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The hormonal regulation of cadherin-11 expression in the human endometrium Chen, George Tzer-Chou 1999

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THE HORMONAL REGULATION OF CADHERIN-11 EXPRESSION IN THE HUMAN ENDOMETRIUM by GEORGE TZER-CHOU CHEN M.D., China Medical College, Taiwan, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Reproductive and Developmental Sciences Program Department of Obstetrics and Gynaecology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1999 © George Tzer-Chou Chen, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, 1 agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of QBSTET^CS ^ C ^ A / f t f i C ^ L C ^ The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT The human endometrium undergoes cyclic proliferation, differentiation, and shedding in response to 17R-estradiol and progesterone. To date, the cellular mechanisms by which these gonadal steroids regulate the formation and organisation of this dynamic tissue remain poorly understood. We have recently determined that cadherin-11, a type 2 classical cadherin, is spatiotemporally expressed in the human endometrium during the menstrual cycle. In particular, cadherin-11 is first detected in the endometrial stroma during the secretory phase of the menstrual cycle with maximum expression levels being observed in the decidua of pregnancy. As the cadherins play critical roles in the formation and organisation of tissues during embryonic development, we hypothesised that the morphogenetic effects exerted by gonadal steroids on the endometrium may be mediated, at least in part, by their ability to regulate stromal cadherin-11 expression levels. In these studies, we have examined the ability of 176-estradiol, progesterone, and dihydrotestosterone, alone or In combination, to regulate cadherin-11 mRNA and protein expression levels in cultured human endometrial stromal cells. Progesterone, but not estradiol or dihydrotestosterone, increased stromal cadherin-11 mRNA and protein expression levels over time in culture. However, 1 7S-estradiol was capable of potentiating the stimulatory effects of progesterone on stromal cadherin-11 expression. Maximum levels of cadherin-11 were detected in endometrial stromal cells which had undergone decidualisation in response to long term culture in the presence of these two gonadal steroids. Cadherin-11 expression in decidualised endometrial stromal cells was effectively reduced by the antiprogestin RU486, the antiestrogen ICI 182,780, and steroid withdrawal suggesting that the progesterone-mediated increase in stromal cadherin-11 expression was dependent on estrogens. Finally, progesterone increased the levels of the mRNA transcript encoding 6 -catenin, a cadherin-associated cytoplasmic protein, in these primary cell cultures. This is the first demonstration that gonadal steroids are capable of coordinately regulating cadherin-11 and S-catenin expression in a mammalian cell type. Collectively, these studies not only add to our understanding of the cell biology of this novel cadherin subtype but give us insight into the cellular mechanisms by which gonadal steroids regulate the remodeling processes that occur in the endometrium during each menstrual cycle. ii TABLE OF CONTENTS ABSTRACT " TABLE OF CONTENTS iii LIST OF ABBREVIATIONS vi LIST OF FIGURES viii ACKNOWLEDGMENTS xi CHAPTER I OVERVIEW 1 1.1: Introduction 1 1.2: Tissue remodeling processes which occur in the endometrium during the menstrual cycle 2 1.3: Hormonal regulation of the decidualisation of endometrial stromal cells 3 1.3.1: Steroids 3 1.3.2: Steroid hormone receptors 4 1.3.3: Cellular localisation of PR and ER in the human endometrium during the menstrual cycle 7 1.3.4: Biological actions of gonadal steroids on the endometrium 7 1.3.5: The effects of anti-steroidal compounds on the endometrium 9 1.3.5-A: Antiestrogens 9 1.3.5-B: Antiprogestins 10 1.3.5-C: Estromedins and progestomedins in the human endometrium 12 iii 1.4: Molecular mechanisms involved in endometrial remodeling 14 1.5: Thecadherins 15 1.51: Type 1 classical cadherins 16 1.5.1- A: Structure of the type 1 classical cadherins 16 1.5.1 -B: The cell biology of classical type 1 cadherins 20 1.5.1 -C: Regulation of type 1 classical cadherin expression 24 1.5.2: Type 2 classical cadherins 25 1.5.2- A: Cell biology of type 2 classical cadherins 25 1.5.3: Protocadherins —27 1.5.4: Desmosomal cadherins 28 1.5.5: Other members of cadherin gene superfamily 29 1.5.5-A: Truncated cadherins 29 1.5.5-B: Unclassified cadherin-releated proteins 30 1.6: Identification of the cadherins present in the human endometrium 30 1.7: Hypothesis and Rationale 31 CHAPTER II: GONADAL STEROIDS ARE KEY REGULATORS OF CADHERIN-11 EXPRESSION IN HUMAN ENDOMETRIAL STROMAL CELLS 34 2.1: 17fc-estradiol potentiates the stimulatory effects of progesterone on cadherin-11 expression in cultured human endometrial stromal cells 36 2.2: Cadherin-11 is a hormonally regulated cellular marker of decidualisation in human iv endometrial stromal cells 65 CHAPTER III: STUDIES EXAMINING THE MECHANISMS BY WHICH GONADAL STEROIDS REGULATE CADHERIN-11 EXPRESSION AND FUNCTION IN HUMAN ENDOMETRIAL STROMAL CELLS 91 3.1: Antisteroidal compounds and steroid-withdrawal downregulate cadherin-11 mRNA and protein expression levels in human endometrial stromal cells undergoing decidualisation in vitro 93 3.2: Progesterone regulates R-catenin mRNA levels in human endometrial stromal cells in vitro 125 CHAPTER IV: GENERAL DISCUSSION 147 SUMMARY ANDCONCLUSIONS 156 REFERENCES 157 APPENDIX I: Immunohistochemical analysis of cul tured endometrial stromal cells used i n these studies 184 LIST OF ABBREVIATIONS A N O V A Analysis o f va r iance A P C Adenomatous polyposis c o l i B S A Bovine serum a lbumin °C Degrees cen t ig rade C a C I 2 Calcium c h l o r i d e C a d Cadherin C A M Cell adhesion mo lecu le cAMP Cyclic adenosine 3 ' ,5 ' -monophospha C A R Cell adhesion r e c o g n i t i o n C A S Cadherin associated s u b s t r a t e c D N A Complementary DNA CP C y t o p l a s m i c DMEM Dulbecco's Modif ied Eagle's med ium DNA Deoxyr ibonucleic ac id D s c Desmoco l l i ns D s g Desmogle ins E2 1 7 6 - e s t r a d i o l EC E x t r a c e l l u l a r ECL Enhanced chemi luminescence ECM Extracel lu lar m a t r i x E D T A Ethylenediamine t e t r a - a c e t a t e EGF Epidermal g rowth f a c t o r ER Estrogen r e c e p t o r FSH Fo l l i c l e - s t imu la t i ng hormone Fn F ib ronec t in g Grams 9 G r a v i t y h Hours hCG Human chorionic gonadot rop in HRE Hormone responsive e lement IGF Insu l i n - l i ke g rowth f a c t o r I G F B P - 1 IGF binding p r o t e i n - 1 I g G Immunoglobul in G k b k i lobases k D a k i l o D a l t o n s L n Lamin in MDCK Madine Darby canine k idney vi m i n Minutes m l M i l l i l i t e r mM Mi l l imolar MMP Matrix metal loproteinase Mr Molecular weight mRNA Messenger RNA MUC1 Episial in ul Micro l i ter uM Micro-Molar N - l i n k e d Asparagine-l inked N a + , K + - A T P a s e Sodium, potassium-adenosine triphosphatase nM Nano-Molar NaCl Sodium chloride P Probabi l i ty P4 Progesterone PA Plasminogen act ivator P A I PA inhib i tor PBS Phosphate-buffered saline Pcdh Protocadherin PGE2 Prostaglandin E2 PMSF Phenylmethyl sulfonyl f lour ide PR Progesterone receptor PRL Prolact in RNA Ribonucleic acid rRNA Ribosomal RNA RT Room temperature RT-PCR Reverse transcriptase-polymerase chain react ion S Svedberg unit of f l o ta t ion SDS Sodium dodecyl su l fa te SEM Standard error of the mean s r c Rouse sarcoma virus SSPE Standard saline phosphate-EDTA TGF-p Transforming growth fac to r -p T IMP Tissue inhibitor o f MMP TM Transmembrane T r i s - H C L Tris (hydroxymethyl)-aminomethane-Hydrochloric acid uPA Urokinase-type PA ZO Zonula occludens vii LIST OF FIGURES Figure 1: Schematic representation of the basic classical cadherin structure 19 Figure 2: Northern blots demonstrating the effects of gonadal steroids on cad-11 in cultured human endometrial stromal cells at different time points 51 Figure 3: Western blots demonstrating the effects of gonadal steroids on cad-11 expression in cultured human endometrial stromal cells at different time points 53 Figure 4: Northern and Western blots demonstrating the effects of estradiol plus progesterone on cad-11 expression in cultured human endometrial stromal cells at different time points 55 Figure 5: Northern and Western blots demonstrating the effects of dihydrotestosterone plus progesterone on cad-11 expression in cultured human endometrial stromal cells at different time points 57 Figure 6: Northern and Western blots demonstrating the effects of varying concentrations of estradiol on the progesterone-mediated increase of cad-11 in cultured human endometrial stromal cells 69 Figure 7: Autoradiograms of a Northern blot demonstrating the effects of progesterone on cad-11 and insulin-like growth factor binding protein-1 in long term cultured human endometrial stromal cells 79 Figure 8: Northern blots demonstrating the effects of vehicle viii and estradiol on cad-11 in long term cultured human endometrial stromal cells 81 Figure 9: Autoradiograms of a Northern blot demonstrating the effects of estradiol on progesterone-mediated cad-11 and insulin-like growth factor binding protein-1 increasing in long term cultured human endometrial stromal cells 83 Figure 10: Western blots demonstrating the effects of gonadal steroids on cad-11 expression in long term cultured human endometrial stromal cells at different time points 85 Figure 11: Northern blots demonstrating the time- and dose-dependent effects of antiprogestin (RU486) on cad-11 in human endometrial stromal cells undergoing decidualisationb 109 Figure 12: Western blots demonstrating the time- and dose-dependent effects of antiprogestin (RU486) on cad-11 in human endometrial stromal cells undergoing decidualisation 111 Figure 13: Northern and Western blots demonstrating the dose-dependent effects of antiestrogen (ICN 82,780) on cad-11 in human endometrial stromal cells undergoing decidualisation 113 Figure 14: Northern and Western blots demonstrating the comparison of the effects of antiprogestin (RU486) and antiestrogen ICI 182,780 on cad-11 in human endometrial stromal cells undergoing decidualisation 115 ix Figure 15: Northern blots demonstrating the effects of steroid-withdrawal on cad-11 in human endometrial stromal cells undergoing decidualisation 117 Figure 16: Western blots demonstrating the effects of steroid-withdrawal on cad-11 in human endometrial stromal cells undergoing decidualisation 119 Figure 17: Autoradiograms of a Northern blot demonstrating the effects of vehicle on 6-catenin in cultured human endometrial stromal cells 136 Figure 18: Autoradiograms of a Northern blot demonstrating the time-dependent effects of progesterone on 6-catenin in cultured human endometrial stromal cells 138 Figure 19: Autoradiograms of a Northern blot demonstrating the dose-dependent effects of progesterone on 6-catenin in cultured human endometrial stromal cells 140 Figure 20: Autoradiograms of a Northern blot demonstrating the effects of varying concentrations of estradiol on the progesterone-mediated increase of 6-catenin in cultured human endometrial stromal cells 142 X ACKNOWLEDGMENTS I would like to take this opportunity to express my indebtedness to Colin D. MacCalman for the immeasurable encouragement, voluminous advice, and excellent guidance afforded me in these studies. In addition, I would like to extend my appreciation to Mary D. Stephenson for her generous support and providing endometrial biopsy specimens. Furthermore, I am grateful to the faculty members and colleagues in the Dept. Ob/Gyn during the course of my studies, especially, the constructive suggestions offered by Spiro Getsios. Finally, I would like to thank ICOS Corp. for their kind gifts of reagents used in these studies. xi CHAPTER I: OVERVIEW 1.1: I n t r o d u c t i o n Infert i l i ty affects more than 4.9 million couples in North America (Mosher and Pratt 1991). We stil l do not fully comprehend many of the molecular defects responsible for this reproductive health problem. However, it is believed that implantation failure may contribute to more than 20% of these cases. In addition, despite increasing experience with assisted reproductive technologies, only 22.5% o f women undergoing in vitro fert i l izat ion and embryo t ransfer establishes a viable pregnancy (Anonymous 1998). A limiting factor i n this sett ing may also be the inability of the blastocyst to in teract with the endometrium during implantat ion. In the establishment of a successful pregnancy, human endometrium must have undergone hormonally-dependent proliferation and dif ferentiat ion in preparation for the implanting embryo (Tabibzadeh and Babaknia 1995). To date, the cellular mechanisms mediate these cyclic tissue remodeling processes remain poorly characterised. The main objectives of these studies were to determine whether gonadal steroids were capable of regulating the expression of cadherin-11 (cad-11) a cell adhesion molecule (CAM) which has been detected in endometrial stromal cells undergoing decidualisation during the secretory phase of the menstrual cycle and decidua of ear ly pregnancy. The mechanism(s) by which gonadal steroids may regulate the expression and function of cad-11 during the termina l l differentiation of endometrial stromal cells into decidual cells were subsequently investigated. In this section, the morphogenetic events which occur in the human endometrium during the menstrual cycle will be described and the molecular mechanisms by which gonadal steroids regulate this highly regulated series of developmental processes will be discussed. The cellular mechanisms which are believed to mediate the formation, organisation and differentiation of this dynamic tissue will then be examined. Finally, the structure, function and regulation of the cadherin gene superfamily will be reviewed. This family of CAMs are key morphoregulators and are therefore likely to play a central role in the tissue remodeling processes which occur in the human endometrium in preparation for the implanting embryo. 1.2: Tissue remodeling processes which occur in the human endometrium during the menstrual cyc le During each menstrual cycle, the epithelial and stromal cells of the human endometrium undergo cyclic proliferation, differentiation and shedding under the influence of gonadal steroids, 17B-estradiol (E2) and progesterone (P4) (Noyes et al. 1950). During the proliferative phase of the menstrual cycle, when E2 is the predominant steroid, the human endometrium is comprised of a dense cellular stroma containing narrow tubular glands. The increase in P4 during the secretory phase promotes glandular secretion and the decidualisation of endometrial stromal cells. 2 The process of decidualisation in the endometrial stromal cells is associated with morphological and functional changes (Kearns and Lala 1983). Morphologically, decidualisation is expressed by a change to a polyhedral cell shape with an increase in cell size. Ultrastructurally, there is an extensive development of the organelles involved in protein synthesis (rough endoplasmic reticulum) and secretion (Golgi apparatus) (Kearns and Lala 1983; Wynn 1974), and the formation of desmosomes and gap junctions (Jahn et al. 1995; Lawn et al. 1971). Functionally, decidualisation is characterised by the onset of prolactin (PRL) (Maslar and Riddick 1979) and insulin-like growth factor binding protein-1 (IGFBP-1) (Bell 1991) secretion. Although the morphological and functional characteristics of decidual cells are well characterised, the sequence of molecular and cellular events involved in the differentiation of stromal fibroblasts into decidual cells remain poorly understood. 1.3: Hormonal regulation of the decidualisation o f endometrial stromal c e l l s 7.3.7: Steroids The ovary is the major site of synthesis and secretion of gonadal steroids in women and is responsible for the fluctuations in the levels of these hormones during the menstrual cycle (Ryan 1970;Tsang 1980). Recent studies also suggest that the human endometrium has an intrinsic capacity to synthesise and secrete E2 (Tseng 1984; Tseng et al. 1986; Yamamoto et al 1993). The biological significance/actions of 3 E2 produced and secreted by the endometrium remain poorly understood. 7.3.2; Steroid hormone receptors The actions of steroids on target tissues are mediated by specific receptor proteins (Szego 1984; Graham and Clarke 1997). The steroid hormone receptor superfamily includes glucocorticoid, mineral-corticoid, progestin, estrogen, androgen, and 1a,25-dihydroxyvitamin D3 receptors. All members of this superfamily share similar structural and functional characteristics and have highly conserved structural domains involved in DNA and ligand binding. A detailed description of the structure and function of the classic steroid receptors is beyond the scope of this literature review. Briefly, within the intact cel l , the unoccupied form of the classic steroid receptor resides in the nucleus or in the nucleus and cytoplasm, dependent on the receptor and cell type, as part of a hetero-oligomeric complex that includes the heat shock proteins, hsp90 and hsp70 (Pratt and Toft 1997). Steroid hormones act in the target tissues with a mechanism that may be summarised in the following steps: 1) free steroid enters the target cell by passive diffusion through the plasma membrane, passes through the nuclear membrane (in the case of nuclear receptors), and binds with high affinity to the C-terminus of the inactive receptor; 2) the steroid-receptor complex undergoes activation, a process that involves conformational changes (tert iary and quaternary structure) and enables the receptor to bind to selective sites on the chromatin; 3) the activated steroid-receptor complex 4 interacts with specific DNA sequences known as steroid response elements (HREs), usually located upstream of the steroid-responsive genes; 4) the activated steroid receptor complex acts as a transcription factor modulating the synthesis of specific mRNA and proteins, which in turn, are responsible for the final cellular effect(s) of the hormone. The P4 receptor (PR) is composed of two hormone binding proteins, designated PRA and PRB (Truss and Beato 1993). These two proteins are encoded by a single gene under the control of distinct promoters, each of which generates distinct PR mRNA transcripts (Kastner et al. 1990). PRA and PRB are both capable of binding progestins and interacting with HREs. However, there is increasing evidence to suggest that they are functionally distinct. For example, in transfection studies these two proteins have different abilities to activate progestin responsive promoters (Tora et al. 1989; Vegeto et al. 1993). These differences were promoter- and cell-specif ic suggesting that cellular responsiveness to progestins may be modulated via alterations in the ratio of PRA and PRB expression. Although PRB tends to be a stronger activator of target genes, PRA can act as a dominant repressor of PRB (Tung et al. 1993; Vegeto et al. 1993). These observations suggest that high PRA expression may result in reduced progestin responsiveness and that PRA and PRB may thus be a repressor and activator of P4 action, respectively. In general, PR expression levels are believed be regulated by E2 and P4 which increase and decrease the levels of this receptor in target tissues, respectively (Levy et al. 1980). In agreement with these 5 observations, E2 has been shown to up-regulate PR whereas P4 has been shown to decrease the levels of both protein isoforms in isolated human glandular epithelial cells (Eckert and Katzenellenbogen 1981; Evans and Leavitt 1980; Katzenellenbogen 1980; Kreitmann et al. 1979). In contrast, P4 was shown to be capable of coordinately up-regulating the expression levels of PRA and PRB in human endometrial stromal cells in vitro (Tseng and Zhu 1997). Recently, a second isoform of the ER has been reported in certain E2-responsive tissues in rat (Kuiper et al. 1996) and human (Mosselman et al. 1996). This isoform, termed ER-fc, is highly homologous to the a-isoform of the receptor, particularly in the DNA-binding and ligand-binding domains (Kuiper et al. 1997; Kuiper et al. 1996). In ligand binding assays ER-fc has been shown to bind E2 with an affinity and specificity that is similar to ER-a (Kuiper eta l . 