 30 min, and were incubated with streptavidin-peroxidase for 30 min. The reaction was developed with the supplied aminoethylcarbazole (AEC) to give a bright red reaction product. Positive controls were performed for all antibodies on normal human placental vessels and villous macrophages (= Hofbauer cells). Negative controls were performed for all specimens. Serial sections were examined, and staining patterns were compared. (2) Endometrial cell culture: A sample was washed with Ham’s F12 medium (HFM; Sigma Inc., USA) twice, and then cut into small pieces. They were washed by centrifugation in HFM, and incubated with 0.25% collagenase (Wako Inc., Japan) in HFM, containing 5% fetal calf serum (FCS; Gibco Inc., USA) for 2 h at 37 ° C. Nondigestible tissues were removed with the Millipore Filter (n250 Ìm). Thereafter the cells were washed by centrifugation in HFM, containing 20% FCS. They were suspended in HFM, containing 10% FCS to a final concentration of 1 ! 105 cells/ml. 1 ! 105 cells/well were seeded into Lab-Tak chamber slides (Nunc Inc., USA), and cultured at 37 ° C in 5% CO2 and 95% air. We changed the medium every 48 h. On the 5th to 6th day during incubation, we removed FCS from incubation medium by four HFM washes. Finally the cells were cultured in FCS-free HFM for 24 h. As a result, the adherent endometrial cell monolayers were prepared on glass slides. (3) Immunohistochemical staining of cultured cells: The cells on glass slides were soaked in 3.5% paraformaldehyde for 60 min for fixation. Thereafter the cells were washed with tap water, and soaked in methanol, containing 0.3% hydrogen peroxide, for 5 min, in order to remove intrinsic peroxidases. The cells were labeled by an indirect immunoperoxidase technique with rabbit IgG anti-human fibronectin and rabbit antisera to human X IIIA, as mentioned above for tissue paraffin sections.

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Case 4 The patient was the same as above. In 1989, the patient became pregnant for the fourth time. At 5w6dG, genital bleeding occurred. Two vials of factor X III concentrate were administrated, and bleeding stopped. We started a prophylactic treatment with decreased volume of factor X III concentrate administration (1 V/w). Firstly the patient’s plasma X III ranged from 10 to 39%. Since 23 wG, her plasma X III decreased to 5%; we increased administration volume to 2 V/w. After that the patient’s plasma X III ranged from 10 to 30%. No genital bleeding occurred. At 37 wG, spontaneous onset of labor occurred. Before the delivery, we administrated 4 vials of factor X III concentrate, and she delivered vaginally without any abnormal bleeding. During the delivery the patient’s plasma X III was 52%. The newborn female infant weighed 3,142 g and was in good condition. The infant’s plasma X III was 31%.

Materials and Methods Tissues For immunohistochemistry of the implantation site, two separate samples of human intrauterine implantation tissues in early pregnancy (5 and 7 wG) were obtained from hysterectomy specimens with informed consent, immediately after surgical removal. Patients were hysterectomized for myoma uteri, with nonendometrial pathology. One separate sample of human full-term placenta was obtained from normal delivery with informed consent. Each 10-mm fragment was fixed in 3.5% paraformaldehyde at once, and embedded in paraffin wax. For cell culture, one separate sample of human endometrial tissue on the 9th day of the menstrual cycle was obtained from a hysterectomy specimen with informed consent, immediately after surgical removal. The patient was hysterectomized for myoma uteri, with nonendometrial pathology. A hematoxylin and eosin-stained paraffin-embedded section was prepared from a remaining specimen for routine histology. This specimen’s histological date coincided with the patient’s menstrual date.

39

1a

1b

2a

2b Fig. 1. a 5 wG implantation site: Fg ; Fg was stained strong positively at the decidual stroma around invasive cytotrophoblasts. Orig. magn. !40. b 5 wG implantation site: Fn; Fn was stained positively at the decidual stroma around invasive cytotrophoblasts. Orig. magn. !40. Fig. 2. a 5 wG implantation site: X IIIA; we found the cellular localization at the mononuclear cells in the decidua around invasive cytotrophoblasts. Orig. magn. !40. b 5 wG implantation site: X IIIA; we found the cellular localization at the mononuclear cells in the decidua around invasive cytotrophoblasts. Orig. magn. !200.

Results Clinical Case Reports Fibrinogen Deficiency. Before 4 wG (= 1 week after implantation), no genital bleeding occurred without any replacement therapy. After 4 wG, decidual bleeding occurred without replacement therapy. In case 2, it was sufficient to keep the patient’s plasma Fg Q60 mg/dl for perfectly preventing abortion or other decidual hemorrhage, by weekly intravenous administration of Fg. As the patient’s plasma Fg was 96 mg/dl at delivery, early separation of the placenta occurred. Factor X III Deficiency. Before 5 wG (= 2 weeks after implantation), no genital bleeding occurred without any

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replacement therapy. After 5 wG, decidual bleeding occurred without replacement therapy. In cases 3 and 4, it was sufficient to keep the patients’ plasma X III Q10% for perfectly preventing abortion or other decidual hemorrhage, by weekly intravenous administration of factor X III. At delivery, the patients’ plasma X III was kept Q30% to avoid early separation of the placenta. Immunohistochemistry (1) 5 wG (= 2 weeks after implantation): (A) Fg and Fn: Maternal side: they were stained strong positively at the decidual stroma around the invasive cytotrophoblasts (fig. 1a, b), and Fn was stained weak positively in the whole endometrial stroma. Fetal side: Fn was stained

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3a

3b

3c

4a

Fig. 3. a 7wG implantation site: Fg; Fg was stained strong positively at the decidual stroma around invasive cytotrophoblasts. Orig. magn. !40. b 7 wG implantation site: Fn; Fn was stained positively at the decidual stroma around invasive cytotrophoblasts. Orig. magn. !40. c 7 wG implantation site: X IIIA; we could not find obvious localization at the decidual stroma around invasive cytotrophoblasts. Orig. magn. !40. Fig. 4. a 7 wG implantation site: Fg; Fg was stained strong positively at the decidual stroma around vessels invaded by cytotrophoblasts. Orig. magn. !40. b 7 wG endometrium: X IIIA; we found the cellular localization at the mononuclear cells in the decidua (cytotrophoblast no invasion area). Orig. magn. !200.

4b

weak positively in the whole villous stroma. (B) X IIIA: Maternal side: we found the cellular localization at the mononuclear cells in the whole endometrium, and they were particularly numerous at the decidua around the invasive cytotrophoblasts (fig. 2a, b). Fetal side: we found the cellular localization at Hofbauer cells, which were villous macrophages, in whole villi.