1996). ER-R is able to activate transcription of E2-response element-containing reporter gene constructs (Kuiper et al. 1996). Furthermore, homodimers and heterodimers of these two ER isoforms are capable of activating transcription, in an E2-dependent manner, from reporter gene constructs containing estrogen response elements. The activation of these gene constructs was more efficient with homodimers of ER-a (Pettersson et a I. 1997). However, recent studies have demonstrated that these two ER subtypes have opposite regulatory modes to the natural hormone from the same DNA response element in transfection studies (Paech et al. 1997). In particular, with ERa, E2 activates transcription, whereas with ER6, E2 inhibits transcription at AP-1 site(Paech et al. 1997). 6 ER-ct appears to be the predominant isoform present in the rat uterus although mRNA transcripts encoding the 6-isoform have been detected in this tissue. In contrast, ER-fc is the predominant isoform expressed by the rat ovary. In addition, the ovarian levels of the mRNA transcripts encoding this ER isoform appear to be regulated during the reproductive cycle (Byers et al. 1997). To date, it has not been determined whether the two different isoforms of ER are differentially expressed in the human endometrium during the menstrual cycle. 7.3.3; Cellular localisation of PR and ER in the human endometrium during the menstrual cycle ER and PR have been shown to be spatiotemporally expressed in the endometrium during the menstrual cycle (Garcia et al. 1988; Ingamells et al. 1996; Lessey et al. 1988). In particular, ER and PR levels are high in both epithelial and stromal cells of the endometrium during the proliferative phase and decrease in epithelial cells during the secretory phase of the menstrual cycle (Lessey et al. 1988). Aberrantly, high levels of PR have been detected in the endometrial epithelium of primary infertile women diagnosed with luteal phase deficiency (Lessey et al. 1996). 7.3A: Biological actions of gonadal steroids on the endometrium The ability of gonadal steroids to regulate the cyclic remodeling processes which occur in the endometrium in preparation for the implanting embryo were first demonstrated using ovariectomised 7 rodent model systems (Psychoyos 1976). However, recent studies employing cultures of endometrial explants (Bentin-Ley et al. 1994), isolated endometrial stromal cells (Lockwood et al. 1998), and endometrial carcinoma cell lines (Somkuti et al. 1997) have given us useful insight into the hormonal regulation of the molecular and cellular mechanisms operative in the human endometrium during the menstrual cycle. Endometrial stromal cells have been isolated from human proliferative, secretory and anovulatory endometrium (Irwin et al. 1989). These endometrial tissue specimens have been obtained from women undergoing hysterectomy for a variety of medical conditions including uterine prolapse, fibroid uterus, adenomyosis, stress incontinence, and cancer of the cervix and ovary but not the endometrium (Han et al. 1997; Irwin et al. 1989; Lane et al. 1994; Tseng et al. 1996); women undergoing dilatation and curettage for the termination of pregnancy and excision of tubal pregnancy (Grosskinsky et al. 1996; Irwin et al. 1996; Schatz and Lockwood 1993); and women undergoing diagnostic biopsy for reproductive failure including luteal phase defect, habitual abortion and infertility (Bruner e ta l . 1995; Satyaswaroop et al. 1979). Despite the source of the endometrial tissue, isolated endometrial stromal cells grow in monolayers and can be passaged up to nine times without losing their responsiveness to gonadal steroids (Irwin et al. 1989; Irwin et al. 1991). Although E2 does not appear to have any morphological or cellular effect on the cultured endometrial stromal cells, P4 has been shown to promote cellular proliferation with short term culture and the production of cellular and biochemical markers of decidualisation, including IGFBP-1, prolactin and the secretion of extracellular matrix components with prolonged culture in the 8 presence of this gonadal steroid (Bell et al. 1991; Chen et al. 1989; Irwin et al. 1989). The stimulatory effects of P4 on the production of these biochemical markers of decidualisation can be potentiated by the addition of E2 to the culture medium (Bell et al. 1991; Chen et al. 1989; Irwin et al. 1989; Irwin et al. 1991). Consequently, cultured human endometrial stromal cells have become a widely used model system for the study of the complex process of decidualisation. 7.3.5; The effects of anti-steroidal compounds on the endometrium The effects of P4 and E2 on the human endometrium are considered to be paramount for the establishment of a successful pregnancy. This i s highlighted by recent studies examining the effects of synthetic an t i -steroidal compounds on the endometrium. 7.3.5-/4; Antiestrogenic compounds Tamoxifen is a nonsteroidal triphenylethylene derivative, closely related to diethylstilbestrol and clomiphene citrate, w i th predominantly antiestrogenic activity (Daniel et al. 1996). The antiestrogenic effects of tamoxifen are mediated by its ability to bind to the ER, transferring the complex to the nucleus (Murphy et al. 1990) and is widely used in the treatment of women with breast cancer (Fisher e ta l . 1996). This results in competitive inhibition of E2 owing to a reduction in free receptors. However, although tamoxifen acts as an estrogen antagonist in the breast, it also has agonist activity in the vaginal epithelium and endometrium (Timmerman and Vergote, 1996). Several of its metabolites are as effective and as potent as tamoxifen but their relevance to the agonistic effects on the endometrium are controversial. These agonistic effects are manifested in an increase in 9 the proliferation and neoplastic transformation of endometrial cells (Daniel et al. 1996). To date, the mechanisms by which tamoxifen exerts its agonist effects on the endometrium have yet to be clarified. 7a-alkylamide analogues of E2, such as ICI 164,384 and ICI 182,720, are commonly referred to as "pure" antiestrogens as they do not appear to exhibit any agonistic effects in E2-responsive tissues (Nicholson. 1996). The mechanism(s) by which 7a-alkylamide analogues of E2 exert their antiestrogenic effects in target cells remain poorly understood. However, these compounds have been shown to reduce ER protein expression levels by increasing protein turnover without affecting ER mRNA levels (Gibson et al., 1991; Dauvois et al., 1991). Other possible mechanisms by which 7a-alkylamide analogues of E2 are believed to act as antiestrogens include the disruption of ER dimerisation and subsequent DNA binding (Fawell et al., 1990), a reduction in transcriptional activation by the DNA-bound ER-antiestrogen complexes (Arbuckle et al., 1992) and/or blockade of ER entry into the nucleus (Reese et al., 1992) 7a-alkylamide analogues of E2 have been shown to inhibit the E2 mediated development of the primate and rodent uterus (Branham et a I 1996; Dukes et al. 1992; Dukes et al. 1993). 1.3.5-B: Antiprogestins Although a number of antiprogestin compounds including onapristone (ZK 98299), lilopristone (ZK 98734), ORG-31710, ORG-31806, and CDB-2194 (RTI3021-012) have been synthesised and characterised 10 (Chwalisz et al. 1997), none of these compounds have been studied as extensively as mifepristone, commonly known as RU486. RU486 is an 11 ft-dimethyl-amino-phenyl derivative of norethidrone which binds PR and glucocorticoid receptors with a higher affinity than their naturally occurring ligands (Baulieu 1997). RU486 effectively excludes the corresponding agonist from receptor binding which in turn, is eliminated from the target cells or metabolised in situ. Af ter RU486 binds the PR, interactions between the receptor and i ts associated heatshock protein, hsp90, are strengthened. Although the increased stability of the PR-hsp90 complex is believed not to interfere with interactions between the PR and the corresponding hormone response elements, RU486 effectively inhibits the transcription of P4-regulated genes (Baulieu 1988; Baulieu 1997). In addition, it is believed that RU486 may decrease PR levels and simultaneously decrease ER levels in the human endometrium (Zaytseva et al. 1993). Since P4 is an essential hormone for the establishment and maintenance of pregnancy, RU486 acts as an effective contragestion when administered during the luteal phase of the menstrual cycle and/or as an abortificant when administered during pregnancy (Baulieu 1988; Baulieu 1997). The precise cellular mechanism(s) by which RU486 induces abortion is not well understood. However, administration of RU486 during the early luteal phase of the menstrual cycle retards the development of a secretory endometrium without affecting the function of the corpus luteum or the length of the menstrual cycle (Gemzell-Danielsson et al. 1994; Swahn et al. 1990). In addition, RU486 has been shown to induce degenerative l l morphological changes in decidua before the onset o f bleeding and abor t ion (Schindler et al. 1985) Col lect ive ly , these observa t ions suggest t ha t RU486 has a d i rect e f fec t on the human e n d o m e t r i u m 1.3.5-C: Estromedins and progestomedins in the human endometrium Growth fac to rs have been shown t o mediate, at least in part , t he regula tory e f fec ts o f gonadal s tero ids on ta rge t t issues, including the human endometr ium (Giudice 1 9 9 4 ; Tabibzadeh and Babaknia 1995) . These polypept ide g rowth fac to rs , in tu rn , may act in an autocr ine or paracrine manner t o modulate cel lular p ro l i fe ra t ion a n d / o r d i f f e r e n t i a t i o n . Epidermal g rowth fac to r (EGF) and the insu l in- l ike g rowth f a c t o r - 1 (IGF-I) have been impl ica ted as est romedins (media tors o f E2 act ion) i n the uterus (Giudice 1994) . EGF is capable o f p romot ing p ro l i fe ra t ion in many cell t ypes including f ib rob las ts , kera t inocy tes and e p i t h e l i a l cells (Giudice 1994) . This potent mi togen has also been shown t o s t imu la te cel lular p ro l i fe ra t ion in human glandular epi thel ia l cells but not endometr ia l s t romal cells cu l tured in the presence of E2 (Haining e t al. 1991 ) . In con t ras t , IGF-1 has been shown t o s t imu la te s t r o m a l cell p ro l i fe ra t ion (Saji e t a l 1990) . However, both IGF-I and IGF-II a re capable o f p romot ing cel lular p ro l i fe ra t ion in endometr ia l s t r o m a l cells undergoing decidual isat ion in response t o t r e a t m e n t w i t h a combinat ion of E2 and P4 (Rosenberg et al 1991) . In the human endomet r ium, moderate EGF immunosta in ing has been de tec ted i n glandular epi thel ia l and s t romal cells o f the human endomet r ium dur ing the p ro l i fe ra t i ve phase of the menstrual cycle. There is a marked 12 increase in the endometrial stromal cells surrounding the sp i ra l arteries (the areas of early decidualisation) as the menstrual cycle enters the secretory phase (Hofmann et al. 1991) whereas only moderate staining is observed in the luminal surface of exhausted secretory glands. Similarly, IGF-II mRNA levels are low in the proliferative phase of the menstrual cycle but are readily detectable i n late secretory endometrium and the decidua of early pregnancy (Giudice et al. 1993). Recent studies suggest that TGF-fcl may act as a progestomedin (a mediator of P4 action) i n the human endometrium (Chegini et al. 1994; Giudice 1994). Although TGFfc-1 mRNA levels are equally d is t r ibuted in the glandular epithelium and stroma of the human endometrium, the mRNA and protein expression levels of this growth factor are differential ly regulated during the menstrual cycle (Bruner et al. 1995; Chegini et al. 1994). TGFfc-1 mRNA and protein expression levels are low during the proliferative phase but increase dramatically during the secretory phase w i t h maximum levels being observed in the decidua of early pregnancy. There is a marked decrease in endometrial TGFG-1 expression levels at the onset of menstruation. The regulated expression of TGF6-1 during the menstrual cycle has led to the proposal that this growth factor may contribute to the P4-mediated decrease in cellular proliferation and the concomitant increase in differentiation. In support of this hypothesis, TGFR-1 has been shown to decrease cellular proliferation in primary cultures of human endometrial stromal cells. However, the ability of TGFfc-1 to promote cellular dif ferentiat ion in cultured endometrial stromal cells has yet to be determined. Finally, TGF-&1 produced by cultured human 13 endometrial stromal cells in response to P4 can suppress the expression of matrilysin in glandular epithelial cells (Bruner et al. 1995). The P4-mediated decrease in epithelial matrilysin production can be abolished by antibodies directed against TGFfc-1. The inhibitory effects of P4 on E2-mediated cell proliferation in the endometrium during the menstrual cycle may be mediated through the actions of IGF-binding protein 1 (IGFBP-1). In humans, IGFBP-1 expression is tightly regulated with the greatest levels being observed in the mid-late secretory phase of the menstrual cycle (Giudice et al. 1993). Increased IGFBP-1 secretion has also been observed in endometrial stromal cells cultured in the presence of P4 (Bell et al. 1991). IGFBP-1 may act in a paracrine fashion to prevent epithelial cell proliferation by binding to IGF. This interaction would prevent IGF-1 binding to its receptor, which in turn, would reduce the cellular response to this growth factor. IGFBP-1 may also act in a autocrine fashion by inhibiting the proliferative effects of IGF in cultured human endometrial stromal cells (Zhou et al., 1994) 1.4: Molecular mechanisms involved in endomet r ia l remodel ing The highly regulated developmental processes which occur in the human endometrium in preparation for the implanting embryo are l ikely to involve complex changes in cell-cell and cell-matrix interactions. To date, studies have focused on the differentiation of the endometrial extracellular matrix (ECM) during the menstrual cycle (Aplin et al. 1988; Church et al. 1996), the breakdown of these matrices during 14 menstruation (Salamonsen et al. 1997), and the spatiotemporal expression of integrin subunits, a family of cell surface receptor which mediate cell-matrix interactions (Lessey et al. 1992). As a result, the adhesive mechanisms which mediate cell-cell interactions in the human endometrium during the menstrual cycle remain poorly characterised. 1.5: The Cadherins The cadherins are an expanding gene superfamily of integral membrane glycoproteins which mediate calcium-dependent cell adhesion in a homophilic manner (Takeichi 1991; Takeichi 1995). Recent cloning studies suggest that this family of CAMs can be subdivided into four distinct subfamilies on the basis of their amino acid sequences: classical cadherins (type 1 and type 2), desmosomal cadherins, protocadherins and "cadherin-related" proteins, a diverse subfamily which includes truncated cadherins and as yet, unclassified family members (Suzuki et al. 1996a). In general, members of the cadherin gene superfamily are composed of an extracellular domain, a transmembrane domain and a cytoplasmic domain (Takeichi 1991; Suzuki et al. 1996a). The extracellular domains of the cadherins are comprised of multiple repeats of a cadherin-specific motif (cadherin-repeat). This motif is approximately 110 amino acids in length and contains several conserved sequences which differ from one another within a protein and between subfamilies. Thus, each cadherin-repeat present in the members of a distinct cadherin subfamily is believed to have its own 15 characteristic properties and fulfill discrete biological function(s). The number of cadherin-specific motifs varies from four to more than thirty among the cadherin subfamilies (Suzuki 1996a). In contrast to the extracellular domains, the cytoplasmic domains of cadherin gene superfamily members are highly variable in length and amino acid composition (Suzuki 1996a) have led to the subclassification of the cadherin gene superfamily described below. 7.5.7: Type 7 classical cadherins The type 1 classical cadherin subfamily including the three originally identified cadherins, E-cadherin (E-cad), N-cadherin (N-cad) and P-cadherin (P-cad), are the best characterised representatives of this CAM gene superfamily (Suzuki 1996a; Takeichi 1995). 7.5.7-A; Structure of type 7 classical cadherins The type 1 classical cadherins are composed of five extracellular, a transmembrane, and two cytoplasmic subdomains (Geiger 1992) (Fig 1). The first four extracellular subdomains contain the calcium binding regions. In addition, the first extracellular domain of each classical cadherin contains the cell adhesion recognition (CAR) amino acid sequence, His Ala Val (HAV) (Blaschuk 1990). The non-conserved amino acid residues immediately adjacent to the CAR site of distinct cadherin subtypes modulate the homophilic interactions between identical cadherins (Nose 1990). Recently, the three-dimensional structures o f the extracellular domains of E-cad (Overduin 1995) and N-cad (Shapiro 1995) have been determined. These studies suggest that the identical cadherin subtypes expressed on the membrane of 16 adjacent cells are dimerised to form a zipper-like interactions during cadherin-mediated cell adhesion. The cytoplasmic subdomains of the type 1 classical cadherins are highly conserved and capable of interacting with at least four intracellular proteins known as a - , IS-, y-catenin and p120 c a s (Kemler 1993; Knudsen 1992; Reynolds 1996). The catenins mediate the interactions between the type 1 classical cadherins and the actin microfilaments of the cytoskeleton (Kemler 1993; Knudsen 1992). The type 1 classical cadherins cannot promote cell adhesion without forming a functional complex with catenins (Kemler 1993; Aberle 1996). These cadherins bind directly to fc-catenin and y-catenin in a mutually exclusive manner (Nathke 1994). a-catenin interacts w i th both cadherin-6- or y-catenin complexes and links them to the cytoskeleton either by direct interaction (Nagafuchi 1991) or via a -actinin (Knudsen 1995). As the two cadherin-catenin complexes are differentially expressed during cellular differentiation and development, it has been proposed that they generate two structurally and functionally distinct membrane domains (Aberle 1996; Nathke 1994). In addition, S-catenin is associated with EGF (Hoschuetzky 1994), and APC (Su 1993), suggest that the catenins not only modulate cadherin-mediated cell adhesion but may also link the cadherins to several receptor-mediated intracellular signalling pathways (Aberle 1996). Elucidating specific functions for the distinct cadherin-catenin complexes will give us a better understanding of how catenins link the process of cadherin-mediated cell adhesion with signal transduction. 17 Figure 1: Diagram of the overall basic structure of type 1 and type 2 classical cadherins. The cadherins are comprised of five extracellular (EC1-EC5), a single transmembrane (TM) and two cytoplasmic (CP1 and CP2) subdomains. The first four extracellular subdomains (ECI-EC4) contain the acidic amino acid motifs, DXNDN and DXD (a and b, respectively), which are capable of binding calcium. The CAR sequence identified in type 1 classical cadherins (HAV; single acid amino code), is indicated in the most distal extracellular subdomain (EC1) of type 1 classical cadherins. The cytoplasmic subdomains of the classical cadherins interact with a family of cytoskeletal-associated proteins, known as the catenins. These intracellular interactions link the cadherins to the microfilaments of the cytoskeleton. 18 a a a < < < _j _ J _ J * * ^ LL LL LL "O "O "O G3 CO CO 0 o o 1 I • Z Hi CL 19 7.5.7 -B: The cell biology of type 7 classical cadherins The type 1 classical cadherins have been shown to play key roles in the morphogenetic events such as cell migration, cell sorting, tissue remodeling, and organogenesis during the embryonic development (Takeichi 1991; Takeichi 1995; Gumbiner 1996). The spatiotemporal expression of these cadherin subtypes is highly regulated during development, in particular, E-cadherin is expressed in the mouse embryo at the one cell stage (Hyafil 1981) and the later derived epithelial cells, P-cad is first expressed in the mural trophectoderm of 4.5-day blastocysts (Kadokawa 1989), and N-cad is first expressed by the mesodermal cells of the gastrula (Takeichi 1988). In general, the expression pattern of these cadherin subtypes appears to be complementary. The regulated expression of classical type 1 cadherins during embryonic development i s believed to govern the developmental fate of cells and mediate the formation and organisation of discrete tissues (Suzuki 1990; Gumbiner 1996). For example, the central role of E-cad in early embryonic development has been confirmed by the embryonic lethality of the E-cad-null mouse (Larue 1994). In particular, E-cad-deficient embryos develop into abnormal blastocysts which cannot form trophectoderm epithelium (Larue 1994; Reithmacher 1995). Similary, N-cad null mutant mice fail to form proper yolk sacs and exhibit neural and cardiac defects (Radice 1997). Furthermore, the transfection of E-cad null mutant embryonic stem cells with E-cad cDNA resulted in the formation of epithelium whereas the transfection of N-cad cDNA promoted the formation of cartilage and neuroepithelium. 20 In the adult mouse, the type 1 classical cadherins are expressed in different tissues (Takeichi 1988). E-cad is expressed by epithelial but not muscle or most neural cells. In contrast, N-cad has been localised primarily to muscle and neural tissues. P-cad can be detected in significant levels in the murine placenta and decidua. Although E-cad and N-cad have been localised to epithelial and neural tissues in the human (Suzuki 1990; Shimoyama 1989; Shimoyama 1991), P-cad has not been detected in the human placenta and decidua (Shimoyama 1989). Type 1 classical cadherins are capable of mediating the formation of the intercellular junctional complex before themselves being restricted to the protein plaques of the adherens junction (Gumbiner 1988; Marrs 1995). In particular, E-cad has been shown to promote the formation of tight junctions, belt desmosomes and spot desmosomes in cultured Madin-Darby canine kidney (MDCK) cells (Gumbiner 1988). E-cad and N-cad have also been shown to mediate the formation of gap junctions in cardiomyocytes (Hertig 1996) and keratinocytes (Wheelock 1992). Collectively, these observations suggest that the formation of the intercellular junctions is dependent on an early mebrane-mediated event which is governed by the type 1 classical cadherins. Type 1 classical cadherins play a key role in the establishment of epithelial cell polarity (McNeill et al. 1990; Drubin et al. 1996). For example, E-cad has been shown to restrict the distribution of Na+K+-ATPase, an ubiquitous protein whose distribution is normally restricted to the basolateral membrane domain of the polarised epithelial cells, including the mural trophectoderm of the mammalian 21 blastocyst (McNeill et al. 1990; Watson et al. 1990). During the early stages of mammalian development Na+K+-ATPase is found in discrete intracellular foci of cells comprising the morula. It then disappears from the cells of the inner cell mass as cavitation commences, and becomes localised to the basolateral plasma membranes of the polarised cells that comprise the mural trophectoderm. This sequence of events is disrupted when 4-cell stage embryos are treated w i th antibodies directed against E-cad. These antibodies inhibit compaction at 8-cell stage embryo, thereby preventing the formation of the inner cell mass and the polarised trophectoderm. These antibodies also cause the blastomeres of the uncompacted embryo to develop f lu id -filled cavities lined by membranes rich in Na+K+-ATPase. These observations suggest that the polarised trophectoderm of the mammalian blastocyst arises as a consequence of E-cad-mediated events between the blastomeres of the developing embryo. Further evidence to support the hypothesis that E-cad can act as an inducer of cell surface polarity, was obtained by transfecting fibroblasts with a full-length E-cad cDNA. In the transfected cells, Na+K+-ATPase and cytoskeletal component, known as fodrin, become localised to areas of cell-cell contacts which resembled the expression pattern in the polarised epithelial cells. In contrast, Na+K+-ATPase was not redistributed in fibroblasts transfected wi th truncated E-cad lacking the catenin-binding region (McNeill et al. 1990). In view of these observations, it has been proposed that the homophilic binding of E-cad on adjacent fibroblasts initiated a cascade of molecular events that result in the assembly of a membrane complex 22 composed of E-cad and Na+K+-ATPase, which in turn was linked to the cytoskeleton byfodrin (McNeill 1990). E-cad expression also maintains the differentiated, non-invasive phenotype of epithelial cells (Behrens 1989; Birchmeier 1994; Blaschuk 1994). A landmark study by Behren et al. (1989) demonstrated that antibodies directed against E-cad disrupted the intercellular junctional complexes which had formed between adjacent MDCK cells. Loss of E-cad function in this mature epithelial cell type promoted the development of a fibroblast-like morphology. These fibroblastic-like MDCK cells become motile and acquire the ability to invade collagen gels and embryonic chick heart tissue. Loss of E-cad expression and/or function has also been associated with the neoplastic transformation of epithelial cells in vivo (Birchmeier 1994; Blaschuk 1994). In general, poorly differentiated, highly metastatic carcinoma cells express low, or undetectable amounts of E-cad whereas well-differentiated, poorly metastatic carcinoma cells frequently express this CAM. These observations have led to the hypothesis that E-cad is the product of a tumor suppressor gene (Birchmeier 1991). 7.5.7-C: Regulation of type 7 classical cadherin expression Recent studies have demonstrated that gonadal steroids are capable of regulating type 1 classical cadherin expression in various mammalian tissues. In particular, E2 is a key regulator of E-cad mRNA levels in the mouse ovary and uterus in vivo (MacCalman et al. 1994b) and human breast carcinoma cell lines in vitro (Jednak et al. 1993). E2 is also 23 capable of regulating N-cad expression in rat granulosa cells, in v i t ro (Blaschuk and Farookhi 1989) and N-cad mRNA levels in the immature mouse ovary and testis in vivo (MacCalman and Blaschuk 1994; MacCalman et al. 1995). Finally, P4 appears to be capable of regulating E-cad mRNA levels in the immature mouse uterus in vivo (MacCalman et al. 1994a). FSH and its secondary intracellular messenger cAMP have also been shown to regulate type 1 classical cadherin expression in mammalian tissues and cells. For example, FSH and cAMP have been shown to increase N-cad mRNA levels in isolated mouse Sertoli cells (MacCalman et al. 1997a). However, E2 was capable of potentiating the cAMP-mediated increase in the transcript encoding N-cad in these primary cell cultures. Finally, cAMP regulates E-cad expression in dog and human thyrocytes in vitro (Brabant et al. 1995) and human choriocarcinoma cell lines (Coutifaris e ta l . 1991). 7.5.2; Type 2 classical cadherins. The type 2 classical cadherin subfamily includes human cadherin-5,-6,-8,-11, -12 and-14 (Suzuki 1990; Tanihara 1994; Shibata 1997) as well as other cadherin subtypes identified in the rodent (Korematsu 1997), chicken (Nakagawa 1995) and Xenopus (Espeseth 1995). Type 2 classical cadherins, like the type 1 classical cadherins, are comprised of five extracellular subdomains, a transmembrane domain and two cytoplasmic subdomains (Suzuki 1990; Tanihara 1994; Takeichi 1995). Similar to classical cadherins, the first four extracellular domains of the type 2 cadherins contain the conserved calcium binding sites but 24 none of them harbor the CAR sequence, HAV. Although the CAR sequence for type 2 cadherins has not been determined, these CAMs are capable of mediating cell adhesion in a homophilic manner (Nakagawa 1997; Kimura 1995). Type 2 classical cadherins contain the highly conserved cytoplasmic domains specific for most cadherins and have been shown to interact with G-catenin (Shibata 1996;Shibata 1997). 7.5.2-A: Cell Biology of Type 2 Classical Cadherins The cell biology of the type 2 classical cadherins remains poorly characterised. However, there is increasing evidence that these CAMs play a central role in tissue morphogenesis and tumourigenesis. For example, the cad-11 expression has been associated with bone formation in the mouse and human (Okazaki et al. 1994; Cheng et al. 1998) and the formation and organisation of the human placenta (MacCalman eta l . , 1996;1997). Cad-11 expression also appears to be highly regulated during tumourigenesis. In particular, this CAM is believed to mediate carcinoma-stromal cell interactions during invasion (Shimazui 1996). In addition, cadherin-6 (cad-6), is believed to play a central role in the formation of the human and rodent kidney. Although cad-6 is not present in the adult kidney, cad-6 has been detected in kidney carcinomas suggesting that this CAM may play a role in the neoplastic transformation of renal cells. The spatiotemporal expression of three type 2 classical cadherins, cad-6, cad-11, and cadherin-8 in the embryonic mouse brain suggest that this cadherin subfamily may play specific role(s) in the formation and organisation of the of the central nervous system during development (Inoue et al. 1997; Korematsu and Redies 1997;Redies and Takeichi 1996). 25 Similar to the type 1 classical cadherins, the expression of type 2 cadherins appears to be complementary during cellular differentiation. For example, cad-6 mRNA levels decrease and the levels of the cad-11 mRNA transcript increase in human granulosa cells undergoing spontaneous luteinisation in culture (MacCalman et al. 1997b). Similarly, cad-6 is present in undifferentiated human myoblasts but not in myotubes (Symonds et al. 1996). There is a marked increase in cad-11 mRNA levels following the formation of the terminally differentiated myotube. To date, the role of cad-11 in cellular differentiation has not been determined. Finally, recent studies indicate that the regulated expression of type 2 cadherin subtypes mediate cell sorting in Xenopus embryos (Hadeball et al. 1998). To date, the factors capable of regulating type 2 cadherins have not been identified. 7.5.3: Protocadherins Five members of the protocadherin subfamily, known as Pcdhl, Pcdh2, Pcdh3, Pcdh8, and Pcdh9, have been identified in the rodent and human (Tanihara et al. 1994; Sago et al. 1995). Members of this cadherin subfamily have also been identified in a variety of multicellular, invertebrate organisms including Xenopus, Drosophila, and Caenorhabditis (Sago et al. 1995). This is in contrast to classical cadherins which have only been identified in vertebrates. In view of these observations, it has been proposed that protocadherins represent the primordial members of the cadherin gene superfamily and that the 26 classical cadherins detected in vertebrates may have evolved from a protocadherin subtypes (Pouliot 1992). The overall structure of the protocadherins is similar to that of classical cadherins (Sano et al. 1993). However, members of this cadherin subfamily share characteristic features which are not found in classical cadherins. In particular, the extracellular domains of protocadherins contain more than five cadherin motif repeats (Suzuki 1996a). Although these repeats contain the calcium-binding motifs found in the corresponding classical cadherin motifs, the cysteine rich repeats observed in the EC5 domain of the classical cadherins are not observed in this cadherin subfamily (Suzuki 1996b). Although protocadherins have been shown to mediate eel I-eel I interactions in a homophilic manner, protocadherins do not interact with the catenins (Suzuki 1996b). Instead, protocadherins are believed to interact with as yet, unidentified cytoplasmic protein(s). These distinctive cytoplasmic interactions may reflect the discrete biological function(s) attributed this subfamily of CAMs. As protocadherins appear to be expressed predominantly in the brain during embryonic development, it has been proposed that this cadherin subfamily may play a key role in the formation and organisation of the central nervous system which requires intricate and highly specific cell-cell interactions (Sago et al. 1995; Sano et al. 1993). 7.5.4; Desmosomal cadherins 27 The major desmosomal glycoproteins, known as the desmocollins (Dsc) and the desmogleins (Dsg), form a distinct cadherin subfamily. To date, three Dsc isoforms (Dscs 1, 2 and 3) and three Dsg isoforms (Dsgs 1, 2 and 3) have been identified (Collins et al. 1991; Garrod et al. 1996). The overall structure of the desmosomal cadherins is very similar to that of classical cadherins. In particular, Dscs and Dsgs contain the conserved calcium-binding amino acid sequences observed in the classical cadherins (Burdett 1998). However, the cytoplasmic domains of the desmosomal cadherins are longer in length and exhibit l i t t l e amino acid sequence homology with the classical cadherins. The unique cytoplasmic sequences appear to cause these proteins to interact w i th the intermediate filaments, either directly or indirectly, via members of the desmoplakin gene superfamily. In addition, Dscs have alternatively spliced isoforms, a phenomenon that is not observed in other cadherin subfamilies (Theis et al. 1993). Amino acid sequence analysis of the Dsc and Dsg isoforms has identified the putative CAR sites of the desmosomal cadherins. Three distinct CAR sites have been identified in the three Dsc isoforms: YAT in Dscl, FAT in Dsc2 and YAS in Dsc3 (single letter amino acid code). In contrast, the CAR site is the same (RAL; single letter amino acid code) in all but one of the Dsg isoforms examined (Garrod et al. 1996). Although it is widely believed that Dscs and Dsgs promote cellular interactions, there is little direct evidence to support this hypothesis at the present time. Recent studies indicate that the expression of 28 full-length DScs is not sufficient to promote cellular interactions in transfected L-cells (Chidgey et al. 1996). 7.5.5; Other members of the cadherin gene superfamily 7.5.5-A: Truncated cadherins T-cadherin (T-cad, also known as cadherin-13) (Suzuki 1996a), H-cadherin (Lee 1996) and Ll cadherin (Kreft et al. 1997) constitute a unique and diverse subfamily of cadherins which contain the conserved cadherin motifs, in their extracellular domain but lack a cytoplasmic domain. In addition, T-cad lacks a portion of the transmembrane domain and is linked to the cell membrane via a phosphoglycolipid (Ranscht and Dours-Zimmermann 1991; Tanihara eta l . 1994). To date, the ability of the truncated cadherin subtypes to promote cell adhesion in a homophilic manner has yet to be clarified. However, there is evidence to suggest that T-cad may modulate the adhesive properties of cells by interacting with N-cad or other CAMs (Ranscht and Dours-Zimmermann 1991; Vestal and Ranscht 1992). Furthermore, it has been proposed that Ll-cadherin may not mediate cel l -cel l interactions but may function as a epithelial transport protein (Kreft et al. 1997). 1.5.5-B: Unclassified cadherin-related proteins Recent studies suggest that the oncogene, known as c-ret, encodes a gene product which contains cadherin-sepcific motifs in i ts 29 extracellular domain (Schneider 1992). However, the amino acid sequence similarity is very low, and the sequence lacks the many highly conserved amino acid sequences found in the classical cadherin motifs. 1.6: Ident i f icat ion of the cadherins present in the human endometr ium Two type 1 classical cadherin subtypes, E-cad and P-cad, have been detected in the human endometrium. In particular, both cadherin subtypes are expressed in the surface and glandular epithelium of the endometrium at all stages of the menstrual cycle (Beliard et al. 1997; van der Linden et al. 1994; van der Linden et al. 1995), and menstrual effluent or endometriosis (van der Linden et al. 1994; van der Linden et al. 1995). In view of the constant expression patterns of E-cad and P-cad, these two cadherin subtypes are unlikely to play a role in the highly regulated developmental processes which occurs in this tissue during each menstrual cycle. In addition, type 1 classical cadherins have not been detected in the human endometrial stroma. Our laboratory has recently determined that two type 2 classical cadherins, cad-6 and cad-11, are spatiotemporally expressed in the human endometrium (MacCalman eta l . 1996a; MacCalman et al. 1997b). During the proliferative phase, both cad-6 and cad-11 are expressed by the glandular epithelium. However, there is a marked reduction in the levels of epithelial cad-6 during the secretory phase. In contrast, the levels of cad-11 in the glandular epithelium remain constant throughout the menstrual cycle. In the endometrial stroma, the expression of cad-6 and cad-11 is coordinately regulated. Only cad-6 30 is present in the stroma during the proliferative phase. Cad-11 is f i r s t detected around the spiral arteries of the stroma (the areas of early decidualisation) during the late secretory phase. Cad-11 expression in the stroma is concomitant with a decrease in cad-6 expression. Cad-11 levels increase as the stroma continues to undergo decidualisation and maximum levels are observed in the decidua of early pregnancy. In view of these observations, it is tempting to speculate that cad-11 may be involved in the hormonally regulated process of decidualisation which occurs in the endometrium during each menstrual cycle. 1.7: Hypothesis and Rat ionale To date, the factors capable of regulating the expression of type 2 classical cadherins have not been identified. As cad-11 is f i r s t detected in the endometrial stroma during the secretory phase of the menstrual cycle when this cellular component is undergoing decidualisation in response to increasing levels of P4, we hypothesize that the gonadal steroids are capable of regulating the expression of this endometrial CAM. To address these issues, we have examined the ability of gonadal steroids, alone or in combination, to regulate cad-11 expression in cultured endometrial stromal cells. This model system will not only allow us to identify factors capable of regulating cad-11 expression in endometrial stromal cells but will gave us useful insight into the role of cad-11 in the steroid-mediated differentiation of stromal cells into decidual cells. Cadherin function is dependent on these CAMs interacting with the family of cytoskeletal-associated proteins, known as the catenins. 31 Cad-11 has been shown to interact with 6-catenin in several cancer cell lines. Furthermore these two proteins have been localised to the same endometrial cell type. In view of these observations, we hypothesised that the steroid-mediated increase in cad-11 expression would be associated with an increase in the function of this CAM mediated by a corresponding increase in stromal 6-catenin levels. To address this issue, we have examined 6-catenin mRNA levels in endometrial stromal cells cultured in the presence of estrogens, progestins and androgens. Finally, if cad-11 expression is highly regulated during the steroid-mediated terminal differentiation of endometrial stromal cells, then the antisteroidal compounds commonly used as contragestions and/or abortificant agents are likely to have a profound effect on the expression of endometrial CAM. In view of these observations, we have examined the ability of the antisteroidogenic compounds, RU486 (an antiprogestin) and ICI 182,780 (an antiestrogen) to regulate the P4-mediated increase in cad-11 mRNA and protein expression levels in cultured endometrial stromal cells. These studies not only allow us to better define the mechanism(s) by which gonadal steroids regulate stromal cad-11 expression levels but add to our understanding of the cellular mechanisms by which these antisteroidogenic compounds may, at least in part, modulate the maturation and function of the human endometrium. The results of these studies are presented in the following section. 