(2) 7 wG (= 4 weeks after implantation): (A) Fg and Fn: Maternal side: they were stained strong positively at the decidual stroma around the invasive trophoblast (fig. 3a, b), and Fn was stained weak positively in the whole endometrial stroma. Fg was stained strong positively at the stroma around the vessels (fig. 4a). (B) X IIIA: Maternal side: we found the cellular localization at the mononuclear

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41

5a

5b

6a

6b

7a

7b

Fig. 5. a Full-term placenta: Fg; Fg was stained strong positively at the decidual layer. Orig. magn. !40. b Full-term placenta: Fn; Fn was stained positively at the decidual layer. Orig. magn. !40. Fig. 6. a Full-term placenta: X IIIA; we found the cellular localization at the mononuclear cells in the decidual layer. Orig. magn. !40. b Full-term placenta: X IIIA; we found the cellular localization at the mononuclear cells in the decidual layer. Orig. magn. !200. Fig. 7. a Endometrial culture cells: Fn; Fn was stained positively at the spindle-like shaped mononuclear cells’ surface with reticular

8

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Table 2. Immunohistochemical staining in implantation area and placenta

Fetal side (villi)

epithelium stroma (Tr. inv.)

stroma (No inv.)

cytotrophoblast

stroma

5 wG

Fg Fn X IIIA

– – –

++ ++ ++1

– + +2

– – –

– + +2

7 wG

Fg Fn X IIIA

– – –

++ ++ –

– + +2

– – –

– + +2

Placenta

1 2

Fg Fn X IIIA

Maternal side

Fetal side (villi)

decidual stroma

cytotrophoblast

stroma

++ ++ ++1

– – –

– + +2

Tr. inv. = Trophoblast invasion area; No inv. = no invasion area. At many macrophages. At macrophages.

cells in almost the whole endometrium (fig. 4b). But, strange to say, at the decidua around the invasive cytotrophoblasts we did not find obvious localization at all (fig. 3c). (3) Full-term placenta: (A) Fg and Fn: Maternal side: they were stained strong positively in the whole decidual stroma which faced the cytotrophoblast layer (fig. 5a, b). Fetal side: Fn was stained weak positively in the whole villous stroma. (B) X IIIA: Maternal side: we found the cellular localization at the mononuclear cells in the whole decidua which faced the cytotrophoblast layer (fig. 6a, b). Fetal side: we found the cellular localization at Hofbauer cells in whole villi. (4) Endometrial culture cells: (A) Fn was detected at the surface of spindle-like shaped mononuclear cells, and stained strong positively at some nuclei of these cells (fig. 7a). (B) X IIIA was detected mainly at the cytoplasm

structure, and stained strong positively at some nuclei of these cells. Orig. magn. ! 100. b Endometrial culture cells: X IIIA; X IIIA was stained positively at the round-shaped mononuclear cells’ cytoplasm, nuclei and at the extracellular matrix of fibroblasts. Orig. magn. !100. Fig. 8. Mirror sections: X IIIA-positive cells coincide with LN5-positive macrophages in the place of endometrial stroma. Orig. magn. !400.

Adhesive Protein in Maintaining Pregnancy

Maternal side (endometrium)

and nuclei of round-shaped mononuclear cells, and at extra-cellular matrix (fig. 7b). But X IIIS was not detected at all. It was just proof that plasma was not concerned with this result. (5) A pair of mirror sections: X IIIA-positive cell coincided with LN-5-positive cell in the place of endometrial stroma (fig. 8). The results are summarized in table 2.

Discussion Fg is a famous glycoprotein as an important blood coagulating factor present in plasma. It has the dimer structure which contains three polypeptides (A·, Bß, and Á chain). In the clotting process, firstly Fg releases fibrinopeptide A from A· chain under thrombin action, and changes to fibrin monomer. Secondly fibrin monomers aggregate one another. Fibrin aggregates release fibrinopeptide B from Bß chain under thrombin action, adhere one another and change to fibrin polymer. Fibrin polymers are cross-linked between Á-Á chain and ·-· chain under factor X III and calcium ion, and change to final, stable fibrin. In the absence of factor X III, fibrin formation is neither delayed nor reduced, but the fibrin polymers are aggregated only by weak intermolecular hydrogen bonds. On the other hand, fibrin is a kind of adhesive

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Fig. 9. Adhesion system model at the implantation site.

protein. It has adhesive sites for another fibrin and fibronectin. Normal plasma Fg ranges from 200 to 400 mg/dl. When pregnancy occurs, the concentration increases gradually to 1.1 times (4–7 wG), 1.2 times (8–11 wG), 1.3 times (12–35 wG) and 1.5 times (35 wGF). A minimum concentration for hemostasis is 100 mg/dl. Plasma Fg half-life is 96 h. Blood coagulation factor X III is a transglutaminase which works at the final stage of blood coagulation. It is present in plasma, platelet and monocyte/macrophage. Plasma type factor X III consists of two subunit As and two subunit Ss. Subunit A is the enzymatic active part and subunit S is the plasma carrier protein. Platelet and monocyte/macrophage type factor X III consists of only two subunit As. Factor X III promotes cross-linking between not only fibrin-fibrin but also fibrin-Fn and Fncollagen. Normal plasma X III ranges from 80 to 100%. When pregnancy occurs, the activity rather decreases to 0.80–0.75 times (8 wGF). A minimum activity for hemostasis is 2–3%. Plasma X III half-life is 96 h. Fn is a high-molecular glycoprotein present in plasma, basement membrane and stromal connective tissue of various organs, and famous as adhesive protein. It is concerned with cell adhesion, outgrowth, migration, differentiation, proliferation and phagocytosis. Plasma type Fn consists of two monomers, on the other hand, tissue type Fn consists of many monomers. A monomer has each adhesive sites for fibrin, collagen, actin and X IIIA. In

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mouse uterus, Grinnell et al. [7] reported that Fn was present at the decidual stroma. Congenital afibrinogenemia and factor X III deficiency are both autosomal recessive disorders, and their pregnancy cases are so-called experiment of nature. From the analysis of these cases, the following facts were made clear. (1) Even if without Fg and factor X III, the patients become pregnant spontaneously; (2) before 5–6 wG, maternal Fg and factor X III are not essential for maintaining pregnancy; (3) but after this period, without these factors, abnormal decidual bleeding or abortion always occurs. Thus, maternal Fg and factor X III become to be essential to the embryonal development after 5–6 wG. What anatomical change takes place in this period? Usually by the 7th day after ovulation (= 3w0dG), implantation is initiated. Firstly blastocyst adheres the surface epithelium of endometrium. Secondly giant trophoblasts begin to penetrate it. After penetration, the blastocyst intrudes into the underlying decidual stroma. Thereafter, giant trophoblasts expand into masses of both syncytiotrophoblasts and cytotrophoblasts. Within a week after implantation (= 4w0dG), firstly, syncytiotrophoblasts invade decidual stroma. They form the inner surface, and decidual stroma forms the outer surface of the implantation site. The fetus and mother bind with each other at this surface in this period. Since 4w1dG, cytotrophoblasts proliferate actively and begin to invade the syncytiotrophoblast layer. Since 5w0dG, 2 weeks after im-