32 CHAPTER II: GONADAL STEROIDS ARE KEY REGULATORS OF CADHERIN-11 EXPRESSION IN HUMAN ENDOMETRIAL STROMAL CELLS Preface In this chapter, we have examined the ability of estrogens, progestins, and androgens, alone or in combination, to regulate cad-11 mRNA and protein expression levels in isolated endometrial stroma cells using Northern and Western blot analysis, respectively. In these studies, we have determined that P4, but not E2 or DHT, is capable of regulating cad-11 expression in human endometrial stroma cells. However, maximum levels of cad-11 mRNA and protein levels were detected in stroma cells cultured in the presence of E2 and P4, suggesting that E2 enhances the P4-mediated increase in stromal cad-11 expression. In contrast, DHT was not capable of potentiating the stimulatory effects of P4 on stromal cad-11 expression. These studies were the first to demonstrate that progesterone is a key regulator of cad-11 mRNA and protein expression levels in cultured endometrial stromal cells. The results of these studies are presented in a manuscript entitled "1 7 6-estradiol potentiates the st imulatory effects of progesterone on cadherin-11 expression in cultured human endomet r ia l stromal c e l l s " published in Endocrinology(1998; 139:3512-3519). © 1998 by The Endocrine Society Previous studies have demonstrated that isolated endometrial stromal cells undergo morphological and biochemical changes that are characteristic of decidualisation with long term culture in the 33 presence of gonadal steroids. In view of these observations, we have examined the expression of the novel cell adhesion molecule, known as cadherin-11, in endometrial stromal cells undergoing steroid-mediated decidualisation. In addition, we have correlated stromal cad-11 mRNA and protein expression with the levels of the mRNA transcript encoding insulin-like growth factor binding protein-1 (IGFBP-1), an established biochemical marker of decidualisation. Progesterone or a combination of progesterone plus 17fc-estradiol increased cad-11 mRNA with time in culture. Maximum levels of cad-11 mRNA and protein expression levels correlated with a marked increase in IGFBP-1 mRNA levels in these cell cultures. In contrast, 17fc-estradiol alone had no effect on cad-11 expression or IGFBP-1 mRNA levels in endometrial stromal cells in vitro. These studies are the first to demonstrate that cad-11 mRNA and protein expression levels are up-regulated during the terminal differentiation of endometrial stromal cells into decidual cells in vitro. These findings are published in a manuscript entit led "Cadherin-11 Is A Hormonally Regulated Cellular Marker o f Decidualisation in Human Endometrial Stromal C e l l s " , in Molecular Reproduction and Development (1999, 52: 158-165) © 1999 Wiley-Liss, INC. The cDNA probes used in these studies were characterised and prepared by S. Getsios. The remainder of the research was conducted by G.T.C. Chen under the supervision of Dr. C. D. MacCalman. 34 2 . 1 : 17B-ESTRADI0L POTENTIATES THE STIMULATORY EFFECTS OF PROGESTERONE ON CADHERIN-11 EXPRESSION IN CULTURED HUMAN ENDOMETRIAL STROMAL CELLS George T.C. Chen, Spiro Getsios, and Colin D. MacCalman Department of Obstetrics and Gynaecology, University of Brit ish Columbia, Vancouver, B.C. Canada, V6H 3V5 A b s t r a c t Cadherin-11 is a novel member of the cadherin gene superfamily of calcium-dependent cell adhesion molecules. To date, the factors capable of regulating this cell adhesion molecule remain poorly characterised. We have recently determined that cadherin-11 expression in the human endometrium is tightly regulated during the menstrual cycle. The spatiotemporal expression of cadherin-11 in the stromal cells of the human endometrium during the menstrual cycle suggests that gonadal steroids regulate the expression of this endometrial cell adhesion molecule. In view of these observations, we have examined the ability of progestins, estrogens, and androgens, alone or in combination, to regulate cadherin-11 expression in isolated human endometrial stromal cells using Northern and Western blot analysis. In these studies, we have determined that progesterone, but not 17B-estradiol or dihydrotestosterone, is capable of regulating cad-11 mRNA and protein expression levels in isolated endometrial stromal 35 cells. In addition, 17G-estradiol, but not dihydrotestosterone, was capable of potentiating the stimulatory effects of progesterone in a dose-dependent manner. Taken together, these observations suggest that both 17B-estradiol and progesterone are required for maximal cadherin-11 expression in human endometrial stromal cells in vitro. I n t roduc t i on Gonadal steroids are key regulators of the cyclic remodeling processes which occur in the human endometrium during the menstrual cycle. I t has been well established that 176-estradiol (E2) promotes cellular proliferation in the stroma and glandular epithelium of the endometrium [1,2]. Progesterone (P4) in turn, is believed to act upon the E2-primed endometrium, thereby initiating glandular secretion and the terminal differentiation of stromal cells into decidual cells [1,2]. However, recent studies also suggest that the non-aromatisable androgen, dihydrotestosterone (DHT), is capable of mediating decidualisation in human endometrial stromal cells in vitro [3,4]. The molecular and cellular mechanisms by which gonadal steroids regulate the differentiation of endometrial stromal cells into decidual cells remain poorly understood. We have recently determined that cadherin-11 (cad-11), a novel member of the gene superfamily of calcium-dependent cell adhesion molecules (CAMs) known as the cadherins, is spatiotemporally expressed in the human endometrium [5]. In particular, cad-11 is f i r s t detected in the endometrial stroma during the late secretory phase of the menstrual cycle when these cells are beginning to undergo 36 decidualisation. Maximum cad-11 levels are expressed in the decidua of early pregnancy [5,6]. Taken together, these observations suggest that cad-11 expression is associated with the terminal differentiation of endometrial stromal cells into decidua cells. To date, the factors capable of regulating cad-11 expression have not been identified. The spatiotemporal expression of cad-11 in endometrial stromal cells during the menstrual cycle suggests that this CAM is hormonally regulated. We have previously demonstrated that gonadal steroids are key regulators of cadherin expression in murine tissues. For example, P4 and E2 were capable of increasing E-cadherin (E-cad) mRNA levels in the immature mouse uterus [7], whereas only E2 (but not P4, testosterone or DHT) increased E-cad mRNA levels in the immature mouse ovary [8]. Similarly, only E2 was capable of stimulating N-cadherin (N-cad) mRNA levels in the immature mouse ovary and test is in vivo [9]. These observations have led us to hypothesise that the ability of steroids to regulate the developmental processes which occur in reproductive tissues may be mediated, at least in part, by their ability to modulate cadherin expression. Previous studies have demonstrated that gonadal steroids can induce cellular differentiation in endometrial stromal cells in vitro [3, 10, 11]. In view of these observations, we have examined the ability of estrogens, progestins, and androgens, alone or in combination, to regulate cad-11 expression in isolated endometrial stromal cells using Northern and Western blot analysis. In these studies, we have determined that P4, but not E2 or DHT, is capable of regulating cad-11 expression in human endometrial stromal cells. However, maximum 37 levels of cad-11 mRNA and protein levels were detected in stromal cells cultured in the presence of E2 and P4, suggesting that E2 enhances the P4-mediated increase in stromal cad-11 expression. In contrast, DHT was not capable of potentiating the stimulatory effects of P4 on stromal cad-11 expression. Materials and Methods Tissues Endometrial tissue biopsy specimens (n=15) were obtained from women of reproductive age in accordance with a protocol for the use of human tissues approved by the Committee for Ethical Review of Research involving Human Subjects, University of British Columbia, Vancouver, B.C., Canada. Tissues used in this study were obtained during the mid-secretory phase of the menstrual cycle. Cell preparation and culture The endometrial stromal cells were separated from the glandular epithelium by enzymatic digestion and mechanical dissociation using a protocol modified from Shiokawa e ta l . [12]. Briefly, the endometrial biopsy specimens were minced and subjected to 0.1%collagenase (type IA, Sigma Chemical Co., St. Louis, MO) and 0.1%hyaluronidase (type l-S, Sigma Chemical Co.) digestion in a shaking water bath at 37 °C for 1 h. The cell digest was then passed through a nylon sieve (38 um). The isolated glands were retained on the sieve and the elute containing the stromal cells collected in a 50 ml tube. The stromal cells were 38 pelleted by centrifugation at 800 x g for 10 min at room temperature. The cell pellet was washed once in phenol red-free Dulbecco's Modified Eagle's medium (DMEM) containing 10% charcoal-stripped fetal bovine serum (FBS) before being resuspended and plated in DMEM containing 25 mM glucose, 25 mM Hepes, 1%(w/v) L-glutamine, antibiotics (100 U/ml penicillin, 100 /vg/ml streptomycin and 2.5 ug/m\ fungizone), and supplemented with 10% charcoal-stripped FBS. The culture medium was replaced 30 min after plating in order to reduce epithelial cell contamination. The purity of the cell cultures was determined by immunocytochemical staining for vimentin, cytokeratin, muscle actin, and factor VIII (see appendix I). As defined by these criteria, the endometrial stromal cell cultures used in these studies contained <1% of endometrial epithelial or vascular cells. Hormone Treatments The stromal cells (passage 2) were grown to confluence, washed wi th PBS, and cultured in phenol red-free DMEM supplemented with 10% charcoal-stripped FBS and containing either P4 (1 uM), E2 (30 nM), DHT (0.1 uM) or vehicle (0.1% ethanol). The concentrations of hormones used in these experiments were selected on the basis of previous studies [4,11,13]. The cells were cultured in the presence or absence of the steroids for 0-96 h before being harvested for Northern or Western blot analysis. In these and the following studies, the culture medium was changed every 24h. To determine whether a combination of steroids was required for maximal cad-11 expression in endometrial stromal cells, the cells 39 were cultured in the presence of P4 (1 uM) plus E2 (30 nM), or P4 (1 j/M) plus DHT (0.1 uM) for 0-96 h before being harvested for Northern or Western blot analysis. Finally, in order to determine whether the ability of E2 to potentiate the effects of P4 on stromal cad-11 expression was dose-dependent, the cells were cultured in the presence of vehicle (0.1% ethanol), E2 (30 nM), P4 (1 /;M) or P4 (1 pM) plus varying doses of E2 (0.5 nM-100 nM) for 96h. The cells were then harvested for Northern or Western blot analysis. Northern Blot Analysis Total RNA was prepared from the cultured stromal cells by the phenol-chloroform method of Chomczynski and Sacchi [14]. The RNA species were resolved by electrophoresis in 1% agarose gels containing 3.7% formaldehyde. Approximately 20 pg of total RNA was loaded per lane. The fractionated RNA species were then transferred onto charged nylon membranes. The Northern blots were hybridised with a radiolabeled cDNA probe specific for human cad-11 according to the methods of MacCalman and Blaschuk [8]. The blots were then washed twice with 2X SSPE at room temperature, twice with 2XSSPE containing 1%SDS at 55 °C and twice with 0.2 X SSPE at room temperature. To standardise the amounts of total RNA in each lane, the blots were then probed with a radiolabeled synthetic oligonucleotide specific for 18S rRNA as described by MacCalman et al. [15]. The blots were again subjected to 40 autoradiography to detect the hybridisation of the radiolabeled probe to the 18S rRNA. The autoradiograms were then scanned using an LKB laser densitometer. The absorbance values obtained for the cad-11 mRNA transcript were normalised relative to the corresponding 18S rRNA absorbance value. Western Blot Analysis For Western blot analysis, the stromal cells were washed with PBS and incubated in 100 u\ of chilled cell lysis buffer (Tris-HCl, pH 7.5 containing 0.5% Nonidet P-40, 0.5 mM CaCI2 and 1.0 mM PMSF) at 4 °C for 30 min on a rocking platform. The cell lysates were centrifuged at 10,000 x g for 20 min and the supernatant used in the Western blot analyses. Aliquots (20 ug) were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions, as described by Laemmli [16]. The stacking gels contained 5% acrylamide and the separating gels were composed of 7.5% acrylamide. The proteins were electrophoretically transferred from the gels onto nitrocellulose paper according to the procedures of Towbin et al. [17]. The nitrocellulose blots were probed with a mouse monoclonal antibody (C11-113H) directed against human cad-11 (ICOS Corporation, Bothell, WA). The Amersham ECL system was used to detect antibody bound to antigen. The autoradiograms were then scanned using an LKB laser densitometer. Statistical Analysis 41 The results are presented as the mean relative absorbance (± SE) for at least three independent experiments. Statistical differences between time points and treatments were assessed by the analysis of variance (ANOVA). Differences were considered to be significant for p < 0.05. Significant differences between the means were determined using the least significant test. Resul ts The effects of gonadal steroids on stromal cad-n mRNA and protein levels A single cad-11 mRNA transcript of 4.4 kb was detected in all of the total RNA extracts prepared from the cultured endometrial stromal cells. The addition of vehicle (0.1%ethanol) to the culture medium had no effect on the levels of the cad-11 mRNA transcript present in these endometrial stromal cell cultures (Fig IA). In contrast, P4 caused a significant increase in the stromal cad-11 mRNA levels after 24 h of culture in the presence of this steroid (Fig. IB). The levels of the cad-11 mRNA transcript continued to increase until the duration of these studies at 96 h. E2, or DHT alone did not significantly increase cad-11 mRNA levels at any of the time points examined in these studies (Figs. 1 C and D, respectively). Western blot analysis, using extracts prepared from endometrial stromal cells cultured in the presence or absence of gonadal steroids and a mouse monoclonal antibody directed against human cad-11, revealed a single cad-11 protein species (Mr 125 kDa) in all of the 42 cellular extracts. In agreement with the Northern blot analysis, the addition of vehicle to the culture medium did not significantly alter cad-11 expression levels in the endometrial cell cultures (Fig. 2A). Similarly, P4 caused an increase in cad-11 expression after 24 h of culture in the presence of this steroid (Fig. 2B). The expression levels of cad-11 continued to increase until the duration of these experiments at 96h. In addition, we failed to detect a significant increase in cad-11 expression levels in endometrial stromal cells cultured in the presence of vehicle, E2, or DHT at any of the time points examined in these studies (Figs 2 C and D, respectively). The effects of P4 plus E2 or DHT on cad-11 mRNA and protein levels in endometrial stromal cells There was a significant increase in cad-11 mRNA and protein expression levels in endometrial stromal cells cultured in the presence of E2 plus P4 for 12h (Fig. 3 A). Similarly, stromal cad-11 protein expression levels were significantly increased after 12h of culture under these conditions (Fig. 3B). Cad-11 mRNA and protein expression levels continued to increase until the duration of these studies at 96h (Figs 3 A and B, respectively). The cad-11 mRNA and protein levels detected in the endometrial stromal cells cultured in the presence of E2 plus P4 for 12-96h were significantly greater than those observed in cells cultured in P4 for the same periods of time (p <0.05). In contrast, there was no significant difference between the cad-11 mRNA and protein levels observed in cells cultured in the presence of P4 plus DHT and those detected in cells cultured in P4 alone at any 43 time points examined in these studies (p <0.05; Figs 4 A and B, respectively). The effects of varying doses of E2 to potentiate the P4-mediated increase in stromal cad-11 mRNA and protein levels. To determine whether the ability of E2 to potentiate the P4-mediated increase in cad-11 expression was dose-dependent, the stromal cells were cultured in the presence of P4 plus varying doses of E2 (0.5-100 nM; Fig. 5). Increasing doses of E2 progressively enhanced the effects of P4 on stromal cad-11 mRNA and protein levels (Figs. 5 A and B, respectively). Maximum cad-11 mRNA and protein expression levels were observed in cells cultured in the presence of 30 nM E2. There was no further enhancement in stromal cad-11 mRNA and protein expression levels when the concentration of E2 was increased to 100 nM (Figs. 5 A and B, respectively). Discussion A single cad-11 mRNA transcript of 4.4 kb was detected in all of the total RNA extracts prepared from the cultured stromal cells. This cad-11 mRNA transcript has been previously detected in RNA extracts prepared from a range of human tissues including endometrium, placenta, and ovary [5,6,18]. Similarly, the single cad-11 protein species (Mr 125 kDa) detected in the stromal cell extracts using Western blot analysis has been observed in total cellular extracts prepared from choriocarcinoma cells [5]. The present studies demonstrate that P4, but not E2 or DHT, is capable of regulating cad-11 44 mRNA and protein levels in endometrial stromal cells in vitro. In addition, the P4-mediated increase in stromal cad-11 mRNA and protein expression levels can be further enhanced by the addition of E2 to the culture medium. These observations indicate that both gonadal steroids are required for maximal cad-11 expression in human endometrial stromal cells. Long term culture of endometrial stromal cells in the presence of P4 has been shown to modulate the expression of endometrial proteins associated with the remodeling processes which occur in the endometriun during late secretory phase of the menstrual cycle. For example, P4 has been shown to stimulate fibronectin production in endometrial stromal cells [19], a key component of the decidual extracellular matrix [20], and suppress the expression of endometrial metalloproteinses which are believed to play a key role in tissue breakdown during menstruation [21,22,23]. In these studies, P4 increased cad-11 mRNA and protein expression levels in the isolated endometrial stromal cells within 24 h suggesting that this gonadal steroid is a key regulator of this endometrial cell adhesion molecule. In addition, we have previously determined that cad-11 is f i r s t expressed in endometrial stromal cells beginning to undergo decidualisation during the late luteal phase when P4 is the predominant steroid [5]. Collectively, these observations suggest that a P4-mediated increase in stromal cad-11 expression may serve as a useful marker for the early cellular events involved in the process of decidualisation in vivo and in vitro. 4 5 We failed to detect a significant increase in cad-11 expression in endometrial stromal cells cultured in the presence of the non-aromatisable androgen, DHT. Furthermore, DHT was not capable of enhancing the P4-mediated increase in cad-11 expression i n endometrial stromal cells. Collectively, these results demonstrate that androgens are unable to increase stromal cad-11 mRNA or protein expression levels in either a direct or indirect manner. Although androgen receptors have been detected in the human endometrium and decidua [3], it is still unclear whether androgens play a direct role in the process of decidualisation in the human. For example, recent studies have demonstrated that DHT alone can induce prolactin secretion in isolated endometrial stromal cells and potentiate the effects of P4 on the secretion of this endometrial protein in vitro [4]. In addition, pharmacological doses of DHT were able to maintain, but not initiate, decidualisation in the stromal cells of the mouse uterus [24]. However, the actions of DHT on the rodent endometrium could be suppressed by the anti-progestin, RU486, suggesting that the action(s) of this androgen on the murine decidua were mediated by its ability to interact with the P4 receptor [24]. E2 did not increase cad-11 mRNA or protein levels in isolated endometrial stromal cells suggesting that this gonadal steroid does not have a direct effect on stromal cad-11 expression. Similarly, previous studies have failed to demonstrate a direct effect of this gonadal steroid on the production of the two decidual cell markers, prolactin and IGFBP-1 [10], or the secretion of metalloproteinases by endometrial stromal cells in vitro [21]. Furthermore, although E2 is essential for the synthesis of specific proteins in the endometrium, 46 including P4 and E2 receptors, depleted levels of this gonadal steroid during the luteal phase of the menstrual cycle do not appear to effect endometrial development in vivo [25]. To date, the role(s) of E2 in the differentiation of endometrial stromal cells in vivo and in vitro remain poorly understood. E2 was capable of potentiating the stimulatory effects of P4 on cad-11 mRNA and protein levels in isolated endometrial stromal cells suggesting that this gonadal steroid has an indirect effect on stromal cad-11 expression. Maximum levels of IGFBP-1 and prolactin have also been detected in endometrial stromal cells cultured in the presence of both E2 and P4 [11]. However, Grosskinsky et al. [26] failed to detect an increase in the expression of integrin subunits in endometrial stromal cells cultured in the presence of these two gonadal steroids, despite observing an increase in prolactin production in these cell cultures. As the differential expression of several integrin subunits in the human endometrium during the secretory phase of the menstrual cycle appears to be required for successful implantation [27], these observations indicate that the process of decidualisation is a complex series of hormonally-dependent and, -independent events. To date, the cellular mechanisms involved at the different stages of this developmental process remain poorly defined. The mechanism(s) by which E2 enhances the effects of P4 on endometrial stromal cell differentiation have not been determined. However, several mechanisms have been recently proposed. For example, as E2 has been shown to induce P4 receptors in human endometrial stromal cells in vivo and in vitro [28],the effects of E2 on 47 the terminal differentiation of endometrial stromal cells may be mediated by an increase in P4 availability. Although this proposed mechanism could explain the ability of E2 to enhance the P4-mediated increase in stromal cad-11, the effects of gonadal steroids are also believed to be mediated through growth factors [29]. In particular, the effects of E2 on endometrial cells has been shown to be mediated, at least in part, by an increase in the levels of epidermal growth factor (EGF). Furthermore, Somkuti et al. [30] have recently suggested that a combination of gonadal steroids and EGF are required for the spatiotemporal expression of integrin subunits in human endometrial cells. The ability of growth factors, alone or in combination wi th gonadal steroids, to regulate cad-11 in human endometrial cells has not been determined. In view of these observations, we are currently examining potential mechanisms by which E2 enhances the effects of P4 on cad-11 expression in isolated endometrial stromal cells. In summary, our findings demonstrate that P4, but not E2 or DHT, are capable of regulating cad-11 mRNA and protein expression levels in isolated endometrial stromal cells. However, E2 in conjunction wi th P4 appears to be necessary to achieve maximal cad-11 expression in these cells. In view of these observations, it is tempting to speculate that the ability of steroids to regulate the terminal differentiation of endometrial stromal cells into decidual cells is mediated, at least in part, by their ability to regulate cad-11 expression. Acknowledgments 48 The authors thank Dr. M.D. Stephenson, Department of Obstetrics and Gynaecology, University of British Columbia for providing the endometrial biopsy specimens and the ICOS Corporation, Bothell, WA for their kind gift of the monoclonal antibody used in these studies. We are grateful to Dr. Riaz Farookhi, Department of Obstetrics and Gynaecology, McGill University, Montreal, PQ for carefully reading this manuscript and for his helpful comments. S.G. is the recipient of a Graduate Fellowship from the University of British Columbia. 49 Fig. 1: Autoradiograms of Northern blots containing total RNA extracted from isolated stromal cells cultured in the presence of vehicle (Panel A), 1 uM P4 (Panel B), 30 nM E2 (panel C), or 0.1 uM DHT (panel D). The cells were harvested 0, 6, 12, 24, 48, 72, or 96h after treatment (lanes a-g respectively). The blots were probed for cad-11 (top) or 18S rRNA (bottom). The autoradiograms were scanned using a laser densitometer. The absorbance values obtained for the cad-11 mRNA transcript were then normalised to the values obtained for the 18S rRNA. The results derived from this analysis, as well as from two other studies (autoradiograms not shown) were standardised to the Oh control and are represented (mean + SEM; n=3) in the bar graphs (*p <0.05). 50 a b e d e f 9 28 S -B a b c d e f 9 28 s -CAD-11 CAD-11 18 S -18 S 18S rRNA 18S rRNA 12 M 4K Time in culture (h) 12 : J « l ime in culture (H) a b c d e f 9 28 s D 28 S a b e d e f 9 *» «*» *•» am CAD-11 CAD-11 18 S 18 S -18S rRNA 18S rRNA rime in cullurcihi o f. 12 24 -W 72 lime in culture <h> 51 Fig. 2: Western blot analysis of cad-11 expression levels in isolated stromal cells cultured in the presence of vehicle (Panel A), 1 uM P4 (Panel B), 30 nM E2 (panel C), 0.1 uM DHT (panel D) or vehicle. Twenty ug of protein extracted from endometrial stromal cells cultured for 0, 6, 12, 24, 48, 72, or 96 h in the presence or absence of steroids were loaded in each lane (lanes a-g respectively). Western blot analysis was performed using a mouse monoclonal antibody directed against human cad-11. The Amersham ECL system was used to detect antibody bound to antigen. The autoradiograms were then scanned using an LKB laser densitometer. The results derived from this analysis, as well as from two other studies (autoradiograms not shown) were standardised to the Oh control and are represented (mean + SEM; n=4) in the bar graphs (*p <0.05). 52 B a b c d e f g 220kDa 97kDa 66kDa a b c d e f g 220kDa -97kDa -66kDa -2£ 0.0 0 6 12 24 48 72 96 Time in culture (h) 0 6 12 24 48 72 < Time in culture (h) a b c d e f g 220kDa -97kDa -66kDa -a b c d e f g 220kDa 97kDa 66kDa — — u 1.2 = 1.0 2 0 ™ 0.2 6 12 24 48 72 Time in culture (h) 96 0 6 12 24 48 72 9 Time in culture (h) 53 Fig 3: The effects of E2 on the P4-mediated increase in stromal cad-11 mRNA levels (Panel A) or protein expression levels (Panel B). Stromal cells were cultured in the presence of I^M P4 plus 30 nM E2 for 0, 6, 12, 24, 48, 72, or 96 h (lanes a-g) before being harvested for Northern or Western blot analysis. Panel A: Autoradiograms of a Northern blot containing total RNA extracted from treated endometrial stromal cells and probed for cad-11 (top) or 18S rRNA (bottom). The absorbance values obtained from this study, as well as from two other studies (autoradiograms not shown) were standardised to the Oh control as described earlier and are represented (mean + SEM; n=3) in the bar graphs (*p <0.05). Panel B: Autoradiogram of a Western blot containing protein extracted from the treated endometrial stromal cells and probed with a mouse monoclonal antibody directed against human cad-11. The Amersham ECL system was used to detect antibody bound to antigen. The absorbance values obtained from this study, as well as from two other studies (autoradiograms not shown) were standardised to the Oh control as described earlier and are represented (mean ± SEM; n=4) in the bar graphs (*p <0.05). 54 a b c d e f g 28 S -»-* CAD-11 18 S -18S rRNA 0 ft 12 24 48 72 % Time in culture (h) a b c d e f g 220kDa -97kDa -66kDa -12 24 48 72 96 Time in culture (h) 55 Fig 4: Effects of DHT on the P4-mediated increase in stromal cad-11 mRNA levels (Panel A) or protein expression levels (Panel B). Stromal cells were cultured in the presence of 1 uM P4 plus 0.1 uM DHT for 0, 6, 12, 24, 48, 72, or 96 h (lanes a-g) before being harvested for Northern or Western blot analysis. Panel A: Autoradiograms of a Northern blot containing total RNA extracted from the treated endometrial stromal cells and probed for cad-11 (top) or 18S rRNA (bottom). The absorbance values obtained from this study, as well as from two other studies (autoradiograms not shown) were standardised to the Oh control as described earlier and are represented (mean + SEM; n=3)in the bar graphs (*p <0.05). Panel B: Autoradiogram of a Western blot containing 20 ug of protein extracted from the treated endometrial stromal cells and probed wi th a mouse monoclonal antibody directed against human cad-11. The Amersham ECL system was used to detect antibody bound to antigen. The absorbance values obtained from this study, as well as from two other studies (autoradiograms not shown) were standardised to the Oh control as described earlier and are represented (mean + SEM; n=4) in the bar graphs (*p <0.05). 56 a b c d e f g 28 S -— — — 1 CAD-11 18 S 18S rRNA 0 6 12 24 4R 72 96 Time in culture (h) a b c d e f g 220kDa -97kDa -66kDa -0 6 12 24 48 72 96 Time in culture (h) 57 Fig 5: Effects of varying concentrations of E2 on the P4-mediated increase in stromal cad-11 mRNA levels (Panel A) or protein expression levels (Panel B). Stromal cells were cultured in the presence of vehicle, 30 nM E2, 1 uM P4, or 1 uM P4 plus 0.5, 1,5, 10, 30, or 100 nM E2 (lanes a-i, respectively) for 96h before being harvested for Northern or Western blot analysis Panel A: Autoradiograms of Northern blots containing total RNA extracted from the treated stromal cells were probed for cad-11 (top) or 18S rRNA (bottom). The absorbance values obtained from this study, as well as from two other studies (autoradiograms not shown) were standardised to the vehicle control as described earlier and are represented (mean + SEM; n=3) in the bar graphs (*p < 0.05). Panel B: Autoradiograms of Western blots containing 20 ug of protein extracted from the treated stromal cells and probed with a mouse monoclonal antibody directed against human cad-11. The Amersham ECL system was used to detect antibody bound to antigen. The absorbance values obtained from this study, as well as from two other studies (autoradiograms not shown) were standardised to the vehicle control as described earlier and are represented (mean + SEM; n=4) in the bar graphs (*p < 0.05) 58 a b c d e f g h i 28 S -CAD-11 18 S -Treatment a b c d e f g h i 220kDa -97kDa -66kDa -59 Treatment References 1. Noyes RW, Hertig AT, Rock J. 1950 Dating the endometrial biopsy. Ferti l Steril 1:3-25 2. 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J Clin Endocrinol Metab 82:2192-2197 63 2.2: CADHERIN-11 IS A HORMONALLY REGULATED CELLULAR MARKER OF DECIDUALISATION IN HUMAN ENDOMETRIAL STROMAL CELLS George T.C. Chen, Spiro Getsios, and Colin D. MacCalman Department of Obstetrics and Gynaecology, University of Brit ish Columbia, Vancouver, B.C. Canada, V6H 3V5 A b s t r a c t Cultured human endometrial stromal cells respond to the gonadal steroids, progesterone and 17fc-estradiol, with morphological and biochemical changes that are characteristic of decidualisation in vivo. To date, the cellular mechanisms involved in the terminal differentiation of human endometrial stromal cells into decidual cells remain poorly understood. We have recently determined that the novel cadherin subtype, known as cadherin-11, is expressed by endometrial stromal cells undergoing decidualisation during the luteal phase of the menstrual cycle and the decidua of pregnancy. In these studies, we have examined cadherin-11 mRNA and protein expression levels in human endometrial stromal cells undergoing steroid-mediated decidualisation in vitro. Progesterone or a combination of progesterone and 17fc-estradiol increased stromal cadherin-11 mRNA and protein expression levels with time in culture. Maximum levels of cadherin-11 expression in these cell cultures correlated with a marked increase in IGFBP-1 mRNA levels, a biochemical marker of decidualisation. In contrast, 1 7G-estradiol had no effect on stromal cad-11 mRNA and protein expression or the levels of the IGFBP-1 mRNA 64 transcript. Taken together, these observations demonstrate that cadherin-11 mRNA and protein expression levels are up-regulated during the terminal differentiation of endometrial stromal cells suggesting that this cell adhesion molecule may serve as a useful cellular marker for decidualisation. I n t r o d u c t i o n The stromal cells of the human endometrium undergo terminal differentiation into decidual cells during the luteal phase of the menstrual cycle (Noyes et al., 1950). These differentiated endometrial cells play an important role in the establishment and maintenance of pregnancy. In particular, decidual cells are believed to f u l f i l l paracrine, nutritional, immunoregulatory, and embryoregulatory roles (Lala et al., 1984). Decidualisation is associated with morphological and functional changes in endometrial stromal cells (Wynn, 1974; Kearns and Lala, 1983). For example, morphological decidualisation is associated wi th a change in cell shape and size, an extensive development of the organelles involved in protein synthesis and secretion (Wynn, 1974; Kearns and Lala, 1983) and the formation of gap and adherens-like junctions (Lawn et al., 1971; Jahn et al., 1995). Functionally, decidualisation has been characterised by the onset of prolactin (PRL) (Masalar and Riddick, 1979) and insulin-like growth factor binding protein-1 (IGFBP-1) secretion (Waites eta l . , 1988; Bell et al., 1991). Although these differentiation processes have been shown to be regulated primarily by progesterone (P4) and 17G-estradiol (E2) (Noyes 65 et al., 1950), the cellular mechanisms by which gonadal steroids mediate decidualisation remain poorly characterised. In view of these observations, we have begun a series of studies to define the role(s) of the hormonally regulated family of cell adhesion molecules (CAMs), known as the cadherins (MacCalman et al., 1994,1995), in the terminal differentiation of endometrial stromal cells. The cadherins are integral membrane glycoproteins which mediate cell adhesion in a homophilic manner (Takeichi, 1991,1995). The spatiotemporal expression of these CAMs has been shown to play a key role in tissue morphogenesis. In particular, the expression of cadherin subtypes is tightly regulated during embryonic development and it is believed that the differential expression of these CAMs governs the developmental fate of cells (Takeichi, 1991,1995). In addition, the cadherins have been shown to promote the formation of cellular junctions, thereby maintaining tissue integrity (Geiger and Ayalon, 1992). We have recently determined that the novel cadherin subtype, known as cadherin-11 (cad-11), is spatiotemporally expressed in the human endometrium during the menstrual cycle (MacCalman et al., 1996,1997). In particular, we failed to detect cad-11 mRNA and protein expression in the stroma of proliferative endometrium using Northern blot analysis and immunohistochemistry, respectively (MacCalman etal . , 1996;Getsios etal. , 1998). In contrast, cad-11 was readily detectable in the endometrial stroma during the luteal phase of the menstrual cycle. Cad-11 was further localised to the stromal cells surrounding the spiral arteries (the areas of early decidualisation) with extensive cad-11 expression being observed in the decidua of 66 early pregnancy (MacCalman et al., 1996). In addition, we have determined that P4 is capable of regulating cad-11 mRNA and protein expression levels in cultured endometrial stromal cells in a dose-dependent manner (Getsios et al., 1998). These observations have led us to propose that cad-11 plays a central role in the steroid-mediated differentiation of human endometrial stromal cells and that this CAM may provide a useful cellular marker for decidualisation. Previous studies have demonstrated that endometrial stromal cells cultured in the presence of gonadal steroids undergo morphological and biochemical changes that are characteristic of decidualisation in vivo (Irwin etal . , 1989; Giudice, 1997). In view of these observations, we have examined cad-11 mRNA and protein expression levels in human endometrial stromal cells undergoing steroid-induced decidualisation in vitro. In addition, as there is a direct correlation between IGFBP-1 mRNA and protein expression levels in cultured endometrial stromal cells (Tseng et al., 1992; Liu et al. 1997), we have correlated stromal cad-11 mRNA and protein expression levels with the levels of the mRNA transcript encoding this biochemical marker of decidualisation. Materials and Methods Tissues Endometrial tissue biopsy specimens (n = 6) were obtained from women of reproductive age in accordance with a protocol for the use of human tissues approved by the Committee for Ethical Review of Research involving Human Subjects, University of British Columbia, Vancouver, 67 B.C., Canada. Tissues used in this study were obtained during the mid-secretory phase of the menstrual cycle. Endometrial Stromal Cell Preparation and Culture The endometrial stromal cells were separated from the glandular epithelium by enzymatic digestion and mechanical dissociation using a protocol modified from Shiokawa et al. (1996). Briefly, the endometrial biopsy specimens were minced and subjected to 0.1% collagenase (type IA, Sigma Chemical Co., St. Louis, MO) and 0.1% hyaluronidase (type l-S, Sigma Chemical Co.) digestion in a shaking water bath at 37 °C for 1 h. The cell digest was then passed through a nylon sieve (38 um). The isolated glands were retained on the sieve and the eluate containing the stromal cells collected in a 50 ml tube. The stromal cells were pelleted by centrifugation at 800 x g for 10 min at room temperature. The cell pellet was washed once in phenol red-free Dulbecco's Modified Eagle's medium (DMEM) containing 10% charcoal-stripped fetal bovine serum (FBS) before being resuspended and plated in phenol red-free DMEM containing 25 mM glucose, 25 mM HEPES, 2 mM L-glutamine, antibiotics (100 U/ml penicillin, 100 ug/ml streptomycin and 2.5 ug/ml Fungizone; Gibco BRL Life Technologies, Burlington, ON) and supplemented with 10% charcoal-stripped FBS. The culture medium was replaced 30 min after plating in order to reduce epithelial cell contamination. The purity of the cell cultures was determined by immunocytochemical staining for vimentin, cytokeratin, muscle actin, and factor VIII (data not shown). These cellular markers have been used to determine the purity of endometrial stromal cell cultures in previous studies (Irwin et al., 1989). As defined by these 68 criteria, the endometrial stromal cell cultures used in these studies contained <1%of epithelial or vascular cells (see apendix I). Hormone Treatments The stromal cells (passage 2) were grown to confluence, washed wi th phosphate-buffered saline (PBS), and cultured in DMEM supplemented with 10% charcoal-stripped FBS and containing either P4 (1 uM), E2 (30 nM), or vehicle (0.1%ethanol). The concentrations of hormones used in these experiments were selected on the basis of previous studies (Irwin etal . , 1989; Grosskinsky etal . , 1996). The culture medium was replaced every 24 h. The cells were harvested for either Northern or Western blot analysis after 0, 2, 4, 6, 8, 10, or 12 days of culture in the presence or absence of the steroids. As both E2 and P4 are believed to be involved in the decidualisation of human endometrial stromal cells (Noyes et al., 1950), we also examined the combined effects of these two gonadal steroids on cad-11 mRNA and protein expression levels in our stromal cell cultures. The cells were cultured in the presence of P4 (1 uM) plus E2 (30 nM) for 0, 2, 4, 6, 8, 10, or 12 days before being harvested for Northern or Western blot analysis. Northern Blot Analysis Total RNA was prepared from the cultured stromal cells by the phenol-chloroform method of Chomczynski and Sacchi (1987). The RNA species were resolved by electrophoresis in 1 % agarose gels containing 3.7% 69 formaldehyde. Approx imate ly 20 /vg of t o ta l RNA w e r e loaded per lane. The f rac t iona ted RNA species were then t rans fe r red onto charged ny lon membranes. The Northern b lots were hybr id ised w i t h radiolabeled cDNA probes speci f ic for human cad-11 or IGFBP-1 (kind g i f t f rom Dr. A Fazleabas, U. I l l inois, Chicago) according t o the methods of MacCalman et a l . ( 1 9 9 2 ) . The blots were then washed tw ice w i th 2X SSPE (20X SSPE consists o f 0.2 M sodium phosphate monobasic, pH 7.4 conta in ing 25 mM EDTA and 3M NaCl) at room tempera tu re , tw i ce w i t h 2X SSPE con ta in ing 1%SDS at 55 °C and tw ice w i t h 0.2 X SSPE at room tempera ture . The