Asahina/Kobayashi/Okada/Itoh/ Yamashita/Inamoto/Terao

plantation, cytotrophoblasts penetrate syncytiotrophoblastic shell and invade decidual stroma, and cover the syncytiotrophoblasts’ surface. At this time, cytotrophoblasts form a new inner surface of the implantation site instead of syncytiotrophoblasts. The fetus and mother finally bind with each other at this new surface between cytotrophoblasts and decidual stroma. Thus, maternal Fg and factor X III are supposed to be concerned with cytotrophoblasts’ invasion into decidual stroma and formation of the new binding surface. The following facts were proved from our immunohistochemical investigation and cell culture. Firstly, Fg and factor X III are surely present at the decidual stroma, which is the outer surface of the implantation site, at 5 wG. Fn, a famous adhesive protein, is abundantly present at the same place. Secondly, decidual factor X III is supplied by not only plasma but also decidual macrophages. Cellular factor X III may support the role of plasma factor X III at the implantation site. That may be the reason why plasma X III does not need to increase during pregnancy. However, cellular factor X III accumulated to the outer surface of the implantation site at 5 wG, and disappeared at that place at 7 wG, and in full-term placenta accumulated again there. Thus, the reason why the cellular factor X III behaves like this remains to be clarified. Thirdly, Fn, an important decidual adhesive protein, is

produced by endometrial stromal fibroblasts. X IIIA is also produced by round-shaped endometrial cells. We suppose that these cells are endometrial macrophages which steal into endometrial stromal and epithelial cells. As to the role of Fg and factor X III, previous authors, except those of our department, thought that it was only protection against bleeding during normal development of placenta. From our results, however, we came to believe firmly that Fg and factor X III not only worked as coagulation factors, but also worked with Fn as adhesive factors. Fg, Fn and factor X III are abundantly present in the endometrium also before implantation [pers. unpubl. data]. The subunits of ·5ß1 integrin (Fn receptor) have been shown to be present at the surface of cytotrophoblast [8] and type IV collagen to be present at the surface of decidual cell [9]. Therefore we propose one adhesion system as follows: when cytotrophoblasts invade decidual stroma, their surface Fn receptors bind to Fn produced by stromal fibroblasts. Fn binds to type IV collagen present at the surface of decidual cells. Their binding plays the role in cytotrophoblasts’ anchoring. Fn also binds to stromal Fg infiltrated from vessels, and the anchoring becomes stronger. Finally, plasma and cellular factor X III promote their bindings most tightly (fig. 9).

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

References 1 Dube B, Agarwal SP, Gupta MM, Chawla SC: Congenital deficiency of fibrinogen in two sisters: A clinical and haematological study. Acta Haematol 1970;43:120–127. 2 Matsuno K, Mori K, Amikawa H: A case of congenital afibrinogenemia with abortion, intracranial hemorrhage and peritonitis. Jpn J Clin Haematol 1977;18:1438–1443. 3 Inamoto U, Terao T: First report of case of congenital afibrinogenemia with successful delivery. Am J Obstet Gynecol 1985;153:803–804.

Adhesive Protein in Maintaining Pregnancy

4 Fisher S, Rikover M, Naor S: Factor 13 deficiency with severe hemorrhage diathesis. Blood 1966;28:34–39. 5 Kobayashi T, Terao T, Kojima T, Takamatsu J, Kamiya T, Saito H: Congenital factor X III deficiency with treatment of factor X III concentrate and normal vaginal delivery. Gynecol Obstet Invest 1990;29:235–238. 6 Kobayashi T, Asahina T, Maehara K, Itoh M, Kanayama N, Terao T: Congenital afibrinogenemia with successful delivery. Gynecol Obstet Invest 1996;42:66–69.

7 Grinnell F, Head JR, Hoffpauir J: Fibronectin and cell shape in vivo: Studies on the endometrium during pregnancy. J Cell Biol 1982;94: 597–606. 8 Bischof P, Haenggeli L, Campana A: Gelatinase and oncofetal fibronectin secretion is dependent on integrin expression on human cytotrophoblasts. Hum Reprod 1995;10:734–742. 9 Wewer UM, Faber M, Loitta LA, Albrechtsen R: Immunohistochemical and ultrastructural assessment of the nature of the pericellular basement membrane of human decidual cells. Lab Invest 1985;53:624-633.

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Yasunori Yoshimura Kei Miyakoshi Toshio Hamatani Kazuhiro Iwahashi Jun Takahashi Noriko Kobayashi Kou Sueoka Toyohiko Miyazaki Naoaki Kuji Mamoru Tanaka Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Key Words ß1 Integrins Endometrium Decidua Implantation

Role of ß1 Integrins in Human Endometrium and Decidua during Implantation

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Abstract The present study was undertaken to investigate the expression and function of ß1 integrins in human endometrium and decidua. Fluorescence-activated flow cytometry demonstrated the greater expression of the ß1, ·1, ·2, and ·5 subunits of the ß1 integrin family in cultured stromal cells from the midsecretory phase than in those of the early proliferative phase. The addition of estradiol (E2) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of ß1 integrins in vitro. The immunohistochemical distribution of ß1 integrins demonstrated predominantly glandular epithelial staining in the proliferative phase, and stromal and glandular staining in the midsecretory phase. Flow cytometry also demonstrated the expression of ß1, ·1, ·2, ·3, ·5, and ·6 subunits of ß1 integrin family in cultured decidual cells. Immunohistochemistry confirmed the ß1 integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. In the subsequent experiment, the effects of antibodies against specific ß1 integrin heterodimers on mouse embryo attachment and spreading were tested to identify the role of ß1 integrins in early implantation. We developed assays for the attachment of mouse embryos and for trophoblastic spreading on cultured human decidual cells. The addition of antibodies directed against ß1 and · integrin subunits to cultured decidual cells did not affect the rates of hatching or attachment of the blastocysts, whereas the outgrowth of embryos on the decidual cells was inhibited by their antibodies in a dose-dependent manner. Thus, ß1 integrin in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation. OOOOOOOOOOOOOOOOOOOOOO

In recent years, the mechanisms surrounding the interactions of cells with the components of extracellular matrix (ECM) proteins have become clearer with the isolation and characterization of cell surface receptors for ECM components [1, 2]. These receptors, termed integrins, are integral membrane glycoproteins that anchor extracellular adhesion proteins to cytoskeletal compo-