blots were sub jec ted t o autoradiography in order t o de tec t the hybr id isat ion o f the radiolabeled probe t o the cad-11 mRNA species. To standardise the amounts o f t o t a l RNA in each lane, the b lots w e r e then probed w i t h a radiolabeled syn the t i c o l igonucleot ide speci f ic f o r 18S rRNA according t o the protocols descr ibed by MacCalman et a l . ( 1 9 9 2 ) . The blots were again sub jec ted to radioautography t o d e t e c t the hybr id isat ion of the radiolabeled probe t o the 18S rRNA., The radioautograms were then scanned using an LKB laser dens i tomete r . The absorbance values obta ined for the cad-11 and IGFBP-1 mRNA t ransc r ip ts were normal ised re lat ive t o the corresponding 18S rRNA absorbance value. Western Blot A n a l y s i s For Western b lot analysis, the s t romal cells were washed w i th PBS and incubated in 100 /vl o f chi l led cell lysis buf fer (Tr is-HCl, pH 7.5 conta in ing 0.5% Nonidet P-40, 0.5 mM CaCI 2 and 1.0 mM PMSF) at 4 °C 70 for 30 min on a rocking platform. The cell lysates were centrifuged at 10,000 x g for 20 min and the supernatant collected for Western blot analysis. Protein concentration in the cellular extracts was determined using the BCA kit (Pierce Chemicals, Rockford, IL). Aliquots (20 ug) were then taken from the samples and subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions, as described by Laemmli (1970). The stacking gels contained 5% acrylamide and the separating gels were composed of 7.5%acrylamide. The proteins were electrophoretically transferred from the gels onto nitrocellulose paper according to the procedures of Towbin et al. (1979). The nitrocellulose blots were probed with a mouse monoclonal antibody (Cl 1-113H) directed against human cad-11 (ICOS Corporation, Bothell, WA). The Amersham ECL system (Amersham Life Science Inc., Oakville, ON) was used to detect antibody bound to antigen. The radioautograms were then scanned using an LKB laser densitometer. Statistical Analysis The results are presented as the mean relative absorbance (± SE) for three independent experiments. Statistical differences between time points and treatments were assessed by the analysis of variance (ANOVA). Differences were considered to be significant for p < 0.05. Significant differences between the means were determined using the least significance difference test. Resul ts 71 A single cad-11 mRNA transcript of 4.4 kb was detected in all of the total RNA extracts prepared from the cultured endometrial stromal cells. P4 caused a significant increase in stromal cad-11 mRNA levels after 2 days of culture in the presence of this steroid (Fig. IA) . The levels of the cad-11 mRNA transcript continued to increase with t ime in culture and maximum cad-11 mRNA levels were observed in stromal cells cultured for 12 days in the presence of P4. P4 also increased stromal IGFBP-1 mRNA levels after 8 days of culture in the presence of this steroid (Fig. IB). IGFBP-1 mRNA levels continued to increase unti l the duration of these studies at 12 days of culture under these conditions. E2 or vehicle alone (0.1% ethanol) did not significantly increase stromal cad-11 mRNA levels at any of the time points examined in these studies (Figs. 2 A and B, respectively). Furthermore, we failed to detect IGFBP-1 mRNA transcripts in cells cultured under these conditions (data not shown). P4 plus E2 caused a significant increase in stromal cad-11 mRNA levels after 2 days of culture in the presence of these two gonadal steroids (Fig. 3A). Cad-11 mRNA levels continued to increase wi th time in culture with maximum levels being observed after 8 days of culture under these conditions. The levels of the cad-11 mRNA transcript remained elevated until the duration of these studies at 12 days of culture in the presence of these two gonadal steroids. The cad-11 mRNA levels detected in the endometrial stromal cells cultured i n the presence of E2 plus P4 were significantly greater than those 72 observed in cells cultured in P4 for the same periods of time (p <0.05). Maximum cad-11 mRNA levels in these stromal cell cultures correlated with a marked increase in the levels of the mRNA transcript encoding IGFBP-1 (Fig 3B). The levels of the IGFBP-1 mRNA transcript continued to increase until the duration of these studies at 12 days of culture in the presence of these two gonadal steroids. Finally, we examined cad-11 protein expression levels in the stromal cells cultured in the presence or absence of steroids using Western blot analysis. A single cad-11 protein species (Mr 125 kDa) was detected in all of the stromal cell extracts using a mouse monoclonal antibody directed against human cad-11. In agreement with the Northern blot analysis, P4 caused an increase in cad-11 expression after 2 days of culture in the presence of this steroid (Fig. 4A). Maximum cad-11 expression levels were detected in extracts prepared from cells cultured for 10 days in the presence of this steroid. E2 plus P4 also caused a significant increase in cad-11 expression after 2 days of culture with maximum levels being observed after 8 days of culture in the presence of these two gonadal steroids (Fig. 4B). The cad-11 expression levels detected in the endometrial stromal cells cultured i n the presence of E2 plus P4 were significantly greater than those observed in cells cultured in P4 for the same periods of time (p <0.05). We failed to detect a significant increase in cad-11 expression in endometrial stromal cells cultured in the presence of E2, or vehicle at any time point examined in these studies (Figs. 4 C and D, respectively). 73 Discussion Endometrial stromal cells in vitro have been shown to respond to progestins with alterations in enzymatic activity (Tseng, 1984; Schatz et al., 1994), extracellular matrix protein synthesis (Irwin et al., 1989; Zhu et al., 1992), and the secretion of IGFBP-1 (Bell e ta l . , 1991; Giudice, 1997) and PRL (Irwin et al., 1989; Chen et al., 1989). In addition, although E2 alone is unable to regulate these endpoints of decidualisation, synergistic effects have been observed in stromal cells cultured in the presence of E2 and P4 (Irwin et al., 1989; Schatz et al., 1994). Consistent with these observations, P4 significantly increased cad-11 mRNA and protein expression levels in cultured endometrial stromal cells. Furthermore, the P4-mediated increase in stromal cad-11 expression was potentiated by the addition of E2 to the culture medium. Collectively, these observations demonstrate that cad-11 mRNA and protein expression levels are tightly regulated during the steroid-mediated differentiation of endometrial stromal cells and strengthen our hypothesis that this CAM may serve as a useful cellular marker for decidualisation in the human endometrium. Recent studies suggest that the process of decidualisation is a complex series of hormonally-dependent and, -independent cellular events. For example, increased IGFBP-1 mRNA levels and secretion, laminin production, and prolactin secretion have been shown to precede morphological changes, including the formation of cellular junctions, in endometrial stromal cells cultured in the presence of gonadal steroids (Irwin et al., 1989; Lane et al., 1994). In contrast, growth factors and not gonadal steroids appear to be the key regulators of the 74 integrin subunits expressed by endometrial stromal cells undergoing decidualisation during the luteal phase of the menstrual cycle and decidua of early pregnancy (Grosskinsky et al., 1996). To date, our studies suggest that cad-11 expression in human endometrial stromal cells is hormonally regulated and that this CAM may mediate an early, membrane-mediated event which is required for the initiation and maintenance of decidualisation in endometrial stromal cells in vivo and in vitro. The association of cad-11 expression with decidualisation in vivo and in vitro suggests that this CAM plays a key role in the terminal differentiation of endometrial stromal cells. Cad-11 expression has been shown to be tightly regulated during the terminal differentiation and fusion of several other cell types. For example, cad-11 expression levels have been shown to increase during the terminal differentiation of osteoblasts (Okazaki et al., 1994) and after the fusion of human myoblasts (Symonds et al., 1996) and cytotrophoblasts isolated from the human term placenta (MacCalman et al., 1996). Cad-11 may also play a central role in the formation and organisation of the decidua. In particular, as cad-11 expression appears to precede the formation of gap and adherens-like junctions between adjacent endometrial stromal cells undergoing decidualisation in vivo and in vitro (Irwin et al., 1989; Lane et al., 1994; Jahn et al. 1995), it is tempting to speculate that this CAM mediates early cellular interaction(s) between adjacent endometrial stromal cells which are a prerequisite for the formation of these intercellular junctions. Although E-cadherin has been shown to regulate the formation of 75 intercellular junctional complex in epithelial cells before being localised to the adherens junctions (Geiger and Ayalon, 1992), it has not been determined whether cad-11 is capable of mediating the formation of junctions or if it subsequently localised to adherens junctions. The regulated expression of cad-11 in endometrial stromal cells undergoing decidualisation suggests that this CAM may also fulfill an embryoregulatory role during human implantation. As cad-11 expression has been localised to the two types of trophoblast cells (syncytial trophoblast and cytotrophoblasts of the extravillous column) which form intimate contacts with the decidua cells (MacCalman et al., 1996,1997), it is tempting to speculate that this CAM mediates trophoblast-decidual cell interactions in a homophilic manner. These cellular interactions may assist in anchoring trophoblasts to decidua cells, thereby arresting their invasive migration. Conclusions In these studies, we have determined that cad-11 mRNA and protein expression levels are tightly regulated during the steroid-mediated differentiation of cultured endometrial stromal cells into decidua cells. Maximum stromal cad-11 mRNA and protein expression levels correlated with an increase in the levels of the mRNA transcript encoding IGFBP-1, a biochemical marker of decidualisation. Taken together, these observations suggest that this CAM may serve as a useful cellular marker of decidualisation in human endometrial stromal cells. Furthermore, the regulated expression of cad-11 in cultured 76 endometrial stromal cells reflects the spatiotemporal expression of this endometrial CAM in vivo demonstrating that these cell cultures will provide an ideal model system in which to define the role(s) of cad-11 in the complex process of decidualisation. These studies not only add to our understanding of the cyclic remodeling processes which occur in the human endometrium in preparation for the implant ing embryo but give us useful insight into the cell biology of the cadherin gene superfamily. A c k n o w l e d g m e n t s The authors thank Dr. M.D. Stephenson, Department of Obstetrics and Gynaecology, University of British Columbia for providing the endometrial biopsy specimens and the ICOS Corporation, Bothell, WA for their kind gift of the monoclonal antibody used in these studies. S.G. is the recipient of a Doctoral Research Award from the Medical Research Council of Canada 77 Fig. 1: Autoradiograms of a Northern blot containing total RNA extracted from endometrial stromal cells cultured in the presence of P4 for 0, 2, 4, 6, 8, 10, or 12 days (lanes a-g, respectively). The blot was probed for cad-11 (upper panel), IGFBP-1 (middle panel) or 18S rRNA (lower panel). The radioautograms were scanned using a laser densitometer. The values obtained for the cad-11 and IGFBP-1 mRNA transcripts were then normalised to the absorbance values obtained for the 18S rRNA. The results derived from this analysis, as well as from two other studies (radioautograms not shown) were standardised to the 0 day control and are represented (mean + SEM; n=3) in the bar graphs (*p <0.05). 78 a b c d e f g Fig. 2: Autoradiograms of Northern blots containing total RNA extracted from endometrial stromal cells cultured in the presence of E2 (Panel A) or vehicle (Panel B) for 0, 2, 4, 6, 8, 10, or 12 days (lanes a-g, respectively). The blots were probed for cad-11 (upper), or 18S rRNA (lower). The radioautograms were scanned using a laser densitometer. The values obtained for the cad-11 mRNA transcript were then normalised to the absorbance values obtained for the 18S rRNA. The results derived from this analysis, as well as from two other studies (radioautograms not shown) were standardised to the 0 day control and are represented (mean±SEM; n=3)in the bar graphs (*p <0.05). 80 a b c d e f g 28 S -18S-Z 1.0 0 2 4 6 8 10 12 Time in culture (days) a b c d e f g 18 S ¥ 1.0 0 2 4 6 Time in culture (days) Fig. 3: Autoradiograms of a Northern blot containing total RNA extracted from endometrial stromal cells cultured in the presence of P4 plus E2 for 0, 2, 4, 6, 8, 10, or 12 days (lanes a-g, respectively). The blot was probed for cad-11 (upper panel), IGFBP-1 (middle panel) or 18S rRNA (lower panel). The radioautograms were scanned using a laser densitometer. The values obtained for the cad-11 and IGFBP-1 mRNA transcripts were then normalised to the absorbance values obtained for the 18S rRNA. The results derived from this analysis, as well as from two other studies (radioautograms not shown) were standardised to the vehicle control and are represented (mean ± SEM; n=3) in the bar graphs (*p <0.05). 82 a b c d e f g 28 S 1 8 S 28 S CAD-11 -•;m 18 S IGFBP-1 18S rRNA 15 4 6 8 10 12 Time in culture (days) 15 • < § 10 ' 0. CD u_ O - 5-2 4 6 10 12 Time in culture (days) 83 Fig. 4: Western blot analysis of cad-11 expression levels in endometrial stromal cells cultured in the presence of P4 (Panel A), P4 plus E2 (Panel B), E2 (Panel C), or vehicle alone (Panel D) 0, 2, 4, 6, 8, 10, or 12 days (lanes a-g, respectively). Western blot analysis was performed using a mouse monoclonal antibody directed against human cad-11. The Amersham ECL system was used to detect antibody bound to antigen. The radioautograms were then scanned using an LKB laser densitometer. The results derived from this analysis, as well as from two other studies (radioautograms not shown) were standardised to the 0 day control and are represented (mean + SEM; n=3) in the bar graphs (*p <0.05). 84 A 125kDa -a b c d e f g turn m* wit" -*» B 125kDa a b c d e 4 6 8 10 Time in culture (days) 4 6 8 Time in culture (days) a b c d e f g 125kDa -D 125kDa a b c d e Time in culture (days) 85 References Bell SC, Jackson JA, Asmore J, Zhu HH, Tseng L (1991): Regulation of insulin-like growth factor-binding protein-1 synthesis and secretion by progestin and relaxin in long term cultures of human endometrial stromal cells. J Clin Endocrinol Metab 72:1014-1024. Chen GA, Huang JR, Mazela J, Tseng L (1989): Long-term effects of progestin and RU486 on prolactin production in human endometrial stromal cells. Hum Reprod 4:355-358. Chomczynski P, Sacchi N (1987): Single-step method of RNA isolation by acid guanidine thiocyanate-phenol chloroform extraction. Anal Biochem 162: 56-1 59. 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Biol Reprod 47:441-450. 88 Waites GT, James RFL, Bell SC (1988): Immunohistological localization of the human endometrial secretory protein pregnancy-associated endometrial al-globulin, an insulin-like growth factor-binding protein, during the menstrual cycle. J Clin Endocrinol Metab 67:1100-1104. Wynn RM (1974): Ultrastructural development of the human decidua. Am J Obstet Gynecol 118:652-674. Zhu HH, Huang JR, Mazela J, Elias J, Tseng L (1992): Progestin stimulates the biosynthesis of fibronectin and accumulation of fibronectin mRNA in human endometrial stromal cells. Hum Reprod 7:141-146. 89 CHAPTER III: STUDIES EXAMINING THE MECHANISMS BY WHICH GONADAL STEROIDS REGULATE CADHERIN-11 EXPRESSION AND FUNCTION IN HUMAN ENDOMETRIAL STROMAL CELLS Preface This chapter describes a series of studies undertaken to define the mechanisms by which gonadal steroids regulate the expression and function of cadherin-11 in human endometrial stromal cells. In order to better define the mechanism(s) by which gonadal steroids regulate stromal cad-11 expression levels, we have examined the effects of the antiprogestin RU486, the antiestrogen IC1182,780 and steroid withdrawal on cad-11 mRNA and protein expression levels in decidualised endometrial stromal cells. RU486 and IC1182,780 were both capable of reducing the steroid-mediated increase in stromal cad-11 mRNA and protein expression levels. These observations suggest that cad-11 expression in cultured endometrial stromal cells is dependent on the endogenous production of E2. Similarly, steroid withdrawal from the culture medium reduced stromal cad-11 mRNA and protein expression level, albeit less efficiently than the antisteroids. These observations, however, provide further evidence to suggest that the cultured endometrial stromal cells have an intr insic capacity to synthesise steroids. These studies are presented in a manuscript entitled "Ant isteroidal compounds and s t e r o i d -withdrawal downregulate cad-11 mRNA and p ro te in expression levels in human endometrial stromal c e l l s undergoing decidualisation in v i t r o . " which has been submitted 90 for publication in Molecular Reproduction and Development, (January, 1999) To better understand the mechanism(s) by which gonadal steroids regulate cad-11 function in endometrial stromal cells, we examined the abilities of progestins, estrogens and androgens to regulate the levels of the mRNA transcripts encoding the cadherin associated protein, S-catenin, in these cell cultures. In these studies, we have determined that P4, but not E2 or DHT, are capable of increasing 15 -catenin mRNA levels in cultured in human endometrial stromal cells. In contrast to cad-11, E2 was not capable of potentiating the stimulatory effects of P4 on R-catenin mRNA levels in these primary cells. These studies are the first to demonstrate that gonadal steroids are capable of regulating R-catenin mRNA levels and that cadherin and catenin mRNA levels are coordinately regulated in mammalian cells. The results of these studies are published in a manuscript entitled "Progesterone regulates S-catenin mRNA levels in human endometrial stromal cells in v i t ro" in Endocrine (in press) © 1998 Humana Press Inc. The cDNA probes used in these studies were characterized and prepared by S. Getsios. The remainder of the research was conducted by G.T.C. Chen under the supervision of Dr. C. D. MacCalman. 91 3 . 1 : Ant isteroidal compounds and s tero id-wi thdrawal d o w n -regulate cadherin-11 mRNA and protein expression levels i n human endometrial stromal cells undergoing dec idua l i sa t ion in v i t r o . George T.C. Chen, Spiro Getsios, and Colin D. MacCalman Department of Obstetrics and Gynaecology, University of Brit ish Columbia, Vancouver, B.C. Canada A b s t r a c t The cellular mechanisms by which steroids and antisteroidal compounds modulate the function and/or integrity of the human endometrium remain poorly understood. We have recently determined that the expression of the novel cadherin subtype, known as cadherin-11, is tightly regulated in endometrial stromal cells undergoing decidualisation in vivo and in vitro. In order to determine whether the actions of antisteroids on the endometrium are mediated, at least in part, by their ability to regulate the expression of this cell adhesion molecule, we have examined the effects of the antiprogestin RU486, the antiestrogen ICI 182,780 and steroid-withdrawal on cad-11 mRNA and protein expression levels in human endometrial stromal cells undergoing decidualisation in vitro. RU486 decreased the levels of the cad-11 mRNA transcript and protein species present in these cell cultures in a dose-, and time-dependent manner. Similarly, ICI 182,780 was capable of reducing stromal cad-11 mRNA and protein expression levels in a dose-dependent manner suggesting that the 92 progesterone-mediated increase in cad-11 expression levels in human endometrial cells undergoing decidualisation in vitro is dependent on the presence of estrogens. Cadherin-11 expression levels were also reduced in endometrial stromal cell cultures subjected to progesterone withdrawal, an in vitro model for menstrual breakdown. These studies not only give us useful insight into the mechanism(s) by which progesterone regulates stromal cadherin-11 expression but strengthen our hypothesis that this cell adhesion molecule plays a central role i n the remodeling processes which occur in the human endometrium in response to fluctuations in the levels of gonadal steroids. I n t r o d u c t i o n The epithelial and stromal cells of the human endometrium undergo proliferation and differentiation in response to the gonadal steroids, 17fc-estradiol (E2) and progesterone (P4), during each menstrual cycle (Noyes et al., 1950). This series of hormonally-regulated cellular events are believed to be essential for the establishment of a successful pregnancy (Horta et al., 1977; Klentzeris e ta l . , 1992). At the end of each non-conception cycle, the decline in E2 and P4 levels induces the breakdown and shedding of this dynamic tissue (Noyes et al., 1950). The recognition that gonadal steroids modulate the function and/or integrity of the human endometrium has led to the use of synthetic steroids as antigestional and/or abortificant agents (Croxatto et al., 1997). 