ABC

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nents [2–5]. Integrins constitute a widespread family of ·ß heterodimeric adhesion receptors that mediate cell attachment to ECM proteins and cell-cell interactions [1, 2]. Each integrin subunit has a large extracellular domain, a single membrane spanning region, and usually a short cytoplasmic domain [2–5]. The integrin receptor family is comprised of at least 14 distinct · subunits and 8 or more

Yasunori Yoshimura, MD Department of Obstetrics and Gynecology Keio University School of Medicine 35 Shinanomachi, Shinjuku-ku, Tokyo 160 (Japan) Fax +81 3 3352 1598, E-Mail [email protected]

ß subunits that can associate in various combinations [2, 5, 6]. Each has been sequenced at the cDNA level. Monoclonal antibodies directed to different · and ß subunits of integrin represent a valuable tool for the initial characterization of the integrin profile in human cells [2]. These reagents also allow tracking of qualitative and quantitative alterations in the expression of ECM proteins [2, 5]. Normal morphogenesis and differentiation heavily depend on the coordination of cell-cell and cell-ECM interactions. In human embryo implantation, a large number of trophoblasts infiltrate through the decidua and extend as far as the superficial layer of the myometrium. Earlier studies have demonstrated that the trophectoderm cells of preimplantation mouse blastocysts contain fibronectin as well as laminin [7–9]. These ECM proteins, when individually precoated on tissue culture plates, promote in vitro attachment and outgrowth of mouse blastocysts in serumfree medium [10]. ECM proteins produced by endometrial stromal or decidual cells are also important for endometrial structure and integrity and provide a site for trophoblast attachment [11–13]. Recent studies have demonstrated that human endometrial and decidual cells express components of receptors for ECM proteins, the ß1 integrin family, on their surfaces and that the expression is a dynamic process related to the menstrual cycle [14–17]. In addition, the expression of ß1 integrins in human endometrium increases at the time of implantation and remains high in the decidua during early pregnancy [17– 19]. However, the role of ß1 integrins in human endometrial stromal and decidual cells during implantation remains to be clarified. In the present study, we investigated the expression of ß1 integrin family in human endometrium and decidua by flow cytometry and immunohistochemistry. In addition, we have developed assays for the attachment of mouse embryos and trophoblastic spreading on cultured human decidual cells [20]. These assays were used to investigate the effects of antibodies against specific ß1 integrin heterodimers on embryo attachment and spreading and to investigate the role of the ß1 integrins in early implantation.

(days 5–7) and the midsecretory phase (days 19–22). The mean cycle length in these patients was 28 days (range 26–31), and none had received hormones for at least 3 months before surgery. Cyclic changes in the endometrium were determined by the endometrial dating system of Jones and Jones [21]. Decidual specimens were obtained from 26 women who underwent therapeutic abortion between 7 and 11 weeks’ gestation. The median age of these women was 31 years (range 24–38). Gestational ages were based on the last menstrual period, uterine size, and crown-rump length. All women gave informed consent for collection of the tissues, and the study protocol was approved by the ethics committee of Keio University School of Medicine. The specimens were placed immediately in icecold Medium 199 (Gibco, Grand Island, N.Y., USA) containing 25 mmol/l Hepes (Sigma Chemical Co, St. Louis, Mo., USA) and 1% antibiotic-antimycotic mixture (Gibco) and transported to the laboratory within 1 h after surgery. After blood clots had been removed, the endometrial and decidual tissues were rinsed thoroughly in icecold Medium 199. The tissue was trimmed and cut into approximately 1-mm pieces using a small pair of scissors. A portion of the tissue was fixed in Bouin’s aqueous fixative for histologic examination. The remaining tissue was treated enzymatically to disperse the cells.

Tissue Preparation Endometrium was obtained from 32 women of reproductive age (range 32–40 years) at the time of hysterectomy at the Keio University Hospital. All hysterectomies were performed for abnormalities other than endometrial origin, including uterine leiomyoma and adenomyosis. The tissues were obtained from the early proliferative

Cell Culture Endometrial specimens were treated with 0.1% collagenase (type 1A, Sigma) and 0.1% hyaluronidase (type IS, Sigma) in C·2+-free phosphate buffer saline (PBS) with stirring at 37 ° C for 1 h. The suspension then was filtered through a nylon mesh (pore size, 105 Ìm) to remove undigested tissue debris. The cells were collected from the filtrate by centrifugation at 800 g for 10 min and washed in Medium 199 containing 10% fetal calf serum (Gibco). The suspension was filtered through a 38-Ìm stainless steel sieve (Spectrum, Los Angeles, Calif., USA) to retain the glandular elements as previously described [22, 23]. The stromal cells then were purified by exploiting their more rapid adherence (60 min) to the plastic surfaces of the tissue culture dishes relative to the epithelial and bone marrow-derived elements. The purity of this stromal-enriched fraction was confirmed immunocytochemically by staining for vimentin (1 95% of the cells) and by the absence of staining for cytokeratin. The stromal cells obtained from the early proliferative phase were cultured with or without 30 nmol/l estradiol (E2) and 300 nmol/l progesterone (P). Sterile isolation of the decidual cells was performed according to the methods described by Satyaswaroop et al. [22] and Braverman et al. [24], with minor modifications. The decidual stromal cells were filtered through a sieve using the same method as for the endometrial stromal cell culture, collected by centrifugation, washed, and resuspended. The decidual cells were resuspended in 20% isotonic Percoll solution and layered on top of 20–60 and 40–55% Percoll gradients [24]. The gradients were centrifuged for 15 min at 30,000 g in a Beckman L3-50 ultracentrifuge at 4 ° C using a type 65 fixed-angle rotor (Beckman, Palo Alto, Calif., USA). An enriched fraction of prolactinproducing decidual cells layered as a single band with a cell density of 1.033–1.048 g/ml. The band contained a nearly homogeneous population of large round mononucleated cells (1 25 Ìm diameter). These cells were washed and suspended 3 times in Medium 199 supplemented with 10% fetal calf serum and 1% antibiotic-antimycotic mixture. Aliquots of viable endometrial stromal and decidual cell suspensions were counted by the dye exclusion test using 0.4% (v/v) trypan blue in PBS. The stromal cells were plated at 5 ! 105 cells/ml in a 35 ! 10-mm plastic Petri dish (Falcon 3001; Becton Dickinson,

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Materials and Methods

47

Lincoln Park, N.J., USA). The culture medium was changed every 48 h, and the cultures were maintained in humidified 95% air:5% CO2 at 37 ° C for 10 days. Reagents The following monoclonal antibodies against integrin subunits were used: ACT-T-SET VLA-1 for the ·1 subunit (T Cell Sciences, Inc., Cambridge, Mass., USA), Cdw49b VLA2gpla/IIa for the ·2 subunit (Immunotech SA, France), Cdw49c VLA3 for the ·3 subunit (Immunotech SA), Cdw49d VLA4 for the ·4 subunit (Immunotech SA), Cdw49e VLA5 for the ·5 subunit (Immunotech SA), Cdw49f VLA6 for the ·6 subunit (Immunotech SA), and CD29 VLAß1 for the ß1 subunit (Immunotech SA). A purified mouse monoclonal IgG1 antibody (clone DAK-GO1, Dako A/S, Glostrup, Denmark) or a purified rat IgG antibody (Organon Teknika Corp., West Chester, Pa., USA) was used as a negative control. A fluorescein isothiocyanate (FITC)-conjugated F(ab))2 fragment of affinity-isolated goat anti-mouse immunoglobulins from Dako A/S was also used. Flow Cytometric Analysis Stromal cells in the early proliferative phase and midsecretory phase and decidual cells were cultured for 10 days. Subconfluent monolayer cultures were harvested by washing with PBS and by the addition of 0.02% EDTA in PBS containing 25 mmol/l Hepes buffer (pH 7.4). After the cells had been detached from the dish, 2 vol of serum-free Medium 199 was added and the cells were pelleted. The cells were resuspended in PBS containing 0.1% bovine serum albumin (BSA, Sigma) and 0.02% sodium azide, and then 100 Ìl of the cell suspension (5 ! 105 cells) was mixed with 10 Ìl of the appropriate monoclonal antibody and inclubated at 4 ° C for 30 min. Following centrifugation for 5 min at 800 g, the supernatant was aspirated, and the cells were resuspended in the washing buffer. After washing 3 times, the cells were incubated at 4 ° C for 30 min with FITC-conjugated goat anti-mouse IgG (Dako A/S) while being protected from light. The cells were again washed 3 times, resuspended in 300 Ìl of the washing buffer, and analyzed by flow cytometry (Epics-CS, Coulter Co., Miami, Fla., USA). In the negative control experiments, cells were incubated with normal mouse or rat IgG as the first antibody. At least 5,000 cells were analyzed for each sample. Immunohistochemistry Tissue was embedded in OCT (Miles Inc., Diagnostic Div., Elkhart, Ind., USA) and frozen in liquid nitrogen. Serial cryosections, 4–8 Ìm thick, were placed onto poly-L-lysine-coated slides, fixed in –20 ° C acetone for 10 min, and stained using Strept ABC complex/ HRP kits (Dako A/S). Primary antibody was placed on cryosections after blocking with 0.5% normal goat serum (Dako A/S) in PBS and allowed to bind at room temperature for 30 min. A PBS rinse was followed by the application of a secondary antibody consisting of biotinylated goat antimouse antibody for 1 h. After a PBS rinse, the endogenous peroxidases were quenched with a 30-min incubation with 0.3% H2O2 in absolute methanol, followed by a 30-min rehydration in PBS. Avidin:biotinylated horseradish peroxidase macromolecular complex was then incubated on the sections for 30 min, before diaminobenzadine for 3 min, to complete the reaction. Negative controls consisted of tissue sections in which a mouse monoclonal IgG1 antibody (Dako A/S) was substituted for the primary antibody; procedure-related background staining was consistently absent.

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Assays for Embryo Attachment and Spreading on Human Decidua Embryo attachment and spreading assays were conducted with cultured human decidua. Female ICR mice (10 weeks old; Clea Japan Inc., Tokyo, Japan) were superovulated with an injection of 5 IU of eCG (Teikoku-zoki Co., Tokyo, Japan) followed after 48 h by an injection of 2.5 IU of hCG (Teikoku-zoki). They were then caged with ICR males. Embryos were flushed at the late morula or early blastocyst stage (96 h after hCG) from the uterine horms and rinsed in Medium 199 supplemented with 0.4% BSA. After decidual cells had been cultured in Medium 199 supplemented with 10% fetal calf serum for 10 days, subconfluent monolayer cultures were maintained in serum-free Medium 199. In control cultures, decidual cells were incubated with a purified mouse monoclonal IgG1 antibody at a concentration of 200 ng/ml before addition of the embryos. In experimental cultures, decidual cells were incubated with a monoclonal antibody against the ß1, ·1, ·2, ·4, ·5, or ·6 subunit at concentrations of 2–200 ng/ml. Five to eight embryos then were placed in prepared dishes with a subconfluent monolayer culture of decidual cells and cocultured for 96 h. To identify embryo attachment, a small amount of medium was gently flushed on each embryo by means of a glass pipette pulled to a very fine bore. Embryos that showed no movement while being observed under an Olympus inverted phase-contrast microscope (Olympus Optical Co. Ltd, Tokyo, Japan) were considered to be attached. Embryos were classified as spreading if migration of individual cells or monolayers of trophoblasts from the ectoplacental cone rudiment were observed. To determine the extent of spreading, the embryos were photographed at a magnification of 200 ! and each negative was printed at the same size. The area of outgrowth was measured with a color image-analyzing system (SP500; Olympus Optical) according to the method described by Imamura et al. [25]. The final value for each embryo was calculated from the average of three tracings. The results of each treatment represent the average of the measurements of at least 35 embryos. Measurements of embryo attachment and spreading were made at 24 and 48 h of incubation, respectively. The area of embryo outgrowth was determined between 48 and 96 h of incubation. Statistical Analysis The percentages of embryo hatching, attachment, and spreading and the area of embryo spreading are expressed as the mean B SEM. To obtain a normal distribution, the percentages of embryo attachment and spreading were subjected to arcsine transformation. Statistical analysis was performed by ANOVA with Scheffé’s test. Differences were considered statistically significant if p ! 0.05.

Results The cell surface ß1 integrin profiles were determined in endometrial stromal cells from the early proliferative and midsecretory phases cultured for 10 days. To verify integrin cell surface phenotype, flow cytometry analyses of endometrial stromal cells were performed using monoclonal antibodies recognizing the · and ß subunits [20, 26]. The cultured stromal cells from the midsecretory

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Fig. 1. Cell surface expression of ß1 integrins on human endometrial stromal cells. Stromal cells in the early proliferative phase (A) and midsecretory phase (C) were cultured for 10 days. Stromal cells in the early proliferative phase were also cultured in the presence of 30 nmol/l E2 and 300 nmol/l P for 10 days (B). Flow cytometry analysis was then performed on the cultured stromal cells. Saturating concentrations of the following monoclonal antibodies were used for staining: mouse monoclonal IgG1 antibody (control), anti-ß1 subunit antibody, anti-·1 subunit antibody, anti-·2 subunit antibody, anti-·4 subunit antibody, and anti-·5 subunit antibody. [From 26, with permission.]