93 The synthetic steroid, RU486 (mifepristone), is a potent antagonist of P4 action in the endometrium (Baulieu, 1989,1997). RU486 has been shown to act as an effective antigestional agent when administered during the luteal phase of the menstrual cycle and as an abortificant when administered during pregnancy. In particular, the administration of RU486 during the early luteal phase of the menstrual cycle delays the development of a secretory endometrium without affecting the function of the corpus luteum or the length of the menstrual cycle (Swahn et al., 1990; Gemzell-Danielsson eta l . , 1994). RU486 induces degenerative morphological changes in the decidua of early pregnancy prior to the onset of abortion (Schindler et al., 1990). Similarly, 7a-alkylamide analogues of 176-estradiol such as, ICI 164,384 and ICI 182,780, are potent steroidal antiestrogens which are capable of inhibiting the E2-mediated development of the primate and rodent uterus (Dukes et al., 1992,1993; Branham et al., 1996). To date, the precise cellular mechanism(s) by which these antisteroidal compounds modulate the function and/or integrity of the human endometrium remain poorly understood. The cadherins are a gene superfamily of integral membrane glycoproteins which mediate cell adhesion in a homophilic manner (Takeichi, 1991). The regulated expression of cadherins during embryogenesis has been shown to govern the developmental fate of cells and mediate the formation and organisation of discrete tissues (Takeichi, 1991,1995). This family of cell adhesion molecules (CAMs) also promotes the formation of intercellular j unctions, thereby 94 maintaining the integrity of tissues in the adult (Geiger and Ayalon, 1992). We have recently determined that the expression of the cadherin subtype, known as cadherin-11 (cad-11), is spatiotemporally expressed in the human endometrium during the menstrual cycle (MacCalman et al., 1996; Getsios et al., 1998). In particular, cad-11 expression is restricted to endometrial stromal cells undergoing decidualisation during the luteal phase of the menstrual cycle and the decidua of pregnancy. In addition, gonadal steroids appear to be key regulators of cad-11 mRNA and protein expression levels in human endometrial stromal cells in vitro. P4 increased stromal cad-11 expression levels in a dose-, and time-dependent manner (Getsios et al., 1998; Chen et al., 1998,1999). Although E2 had no effect on the expression of this CAM in cultured endometrial stromal cells, this gonadal steroid was capable of potentiating the stimulatory effects of P4 on stromal cad-11 mRNA and protein expression levels. Maximum cad-11 expression levels in these endometrial stromal cell cultures correlated with a marked increase in the levels of the mRNA transcript encoding insulin-like growth factor binding protein-1 (IGFBP-1), a biochemical marker of decidualisation (Chen et al., 1999). Collectively, these observations have led us to propose that cad-11 may play a central role in the steroid-mediated decidualisation of human endometrial stromal cells and/or that this CAM may maintain the integrity of the human decidua during pregnancy. In these studies, we have examined the effects of RU486 and ICI 182,780 on cad-11 mRNA and protein expression levels in human 95 endometrial stromal cells undergoing steroid-mediated decidualisation in vitro. In addition, as the withdrawal of gonadal steroids from this culture system is believed to mimic many of the cellular events involved in menstruation (Irwin et al., 1996; Lockwood et al., 1996), we have examined cad-11 mRNA and protein expression levels in our endometrial stromal cell cultures following the removal of E2 and P4, alone or in combination, from the culture medium. Materials and Methods Tissues Endometrial tissue biopsy specimens (n=12)were obtained from women of reproductive age in accordance with a protocol for the use of human tissues approved by the Committee for Ethical Review of Research involving Human Subjects, University of British Columbia. Tissues used in this study were obtained during the mid-secretory phase of the menstrual cycle. Cell preparation and culture Endometrial stromal cells were separated from the epithelial glands by collagenase (0.1%)/hyaluronidase (0.1%) digestion as previously described (Chen et al., 1998,1999). The isolated endometrial stromal cells were cultured in phenol red-free Dulbecco's Modified Eagle's medium (DMEM) containing 25 mM glucose, 25 mM Hepes, 2 mM L-glutamine, antibiotics (100 U/ml penicillin, 100 A/g/ml streptomycin 96 and 2.5 ug/m\ fungizone), and supplemented with 10% charcoal-stripped fetal bovine serum (FBS). Endometrial stromal cells (passage 2) were grown to confluence and then cultured in phenol red-free DMEM supplemented with 10% charcoal-stripped FBS containing P4 plus E2 (1 uM and 30 nM, respectively) for 12 days before the addition of antisteriodal compounds or the withdrawal of steroids from the culture medium. The longterm culture of endometrial stromal cells in the presence of gonadal steroids has been shown to increase cad-11 mRNA and protein expression levels during the progressive decidualisation of these cells in vitro (Chen et al., 1999). The purity of the endometrial stromal cell cultures was determined by immunocytochemical staining for vimentin, cytokeratin, muscle actin, and factor VIII (data not shown). As defined by these criteria, the cell cultures used in these studies contained <1%of endometrial epithelial, muscle, or vascular cells (see appendix I). Experimental culture conditions Endometrial stromal cells undergoing steroid-mediated decidualisation in vitro were cultured under the following conditions. To determine the effects of RU486 on stromal cad-11 mRNA and protein expression levels, cells were cultured in the presence of either a fixed concentration of RU486 (2.5 uM; Sigma Chemical Co., St. Louis, 97 MO) for 0-120 h or increasing concentrations of RU486 (0-5 uM) for 72 h before being harvested for Northern or Western blot analysis. To better define the mechanism(s) by which gonadal steroids regulate cad-11 expression in endometrial stromal cells, cultures were treated with increasing concentrations o f the antiestrogen, ICI 182,780 (0-10 uM; gift from Dr. R. Farookhi, McGill University, Montreal, PQ), for 72 h before being harvested for Northern or Western blot analysis. In addition, the inhibitory effects of ICI 182,780 and RU486 on the steroid-mediated increase in stromal cad-11 mRNA and protein expression levels were compared by culturing endometrial stromal cells in the presence or absence of RU486 (2.5 JL/M) or ICI 182,780 (1 uM) for 72 h before being harvested for Northern or Western blot analysis. Finally, in order to determine the effects of steroid-withdrawal on stromal cad-11 mRNA and protein expression levels, E2, P4, or E2 plus P4 was removed from the culture medium. Cells were harvested for Northern or Western blot analysis after 0-120 h of culture under these conditions. Northern Blot Analysis Total RNA was prepared from the cultured stromal cells by the phenol-chloroform method of Chomczynski and Sacchi (1987). The RNA species were resolved by electrophoresis in 1 % agarose gels containing 3.7% formaldehyde. Approximately 20 ug of total RNA were loaded in each 98 lane. The fractionated RNA species were then transferred onto charged nylon membranes. The Northern blots were hybridised with a radiolabeled cDNA probe specific for human cad-11 according to the methods of MacCalman et al. (1992). The blots were then washed twice with 2X SSPE at room temperature, twice with 2XSSPE containing 1%SDS at 55 °C and twice with 0.2 X SSPE at room temperature. The blots were subjected to autoradiography in order to detect the hybridisation of the radiolabeled probe to the cad-11 mRNA species. To standardise the amounts of total RNA in each lane, the blots were also probed with a radiolabeled synthetic oligonucleotide specific for 18S rRNA as described by MacCalman et al. (1992). The blots were again subjected to autoradiography to detect the hybridisation of the radiolabeled probe to the 18S rRNA. The autoradiograms were scanned using an LKB laser densitometer. The absorbance values obtained for the cad-11 mRNA transcript were normalised relative to the corresponding 18S rRNA absorbance value. Western Blot Analysis For Western blot analysis, the endometrial stromal cell cultures were washed with PBS and incubated in 100 /vl of chilled cell lysis buffer (Tris-HCl, pH 7.5 containing 0.5%Nonidet P-40, 0.5 mM CaCI2 and 1.0 mM PMSF) at 4 °C for 30 min. on a rocking platform. The cell lysates were centrifuged at 10,000 x g for 20 min and the supernatant used i n the Western blot analyses. Aliquots (20 /vg) were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions, as 99 described by Laemmli (1970). The stacking gels contained 5% acrylamide and the separating gels were composed of 7.5%acrylamide. The proteins were electrophoretically transferred from the gels onto nitrocellulose paper according to the procedures of Towbin et al. (1979). The nitrocellulose blots were probed with a mouse monoclonal antibody (Cl 1-113H) directed against human cad-11 (ICOS Corporation, Bothell, WA). The Amersham ECL system was used to detect antibody bound to antigen. The autoradiograms were then scanned using an LKB laser densitometer. Statistical Analysis The results are presented as the mean relative absorbance (± SE) for at least three independent experiments. Statistical differences between time points and treatments were assessed by the analysis of variance (ANOVA). Differences were considered to be significant for p < 0.05. Significant differences between the means were determined using the least significance difference test. Resul ts A single cad-11 mRNA transcript of 4.4 kb was detected in all of the total RNA extracts prepared from the human endometrial stromal cell cultures. RU486 caused a significant decrease in stromal cad-11 mRNA levels after 48 h of culture in the presence of this antiprogestin (Fig 1A). The levels of the cad-11 mRNA transcript continued to decrease until the duration of these studies at 120 h. The effect of 100 RU486 was also dose-dependent with a significant reduction in cad-11 mRNA levels being observed in cells cultured in the presence of 2.5 uM RU486 (Fig. IB). There was no further decrease in the levels of the stromal cad-11 mRNA transcript when the concentration of RU486 was increased to 5 uM. A single cad-11 protein species (Mr 125 kDa) was detected in all of the extracts prepared from the endometrial stromal cell cultures. In agreement with the Northern blot analysis, RU486 decreased cad-11 expression in these endometrial stromal cells in a time-, and dose-dependent manner. Stromal cad-11 expression levels were significantly reduced after 48 h of culture in the presence of this antiprogestin and continued to decrease until the duration of these experiments at 120 h (Fig 2A). Similarly, cad-11 expression levels were significantly reduced in endometrial stromal cells cultured in the presence of 2.5 uM RU486 with no further decrease being observed when the concentration of this antiprogestin was increased to 5 uM (Fig. 2B). ICI 182,780 also reduced cad-11 mRNA and protein expression levels in these endometrial stromal cell cultures in a dose-dependent manner (Figs 3 A and B, respectively). Stromal cad-11 mRNA and protein expression levels were significantly reduced by 1 uM ICI 182,780. There was no further decrease in the levels of the cad-11 mRNA transcript and protein species present in these endometrial stromal cell cultures when the concentration of this antiestrogen was increased to 10 uM. Similar results were observed in endometrial 101 stromal cells cultured in the presence of P4 alone for 12 days prior to treatment with these two antisteroidal compounds (data not shown). A comparison of the inhibitory effects of RU486 and ICI 182,780 on stromal cad-11 expression demonstrated that there was no significant difference in the ability of these two antisteroidal compounds to reduce the levels of the cad-11 mRNA transcript and protein species present in these endometrial cell cultures (Figs. 4 A and B, respectively). The removal of E2 plus P4 or P4 from the culture medium significantly reduced the levels of the cad-11 mRNA transcript present in these endometrial stromal cell cultures after 72 h (Figs. 5 A and B, respectively). Stromal cad-11 mRNA levels continued to decrease unti l the duration of these studies at 120 h. In contrast, the levels of the cad-11 mRNA transcript present in endometrial stromal cells maintained in the presence of P4, following E2 withdrawal, or both gonadal steroids continued to increase until the duration of these studies at 120 h (Figs 5C and D, respectively). Similarly, the removal of E2 plus P4 or P4 from the culture medium reduced cad-11 protein expression levels in these endometrial stromal cell cultures after 72 h (Figs. 6 A and B, respectively) whereas there was a significant increase in cad-11 expression levels in cultures maintained in P4, following the withdrawal of E2, or both gonadal steroids at all of the time points examined in these studies (Figs. 6 C and D, respectively). 102 Discussion In the present studies, we have determined that antisteroidal compounds and steroid-withdrawal are both capable of down-regulating the levels of the cad-11 mRNA transcript and protein species present in endometrial stromal cells undergoing decidualisation in vitro. To our knowledge, this is the f i r s t identification of factors/conditions which can effectively decrease human cad-11 expression and confirm our hypothesis that gonadal steroids are key regulators of this endometrial CAM. RU486 binds to the progesterone receptor (PR) with a higher af f in i ty than its naturally occurring ligands (Baulieu, 1989). RU486 prevents P4 binding to the PR, which in turn, results in this gonadal steroid being eliminated from the cell or metabolised in situ. In addition, it i s believed that RU486 decreases PR levels in the human endometrium (Zaytseva et al., 1993). Both of these mechanisms effectively reduce the availability of P4 to the target cell and may explain the inhibitory effects of RU486 on cad-11 mRNA and protein expression levels in human endometrial stromal cells undergoing steroid-mediated decidualisation in vitro. In addition to reducing the availability of P4 to target cells or tissues, the binding of RU486 to the PR effectively inhibits the transcription of P4-regulated genes (Baulieu, 1989,1997). As the promoter region of the human cad-11 gene has not been characterised, it remains unclear whether P4 has a direct effect on the expression levels of this cadherin subtype in human endometrial stromal cells. Alternatively, 103 the stimulatory effects of P4 on stromal cad-11 mRNA and protein expression levels may be mediated by an increase in the secretion of specific growth factor(s). In particular, transforming growth factor-Si (TGF-S1) is believed to mediate the actions of P4 in the human endometrium (Giudice, 1994). Although we have recently determined that TGF-fcl is capable of increasing cad-11 mRNA and protein expression levels in human extravillous cytotrophoblasts in v i t ro (Getsios et al., 1998b), this growth factor does not appear to be capable of regulating the expression levels of this CAM in cultured human endometrial stromal cells (unpublished observations). Taken together, these observations suggest that the regulation of human cad-11 expression is complex and is likely to involve cell-specif ic regulatory factor(s). The antiestrogen, ICI 182,780, was capable of decreasing cad-11 mRNA and protein expression levels in human endometrial stromal cells undergoing decidualisation in vitro. As ICI 182,780 has been shown to reduce ER levels and the E2-induced expression of PR in steroid-responsive cells and tissues (May et al., 1989; Branham et al., 1996), the ability of this antisteroidal compound to reduce stromal cad-11 expression levels is likely to be mediated by a reduction in the availability/actions of E2 and/or P4. We have recently determined that E2 has no effect on cad-11 mRNA and protein expression levels in cultured human endometrial stromal cells but is capable of potentiating the stimulatory effects of P4 on stromal cad-11 expression. Taken together, these observations suggest that the P4-mediated increase in cad-11 mRNA and protein expression levels in human endometrial stromal cells is dependent on the action(s) of 104 estrogens. Cultured human endometrial stromal cells have been shown to be capable of producing endogenous estrogens, primarily through the activities of the two steroidogenic enzymes, aromatase and estrone sulphatase (Tseng, 1984, Benedetto et al., 1990). The activities of these two enzymes also increase during the steroid-mediated decidualisation of endometrial stromal cells in vitro (Benedetto et al., 1990) and have been shown to be inhibited by ICI 182,780 in human breast cancer cell lines (Santner et al., 1993). Although charcoal-stripped FBS was used in these studies, it is possible that some E2 is formed from polar precursors, such as estrone sulphate, that are inefficiently adsorbed from the serum (Nicholson et al., 1994). The effects of low concentrations of estrogens in this culture system may also be mediated and/or potentiated by growth factors. In particular, epidermal growth factor and insulin-like growth factor-1 and -II are believed to mediate E2 actions in the human endometrium (Giudice, 1994). To date, the ability of these growth factors to regulate cad-11 expression in human endometrial stromal cells has not been determined. The breakdown and shedding of the endometrium during menstruation are likely to be mediated by alterations in cell-cell and cel l-matr ix interactions. Recent studies examining the effects of steroid-withdrawal from the culture medium of human endometrial stromal cells undergoing decidualisation have given us useful insight into the central role(s) that matrix metalloproteinases (MMPs) play in the breakdown of the extracellular matrix of the endometrium during menstruation (Irwin et al., 1996; Lockwood et al., 1996). The loss of 105 cad-11 expression in endometrial stromal cells following the removal of P4 from the culture medium suggests that there may also be a corresponding decrease in cell-cell interactions in the human endometrial stroma during menstruation. RU486 caused a more rapid decrease in stromal cad-11 mRNA and protein expression levels than the withdrawal of P4 from the culture medium. Similarly, RU486 has been shown to be more efficient at inducing MMP activity in human endometrial stromal cells than the removal of gonadal steroids from the culture medium (Lockwood et al., 1996). These differential effects may be the result of residual steroids or growth factors being present in the endometrial stromal cells following steroid-withdrawal, the actions of which would be inhibited by the addition of antisteroidal compounds to the culture medium. The accelerated loss of stromal cad-11 expression paralleled with a more rapid increase in MMP activity is consistent with the enhanced tissue degradation and subsequent excess uterine bleeding associated with RU486-induced abortion (Swhan et al., 1990; Gemzell-Danielsson etal . , 1994). Conclusions In these studies, we have determined that RU486 and ICI 182,780 are both capable of decreasing cad-11 mRNA and protein expression levels in human endometrial stromal cells undergoing steroid-mediated decidualisation in vitro. Similarly, cad-11 mRNA and protein expression levels were reduced in these cell cultures following the 106 removal of P4, but not E2, from the culture medium. Taken together, these observations suggest that the P4-mediated increase in cad-11 expression in endometrial stromal cells undergoing decidualisation in vitro is dependent on endogenous estrogens. These studies not only give us useful insight into the cellular mechanisms by which steroids and antisteroidal compounds may modulate the proliferation, differentiation and shedding of the endometrium but strengthen our hypothesis that gonadal steroids are key regulators of cad-11 expression in human endometrial stromal cells. Acknowledgments The authors thank Dr. M.D. Stephenson, Department of Obstetrics and Gynaecology, University of British Columbia for providing the endometrial biopsy specimens and the ICOS Corporation, Bothell, WA for their kind gift of the monoclonal antibody used in these studies. S.G. is the recipient of a doctoral research award from the Medical Research Council of Canada 107 Fig. 1: The effects of RU486 on cad-11 mRNA levels in human endometrial stromal cells undergoing decidualisation in vitro. Panel A: Autoradiograms of a Northern blot containing total RNA extracted from endometrial stromal cells cultured in the presence of 2.5 uM RU486 for 0, 24, 48, 72, 96, or 120 h (lanes a-f, respectively). Panel B: Autoradiograms of a Northern blot containing total RNA extracted from endometrial stromal cells cultured in the presence of vehicle (0.1% ethanol), 0.1, 1, 2.5, or 5 f/M RU486 for 72 h (lanes a-e, respectively). The blots were probed for cad-11 (top) or 18S rRNA (bottom). The autoradiograms were scanned using a laser densitometer. The absorbance values obtained for the cad-11 mRNA transcript were then normalised to the values obtained for the 18S rRNA. The results derived from this analysis, as well as from two other studies (autoradiograms not shown), were standardised to the respective controls and are represented (mean ± SEM; n=3) in the bar graphs (*p < 0.05). 