phase expressed the ß1, ·1, ·2, and ·5 subunits of the ß1 integrin family; however, no measurable expression of the ·4 subunit was detected (fig. 1). Fluorescence-activated flow cytometry demonstrated a greater expression of the ß1 integrins in the stromal cells from the midsecretory phase, compared with the stromal cells from the early proliferative phase (table 1). Addition of E2 and P to the cultured stromal cells from the early proliferative phase resulted in an increased expression of ß1 integrins in vitro (fig. 1, table 1). The distribution of the ß1 and · subunits in the endometrium is shown in figures 2 and 3. In the proliferative phase, these subunits were mainly present on glandular epithelium (fig. 2). The ß1 subunit demonstrated predominantly grandular epithelial staining; however, the expression in the stroma was less pronounced. The ·2 subunit was distributed around the entire circumference of the cells, while the ·3 and ·6 subunits were localized to the basolateral surface, adjacent to the basement membrane of the endometrial gland. In the midsecretory phase, the expression of the ß1 subunit and the · subunits was more pronounced in the stroma as well as in the glandular epithelium (fig. 3). The ·2, ·3, and ·5 subunits were expressed in both epithelial and stromal cells, whereas the ·1 subunit was restricted to the glandular epithelium. The ·4 subunit was not detected in the epithelium or stroma during the menstrual cycle. The cultured decidual cells expressed the ·1, ·2, ·3, ·5, and ·6 subunits of the ß1 integrin family; however, no measurable expression of the ·4 subunit was detected. The decidual cells showed high levels of expression of the ·1 and ·2 subunits (percentage of positive cells: ·1, 91%;

Table 1. Expression of ß1 integrins on human endometrial stromal cellsa in the early proliferative phase and the midsecretory phase (percent positiveb)

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Proliferative Proliferatived + E2, P Secretory

ß1

·1

·2

·4

·5

60c 90 94

28 65 92

15 40 89

0 0 0

6 20 87

a

Stromal cells were cultured for 10 days. Stromal cells were characterized by flow cytometric analysis for the reactivity with monoclonal antibodies directed against · and ß subunits of VLA antigens. c Percent of positive cells. d Stromal cells in the early proliferative phase were cultured with 30 nmol/l E2 and 300 nmol/l P for 10 days. b

·2, 92%). The mean fluorescence intensities were almost identical to those of the ß1 subunit. Moderate levels of ·3, ·5, and ·6 were present (percentage of positive cells: ·3, 61%; ·5, 76%; ·6, 60%). Large mature decidual cells stained intensely for the ß1, ·1, ·3, and ·5 subunits (fig. 4). The expression of the ·2 subunit was observed in a few mature decidual cells but the ·4 subunit was undetectable in any decidual cells. The ß1, ·3, and ·5 subunits were distributed at the pericellular and cytoplasmic regions, whereas the ·1 subunit was distributed intensely around the entire circumference of the mature decidual cells. This immunohistochemical distribution in decidual tissues was almost identical to the ß1

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Fig. 2. Immunohistochemical profile of ß1 integrin subunits in early proliferative endometrium (day 7). Tissues were stained as follows: (A) mouse monoclonal IgG1 antibody (control); (B) anti-ß1 subunit antibody; (C) anti-·2 subunit antibody; (D) anti-·3 subunit antibody; (E) anti-·5 subunit antibody, and (F) anti-·6 subunit antibody. Note the staining of the ·2, ·3, and ·6 subunits on glandular epithelium. The ß1 subunit showed predominant glandular epithelial staining with decreased stromal staining. Bars = 100 Ìm. [From 26, with permission.]

integrin cell surface phenotype observed in cultured decidual cells observed by flow cytometry. Hatched mouse blastocysts cultured in vitro attached and formed outgrowths of trophoblasts on decidual cells, providing a model for implantation [20, 27]. The effects of the ß1 integrin subfamily on embryo attachment and the subsequent spreading of trophoblast were investigated in vitro on decidual cells incubated with monoclonal antibodies against the ß1 and various · subunits. When decidual cell monolayers were treated with the anti-ß1 subunit antibody in concentrations above 1 Ìg/ml, minute flaws were observed in the continuity of the cellular pattern, resulting in a ‘laddered stocking’ appearance. These tiny discontinuities were apparently the result of dissociation

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of adjacent cells and not detachment as observed in human umbilical vein endothelial cells [28]. Increasing concentrations of the anti-ß1 subunit antibody induced progressively larger discontinuities in the decidual cell monolayers, eventuating in substantial holes and resulting in monolayer detachment from the culture dish by 72 h. However, monoclonal antibodies against the ß1 integrins at less than 200 ng/ml had no effect on monolayer integrity. Addition of the anti-ß1 subunit antibody to cultured decidual cells did not affect the hatching ratio (fig. 5). Attachment of hatched blastocysts was slightly, but not significantly, reduced in cultures treated with the anti-ß1 subunit antibody as compared to control cultures (fig. 5). The spreading of trophoblasts from attached blastocysts

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Fig. 3. Immunohistochemical profile of ß1 integrin subunits in midsecretory endometrium (day 20). Tissues were stained as follows: (A) mouse monoclonal IgG1 antibody (control); (B) anti-ß1 subunit antibody; (C) anti-·1 subunit antibody; (D) anti-·2 subunit antibody; (E) anti-·3 subunit antibody, and (F) anti-·5 subunit antibody. Note the staining of the ß1, ·2, ·3, and ·5 subunits on glandular epithelium and the stroma. Bars = 100 Ìm. [From 26, with permission.]

was observed at 48–96 h of coculture with decidual cells when incubation was carried out with a mouse monoclonal IgG1 antibody at a concentration of 200 ng/ml as previously described [20, 27]. Exposure of the decidual cells to the anti-ß1 subunit antibody inhibited the incidence and area of outgrowth of trophoblasts in a dose-dependent manner as measured at 72 h of coculture (fig. 5, 6). Spreading of blastocysts was also inhibited by exposure of the decidual cells to monoclonal antibodies against the · subunits. The incidence of trophoblast outgrowth was significantly reduced in the presence of antibodies against the ·1, ·2, ·5, and ·6 subunits (fig. 7). In contrast, trophoblast outgrowth on decidual cells was not affected by the presence of antibody against the ·4 subunit. The addition

of anti-·1, -·2, -·5, and -·6 subunit antibodies significantly inhibited the extent of trophoblast outgrowth (fig. 8).

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Discussion Several studies confirm the presence of ß1 integrins on human endometrium and demonstrate that integrin expression is a dynamic process related to the menstrual cycle [14–17, 20, 26]. The distribution of different · and ß integrin subunits in human endometrial tissues at different stages of the menstrual cycle has been determined using immunohistochemistry. These studies suggest that some integrins normally undergo spatial and temporal

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Fig. 4. Immunohistochemical profile of ß1 integrin subunits in decidua. Tissues were stained as follows: (A) mouse monoclonal IgG1 antibody (control); (B) anti-ß1 subunit antibody; (C) anti-·1 subunit antibody; (D) anti-·2 subunit antibody; (E) anti-·3 subunit antibody, and (F) anti-·5 subunit antibody. Note the intense staining of the ß1, ·1, ·3, and ·5 subunits in large mature decidual cells. Bars = 100 Ìm. [From 26, with permission.]

changes with expression in the cycling endometrium, and that disruption of this expression pattern may be associated with certain types of infertility in women [16, 17, 29]. The pattern of integrin expression in the endometrium during implantation is most interesting. In the present study, the expression of ß1 integrins in the endometrium coincided with the ovarian changes, allowing a distinction between the early proliferative and midsecretory phases. The expression of ß1 integrins varied throughout the cycle, with predominant expression occurring during the secretory phase. The localization of ß1 integrins in the early proliferative phase was restricted to the glandular epithelium, whereas stromal cells in the midsecretory phase also expressed ß1 integrins. Recent studies have demon-

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strated that the expression of ß1 integrins in human endometrium increases at the time of implantation [16, 17, 20, 26, 29]. Thus, although certain ß1 integrin moieties appear to be regulated throughout the endometrial cycle, the mechanisms responsible for ß1 integrin regulation have yet to be established. The present study demonstrated that treatment of stromal cells in the proliferative phase with E2 and P increased the expression of ß1 integrins in vitro, suggesting that ß1 integrin expression in human endometrium may be progestin-dependent. The differential expression of ß1 integrins within different compartments of the endometrium reflects the distinct nature and function of these compartments.

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5

7

6

8 Fig. 5. Effects of the anti-ß1 subunit antibody on hatching, attachment, and spreading of blastocysts. Mouse embryos were placed on subconfluent cultured decidual cells treated with or without a monoclonal antibody against ß1 subunit at a concentration of 2, 20, 100, or 200 ng/ml. Hatching (cross-hatched bars), attachment (solid bars), and spreading (open bars) of embryos were assessed at 12, 24, and 48 h after the beginning of coculture with decidual cells. Data represent the mean B SEM of at least six different experiments. At least 43 embryos were analyzed in each treatment group. Values with asterisks differ significantly from values on decidual cells treated with monoclonal IgG1 antibody (control); * p ! 0.05, ** p ! 0.01, *** p ! 0.001. [From 27, with permission.] Fig. 6. Quantitative evaluation of the area of outgrowth (Ìm2) of embryos cultured with or without an anti-ß1 subunit antibody at a concentration of 2, 20, 100, or 200 ng/ml. The area of outgrowth was determined at 72 h of coculture with decidual cells. Data represent the mean B SEM of at least 40 embryos. Values with asterisks differ significantly from values on decidual cells treated with a monoclonal IgG1 antibody (control); * p ! 0.01, ** p ! 0.001. [From 27, with permission.]

Fig. 7. Effects of anti-· subunit antibodies on hatching, attachment, and spreading of blastocysts. Mouse embryos were placed on subconfluent cultured decidual cells treated with or without anti-· subunit antibodies at a concentration of 100 ng/ml. Hatching (crosshatched bars), attachment (solid bars), and spreading (open bars) were assessed at 12, 24, and 48 h of coculture with decidual cells, respectively. Data represent the mean B SEM of at least eight different experiments. At least 56 embryos were analyzed in each treatment group. Values with asterisks differ significantly from values on decidual cells treated with a monoclonal IgG1 antibody (control); * p ! 0.05, ** p ! 0.01. [From 27, with permission.] Fig. 8. Quantitative evaluation of the area of outgrowth (Ìm2) of embryos cultured with or without anti-· subunit antibodies at a concentration of 100 ng/ml. The area of outgrowth of embryos was determined at 72 h of coculture with decidual cells. Data represent the mean B SEM of at least 35 embryos. Values with asterisks differ significantly from values on decidual cells treated with a monoclonal IgG1 antibody (control); * p ! 0.05, ** p ! 0.01, *** p ! 0.001. [From 27, with permission.]

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Human decidua differentiates from the uterine stromal lining into actively secreting tissue under hormonal stimulation during early pregnancy. The maternal decidua is in direct contact with the fetal trophoblasts; therefore, it provides a hospitable environment for implantation of the blastocyst. Wewer et al. [12] have identified immunohistochemically and morphologically three separate subpopulations of decidual cells that may represent different stages of the human decidual cell lineage. Since decidualization appears to represent a continuum of cells ranging from stromal cells (!15 mm diameter) to hypertrophied mature decidual cells (125 mm diameter), the expression of ß1 integrins on decidual cells may represent any or all of these stages of differentiation. The most prominent cell type present in decidual tissue has been shown to be large, mature epithelioid cells with a distinct pericellular basement membrane containing laminin, type IV collagen, fibronectin, and possibly other components [12]. The pericellular distribution pattern of ECM around the individual decidual cells suggests that ECM receptors are present on the surface of decidual cells. In the present study, flow cytometric analysis revealed that decidual cells express high levels of the ·1 and ·2 subunits and moderate levels of the ·5 and ·6 subunits. In addition, the biosynthesis of ß1 integrins has been demonstrated by metabolic labeling of cultured decidual cells and immunoprecipitation of the cell extract with a specific antibody followed by SDS-PAGE [26]. The expression of ß1 integrins in human endometrium increases at the time of implantation and remains high in decidua during early pregnancy. This indicates the presence of a certain degree of specificity, potentially mediated by ß1 integrins, which are the major family of ECM receptors present on the surface of decidual cells. The mechanism by which ß1 integrins promote trophoblast outgrowth may be related to their known adhesive role and their participation in cell attachment/detachment and cell migration [1, 2, 5]. ECM proteins are expressed during early embryogenesis. Specifically, laminin B1 and B2 chains appear at the four-cell stage, and fibronectin and type IV collagen are first detected at the blastocyst stage [9, 30, 31]. In addition, trophoblasts isolated in primary culture have been demonstrated to synthesize and secrete fibronectin molecules bearing a unique glycopeptide domain within the type III connecting segment [32]. This oncofetal fibronectin class of molecules may mediate implantation and placental-uterine attachment throughout gestation [32]. ß1 Integrins present on human decidual cells may mediate the organization of ECM proteins derived from embryos during the early

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stage of implantation, thereby connecting the ECM framework to the intracellular cytoskeletal structures [1, 2, 5]. It is also possible that expression of ß1 integrins on decidual cells may prepare the uterine wall for blastocyst invasion. In the present study, mouse blastocysts were capable of attaching to and forming extensive trophoblast outgrowth on human decidua cells in vitro. Because blastocysts grown in vitro on cultured human decidual cells displayed attachment and outgrowth of trophoblasts in the presence of a mouse monoclonal IgG1 antibody, we chose this system to evaluate the factors that regulate trophoblast differentiation. Attachment of the blastocysts to the cultured decidual cells appears to be prerequisite for further outgrowth of trophoblasts. Outgrowth, but not attachment, of embryos on decidual cells was inhibited in a dose-dependent manner by adding an antibody that recognizes the ß1 chain, suggesting that ß1 integrins are important in blastocyst development and differentiation following attachment. Monoclonal antibodies directed against ß1, ·1, ·2, ·5, and ·6 subunits affected embryo outgrowth, but not attachment, suggesting that blastocyst attachment and outgrowth may be mediated by different mechanisms. Subsequent spreading of trophoblasts involves a number of cellular events that are necessary to produce morphologic changes and cell migration [33]. Farach et al. [34] have reported that soluble heparin inhibits mouse embryo attachment and outgrowth on fibronectin and laminin, and that substrates of a heparin-binding protein, platelet factor 4, support attachment, but limited outgrowth. Purified cell-substratum adhesion glycoprotein, GP140, which participates in the attachment of somatic cells to the substratum, has also been demonstrated to mediate trophoblast attachment of mouse blastocysts [35]. In recent reports, increased expression of the epithelial vitronectin receptor, ·vß3, during normal menstrual cycles has been shown to correlate to a putative ‘implantation window’ within the secretory phase [16, 17]. Since both trophoblast and endometrium express ·vß3 on their surface [36, 37], this epithelial integrin may be involved in the endometrial-trophoblast interaction that takes place during early embryonic attachment. These studies imply that blastocyst outgrowth is primarily mediated by mechanisms that involve the expression of ß1 integrins on decidual cells; however, attachment may be mediated through heparin or heparin-sulfate-containing moieties or other cell surface adhesion molecules present on endometrial epithelial cells. In summary, the present results demonstrated that human endometrial stromal and decidual cells expressed

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components of the ß1 integrin family at their cell surface. This expression varied throughout the menstrual cycle, occurring predominantly during the secretory phase. The expression of ß1 integrins in the endometrium may be progestin-dependent. The expression of ß1 integrin in human endometrium increased at the time of implanta-

tion and remained high in decidua during early pregnancy. The outgrowth of embryos on decidual cells, but not the attachment, was inhibited by antibodies recognizing the components of the ß1 integrin family, suggesting that ß1 integrins on decidual cells may be important in development and differentiation following attachment.

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ß1 Integrins in Endometrium and Decidua

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26 Shiokawa S, Yoshimura Y, Nagamatsu S, Sawa H, Hanashi H, Oda T, Katsumata Y, Koyama N, Nakamura Y: Expression of ß1 integrins in human endometrial stromal and decidual cells. J Clin Endocrinol Metab 1996;81:1533–1540. 27 Shiokawa S, Yoshimura Y, Nagamatsu S, Sawa H, Hanashi H, Koyama N, Katsumata Y, Nagai A, Nakamura Y: Function of ß1 integrins on human decidual cells during implantation. Biol Reprod 1996;54:745–752. 28 Lampugnani MG, Resnati M, Dejana E, Marchisio PC: The role of integrins in the maintenance of endothelial monolayer integrity. J Cell Biol 1991;112:479–490. 29 Klentzeris LD, Bulmer JN, Trejdosiewicz LK, Morrison L, Cooke ID: Beta-1 integrin cell adhesion molecules in the endometrium of fertile and infertile women. Hum Reprod 1993;8: 1223–1230. 30 Camegie JA: Immunolocalization of fibronectin and laminin within rat blastocysts cultured under serum-free conditions. J Reprod Fertil 1991;91:423–434. 31 Thorsteinsdottir S: Basement membrane and fibronectin matrix are distinct entities in the developing mouse blastocyst. Anat Rec 1992; 232:141–149. 32 Feinberg RF, Kliman HJ, Lockwood CJ: Is oncofetal fibronectin a trophoblast glue for human implantation? Am J Pathol 1991;138: 537–543. 33 Romagnano L, Babiarz B: Mechanisms of murine trophoblast interaction with laminin. Biol Reprod 1993;49:374–380. 34 Farach MC, Tang JP, Decker GL, Carson DD: Heparin/heparan sulfate is involved in attachment and spreading of mouse embryos in vitro. Dev Biol 1987;123:401–410. 35 Richa J, Damsky CH, Buch CA, Knowles BB, Solter D: Cell surface glycoproteins mediate compaction, trophoblast attachment, and endoderm formation during early mouse development. Dev Biol 1985;108:513–521. 36 Damsky C, Sutherland A, Fisher S: Extracellular matrix 5: Adhesive interactions in early mammalian embryogenesis, implantation, and placentation. FASEB J 1993;7:1320–1329. 37 Vanderpuye OA, Labarrere CA, McIntyre JA: A vitronectin-receptor-related molecule in human placental brush border membranes. Biochem J 1991;280:9–17.

Horm Res 1998;50(suppl 2):46–55

55

Author Index

Asahina, T. 37 Fujimoto, J. 30 Fujiwara, H. 25 Hamatani, T. 46 Hayashi, K. 15 Hirahara, F. 7 Hirose, R. 30 Honda, T. 25 Inamoto, Y. 37 Itoh, M. 37 Iwahashi, K. 46 Kanzaki, H. 1 Kataoka, N. 25 Kobayashi, N. 46 Kobayashi, T. 37 Koshikawa, N. 7 Kuji, N. 46 Maeda, M. 25 Miyake, A. 1 Miyakoshi, K. 46 Miyazaki, K. 7 Miyazaki, T. 46

Mizushima, H. 7 Mori, T. 25 Moriyama, K. 7 Nagashima, Y. 7 Nakamura, K. 25 Nishida, W. 15 Okada, Y. 37 Sakaguchi, H. 30 Sobue, K. 15 Sueoka, K. 46 Suginami, H. 1, 2, 25 Taga, M. 1, 2 Takahashi, J. 46 Takamura, H. 7 Tamaya, T. 30 Tanaka, M. 46 Terao, T. 37 Tsutsumi, O. 1 Ueda, M. 25 Yamada, S. 25 Yamashita, M. 37 Yoshimura, Y. 1, 46

Subject Index

Adhesion molecule 2 Atherosclerosis 15 Cadherin 30 Caldesmon 15 Catenin 30 Cell adhesion 2, 7 Contractile protein 15 Corpus luteum formation 25 Decidua 46 Distributions 7 Early pregnancy 37 Endometrium 46 Extracellular matrix 25 Factor X III 37 Fibrinogen 37 Fibronectin 37 Follicular growth 25 Gene expression 15

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Immunohistochemistry 7 Implantation 46 Integrin 25 ·1 Integrin 15 ß1 Integrins 46 Laminin Á2 chain 7 Laminin-5 7 Macrophage 37 Myosin heavy chain 15 Ovary 25 Phenotypic modulation 15 Reproduction 2 Sex steroids 30 SM22 15 Smooth muscle 15 ·-Smooth muscle actin 15 Splicing 15 Vessel permeability 30