108 109 Fig. 2: The effects of RU486 on cad-11 expression levels in human endometrial stromal cells undergoing decidualisation in vitro. Panel A: Autoradiogram of a Western blot containing total protein extracted from endometrial stromal cells cultured in the presence of 2.5 uM RU486 for 0, 24, 48, 72, 96, or 120 h (lanes a-f, respectively). Panel B: Autoradiogram of a Western blot containing total protein extracted from endometrial stromal cells cultured in the presence of vehicle (0.1% ethanol), 0.1, 1, 2.5, or 5 uM RU486 for 72 h (lanes a-e, respectively). Twenty ug of protein were loaded in each lane. Western blot analysis was performed using a mouse monoclonal antibody directed against human cad-11. The autoradiograms were then scanned using an LKB laser densitometer. The results derived from this analysis, as well as from two other studies (autoradiograms not shown) were standardised to the respective controls and are represented (mean ± SEM; n=3) in the bar graphs (*p <_0.05). 110 220 kDa -125 kDa-97kDa-43 1.2 0 24 48 72 Time in culture (h) B a b c d e 220 kDa -125 kDa- i i — . — 97 kDa -Pt o.o-a Treatment 111 Fig 3: The effects of increasing concentrations of ICI 182,780 on cad-11 mRNA (Panel A) or protein expression levels (Panel B). Endometrial stromal cells undergoing decidualisation were cultured in the presence of vehicle (0.1% ethanol), 0.1, 1, or 10 uM ICI 182,780 for 72 h (lanes a-d) before being harvested for Northern or Western blot analysis. Panel A: Autoradiograms of a Northern blot containing total RNA extracted from the treated cells and probed for cad-11 (top) or 18S rRNA (bottom). The absorbance values obtained from this study, as well as from two other studies (autoradiograms not shown) were standardised to the vehicle control as described earlier and are represented (mean + SEM; n=3) in the bar graphs (*p < 0.05). Panel B: Autoradiogram of a Western blot containing total protein extracted from the treated cells and probed with a mouse monoclonal antibody directed against human cad-11. The absorbance values obtained from this study, as well as from two other studies (autoradiograms not shown) were standardised to the vehicle control as described earlier and are represented (mean ± SEM; n=3) in the bar graphs (*p <0.05). 112 a b e d 28S-Cad-11 18 S 18 S rRNA .2 12 Treatment B a b e d 220 kDa -125 kDa - — ^ 97 kDa-Treatment 113 Fig 4: A comparison of the effects of RU486 and ICI 182,780 on stromal cad-11 mRNA (Panel A) and protein expression levels (Panel B). Endometrial stromal cells undergoing decidualisation were cultured in the presence of vehicle (0.1 % ethanol), RU486 (2.5 uM) or ICI 182,780 (1 JL/M) for 72 h (lanes a-c, respectively) before being harvested for Northern or Western blot analysis. Panel A: Autoradiograms of a Northern blot containing total RNA extracted from treated endometrial stromal cells and probed for cad-11 (top) or 18S rRNA (bottom). The absorbance values obtained from this study, as well as from two other studies (autoradiograms not shown) were standardised to the vehicle control as described earlier and are represented (mean ± SEM; n=3) in the bar graphs (*p < 0.05). Panel B: Autoradiogram of a Western blot containing total protein extracted from treated cells and probed with a mouse monoclonal antibody directed against human cad-11. The absorbance values obtained from this study, as well as from two other studies (autoradiograms not shown) were standardised to the vehicle control as described earlier and are represented (mean + SEM; n=3) in the bar graphs (*p <0.05). 114 A a b c 28 S -Cad-11 18 S 18 S rRNA •a 0.2 Treatment B a b c 220 kDa -125 kDa - mm — — 97 kDa-£ 1.2 Treatment 1 1 5 Fig 5: The effects of steroid withdrawal on cad-11 mRNA levels in human endometrial stromal cells undergoing decidualisation in vitro. Autoradiograms of Northern blots containing total RNA extracted from endometrial stromal cultured for 0, 24, 48, 72, 96, or 120 h (lanes a-f) following the removal of E2 plus P4, (Panel A), P4 (Panel B), E2 (Panel C) or maintained in the presence of E2 plus P4 (Panel D). The blots were probed for cad-11 (top) or 18S rRNA (bottom). The absorbance values obtained from this study, as well as from two other studies (autoradiograms not shown) were standardised to the 0 h control as described earlier and are represented (mean + SEM; n=3) in the bar graphs (*p <0.05). 116 a b c d e f 28S-B 28S Cad-11 a b c d e f Cad-11 18 S- 18 S 18 S rRNA 18 S rRNA 3 0.4 Hi.. . 0 24 48 72 96 120 3 0.4 OS 0.0 III... 0 24 48 72 96 120 Time in culture (h) a b c d e f Time in culture (h)b c d e f 28 S-18 S-Cad-11 28 S -18 S-Cad-11 18 S rRNA 18 S rRNA .••III 24 48 72 96 1ZO Time in culture (h) .mil 0 24 48 72 96 120 Time in culture (h) 117 Fig 6: The effects of steroid withdrawal on cad-11 protein expression levels in human endometrial stromal cells undergoing decidualisation. Autoradiograms of Western blots containing total protein extracted from endometrial stromal cells cultured for 0, 24, 48, 72, 96, or 120 h (lanes a-f) following the removal of E2 plus P4, (Panel A), P4 (Panel B), E2 (Panel C) or maintained in the presence of E2 plus P4 (Panel D) and probed with a mouse monoclonal antibody directed against human cad-11. The absorbance values obtained from this study, as well as from two other studies (autoradiograms not shown) were standardised to the 0 h control as described earlier and are represented (mean + SEM; n=3) in the bar graphs (*p <0.05). 118 a b c d e f 220 kDa -125 kDa-97 kDa-» 1.2 - 0.S 3 0.4 •a 0.2 * 0.0 0 24 48 72 96 120 Time in culture (h) B a b c d e 220 kDa 125 kDa - —* w - — — 97 kDa -ae 0.0 0 24 48 72 T ime in culture (h) a b c d e f 220 kDa -125 kDa -97 kDa -a b c d e 220 kDa -125 kDa - — 97 kDa-References Baulieu EE. 1989. Contragestion and other clinical applications of RU486, an antiprogesterone at the receptor. 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Santner SJ, Ohlsson-Wilhelm B, Santen RJ. 1993. Estrone sulfate promotes human breast cancer cell replication and nuclear uptake of estradiol in MCF-7 cell cultures. Int J Cancer 54:119-124. Schindler AM, Zanon P, Obradovic D, Wyss R, Graff P, Hermann WL 1985. Early ultrastructural changes in RU486 exposed decidua. Gynecol Obstet Invest 20:62-67. Swahn Ml, Bygdeman M, Cekan S, Xing S, Masironi B, Johannisson E. 1990. The effect of RU486 administered during the early luteal phase on bleeding pattern, hormonal parameters and endometrium. Hum Reprod 5:402-408. 122 Takeichi M. 1991. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251:1451-1455. Takeichi M. 1995. Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 7:619-627. Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354. Tseng L. 1984. Effect of estradiol and progesterone on human endometrial aromatase activity in primary cell culture. Endocrinology 115:833-835. Zaytseva TS, Gincharova VN, Morozova MS, Astakhova TM, Manuilova I A, Pankov YA. 1993. The effect of RU486 on progesterone and oestrogen receptor concentration in human decidua of early pregnancy. Hum Reprod 8:1288-1292. 123 3.2: PROGESTERONE REGULATES 8-CATENIN mRNA LEVELS IN HUMAN ENDOMETRIAL STROMAL CELLS IN VITRO George T.C. Chen, Spiro Getsios, and Colin D. MacCalman Department of Obstetrics and Gynaecology, University of Brit ish Columbia, Vancouver, B.C., Canada. A b s t r a c t Cadherin-catenin complexes mediate cell-cell interactions and may play a central role in intracellular signaling. To date, the factors capable of coordinated regulating cadherin and catenin expression levels within a mammalian cell remain poorly characterised. We have recently determined that progesterone is a key regulator of cadherin-11 mRNA and protein expression levels in cultured human endometrial stromal cells. As a first step in determining whether gonadal steroids are also capable of regulating stromal catenin expression, we have examined the ability of progestins, estrogens, and androgens to regulate 6-catenin mRNA levels in these endometrial cell cultures. Here we report that progesterone, but not 176-estradiol or dihydrotestosterone, increased mRNA levels in cultured human endometrial stromal cells. The stimulatory effect of progesterone on the levels of the stromal 6-catenin mRNA transcript could not be potentiated by 176-estradiol. These studies not only demonstrate that gonadal steroids are capable of regulating 6-catenin mRNA levels i n human endometrial stromal cells but may give us useful insight into 124 the cellular mechanisms by which gonadal steroids regulate the cyc l ic remodeling processes which occur in the human endometrium during each menstrual cycle. I n t r o d u c t i o n The cadherins are a gene superfamily of integral membrane glycoproteins which mediate calcium-dependent cell adhesion in a homophilic manner (1-3). The spatiotemporal expression of cadherin subtypes during tissue morphogenesis has been associated with such fundamental biological processes as cell sorting and aggregation, proli feration, and dif ferentiat ion (1, 2, 4). The ability of the cadherins to mediate cellular interactions and govern the developmental fate o f cells is dependent on these cell adhesion molecules (CAMs) in teract ing with at least three cytoskeletal-associated proteins known as a-, B - , andy-catenin (5-7). The cytoplasmic domain of the cadherin in teracts with either p - or y-catenin in a mutually exclusive manner (8, 9). These two catenins, in turn, bind to a-catenin which is responsible fo r anchoring the cadherins to the cytoskeleton either directly (10) or indirectly through interactions with the actin-binding protein, <x-actinin (11). The catenins not only link the cadherins to the underlying actin cytoskeleton but are believed to be involved in activating several intracellular signaling pathways (12, 13). We have recently determined that the cadherin subtype, known as cadherin-11 (cad-11), is spatiotemporally expressed in the stroma of the human endometrium during the menstrual cycle (14, 15). In 125 particular, cad-11 is first detected in the endometrial stroma during the secretory phase of the menstrual cycle when these cells are beginning to undergo decidualisation in response to increasing levels of progesterone (P4). Maximum levels of cad-11 were detected in the decidua of early pregnancy. Similarly, 8-catenin has been detected in the human endometrium (16) and been further localised to the endometrial stroma (17). In view of these observations, it is tempting to speculate that cad-11 may play a central role in the steroid-mediated, differentiation of endometrial stromal cells into decidual cells by associating with 6-catenin. The factors capable^ of regulating cadherin and catenin expression within mammalian cells remain poorly characterised. We have recently demonstrated that progesterone (P4) is a key regulator of cad-11 mRNA and protein expression levels in human endometrial stromal cells in vitro (15,18). In addition, the stimulatory effects of P4 on stromal cad-11 mRNA and protein expression are enhanced by 176-estradiol (E2). As a f irst step in determining whether gonadal steroids coordinately regulate stromal cad-11 and 6-catenin expression, we have examined the ability of E2 and P4, alone or in combination, and the non-aromatisable androgen, dihydrotestosterone (DHT), to regulate 6-catenin mRNA levels in these endometrial cell cultures. Materials and Methods Tissues 126 Endometr ia l t issue biopsy specimens (n = 18) were obtained f r o m women of reproduct ive age in accordance w i t h a pro toco l for the use o f human t i s s u e s approved by the Commi t tee for Ethical Review o f Research involv ing Human Subjects, Univers i ty o f Br i t ish Co lumbia , Vancouver, B.C., Canada. Tissues used in th is s tudy were obta ined during the m id -sec re to ry phase o f the menstrual cyc le . Cell preparat ion and c u l t u r e The endometr ia l s t romal cells were separated f rom the g landu lar ep i the l ium by enzymat ic d igest ion and mechanical d issoc ia t ion using a pro toco l modi f ied f rom t h a t repor ted by Shiokawa et al. (29). B r i e f l y , the endometr ia l biopsy specimens w e r e minced and sub jec ted t o 0 . 1 % collagenase ( type I A, Sigma Chemical Co., St. Louis, MO) and 0 . 1 % hyaluronidase ( type l-S, Sigma Chemical Co.) d igest ion in a shak ing wa te r bath at 3 7 °C for 1 h. The cell d igest was then passed th rough a nylon sieve (38 um). The isolated glands were reta ined on. the s ieve and the eluate contain ing the s t romal cells co l lec ted in a 50 ml tube. The s t romal cells were pel leted by cen t r i f uga t ion at 8 0 0 x g for 10 min at room tempera ture . The cell pel let was washed once in phenol red- f ree Dulbecco's Modif ied Eagle's medium (DMEM) contain ing 10% charcoa l -s t r ipped fe ta l bovine serum (FBS) before being resuspended and plated in phenol red- f ree DMEM contain ing 25 mM glucose, 25 mM HEPES, 2mM L-glutamine, an t ib io t i cs ( l O O U / m l penic i l l in , 100 ug/m\ s t rep tomyc in and 2.5 jug/ml fungizone), and supplemented w i t h 10% charcoa l -s t r ipped FBS. The cu l ture medium was replaced 30 min a f t e r p lat ing in order t o reduce epi thel ia l cell contaminat ion . The pur i ty o f the cell cu l tures was determined by immunocy tochemica l s ta in ing f o r v iment in , cy tokera t in , muscle act in , and fac to r VIII (data not shown) . 127 These cellular markers have been previously used to determine the purity of human endometrial stromal cell cultures (30). As defined by these criteria, the endometrial stromal cell cultures used in these studies contained less than 1%of muscle, epithelial and vascular cells (see appendix I). Hormone treatments The stromal cells (passage 2) were grown to confluence, washed wi th PBS, and cultured in phenol red-free DMEM supplemented with 10% charcoal-stripped FBS under the following conditions. To determine the effects of P4 on B-catenin mRNA levels in human endometrial stromal cells, cultures were exposed to the vehicle (0.1% ethanol) or a fixed concentration of P4 (1 LIM) for 0-96 h. Endometrial stromal cells were also cultured in the presence of vehicle or increasing doses of P4 (0.1-5 uM) for 96 h before being harvested for Northern blot analysis. The ability of other gonadal steroids to regulate stromal B-catenin mRNA levels was determined by culturing the cells in the presence of E2 (30 nM) or DHT (0.1 ^M) for 0-96 h before being harvested for Northern blot analysis. Finally, as a combination of E2 and P4 is required for maximal cad-11 mRNA levels in endometrial stromal cells (18), we examined whether different concentrations of E2 could potentiate the P4-mediated increase in stromal 6-catenin mRNA levels. The stromal cell cultures were cultured in the presence of vehicle (0.1% ethanol), E2 (30 nM), P4 128 (1 uM) or P4 (1 uM) plus varying doses of E2 (0.5-100 nM) for 96 h before being harvested for Northern blot analysis. The concentrations of hormones used in these experiments were selected on the basis of previous studies (30-32). In all of these studies, the culture medium was changed every 24 h. Northern blot analysis Total RNA was prepared from the cultured stromal cells by the phenol-chloroform method of Chomczynski and Sacchi (33). The RNA species were resolved by electrophoresis in 1% agarose gels containing 3.7% formaldehyde. Approximately 20 /yg of total RNA were loaded per lane. The fractionated RNA species were then transferred onto charged nylon membranes. The Northern blots were hybridised with a radiolabeled cDNA probe specific for human B-catenin (kind gift from S.W. Byers, Georgetown University, Washington, D.C.) according to the methods of MacCalman and Blaschuk (34). The blots were then washed twice with 2X SSPE (20X SSPE consists of 0.2 M sodium phosphate monobasic, pH 7.4 containing 25 mM EDTA and 3 M NaCl) at room temperature, twice wi th 2XSSPE containing 1%SDS at 55 °C and twice with 0.2 X SSPE at room temperature. The blots were subjected to autoradiography in order to detect the hybridisation of the radiolabeled probe to the B-catenin mRNA species. To standardise the amounts of total RNA in each lane, the blots were then probed with a radiolabeled synthetic oligonucleotide specific for 18S rRNA as described by MacCalman et al. (35). The blots were again subjected to autoradiography to detect the 129 hybridisation of the radiolabeled probe to the 18S rRNA. The autoradiograms were then scanned with an LKB laser densitometer (LKB, Rockville, MD). The absorbance values obtained for the p-catenin mRNA transcript were normalised relative to the corresponding 18S rRNA absorbance value. Statist ical Analysis The results are presented as the mean relative absorbance (± SE) for three independent experiments. Statist ical differences between t ime points and treatments were assessed by the analysis of variance (ANOVA). Significant differences between the means were determined using the least significance test. Differences were considered to be significant for p < 0.05. R e s u l t s A single p-catenin mRNA transcript of 3.3 kb was detected in all of the total RNA extracts prepared from the cultured endometrial s t romal cells (Fig. 1). The addition of vehicle to the culture medium had no significant effect on the levels of this stromal p-catenin mRNA transcript at any of the time points examined in these studies (Fig. 1). P4 caused a significant increase in stromal p-catenin mRNA levels after 24 h of culture in the presence of this steroid (Fig. 2). The levels of the p-catenin mRNA transcript remained elevated until the duration of these studies at 96 h. The effect of P4 on stromal p-catenin mRNA levels was also dose-dependent with maximal stimulation being 130 observed at 1 uM P4 (Fig. 3). There was no further increase in the levels of the stromal 6-catenin mRNA transcript when the concentration of P4 was increased to 5 uM. E2 or DHT had no significant effect on stromal 6-catenin mRNA levels at any of the time points examined in these studies (data not shown). Finally, there was no significant difference between the 6-catenin mRNA levels observed in endometrial stromal cells cultured in the presence of P4 plus varying doses of E2 and those detected in cells cultured in P4 alone (Fig. 4). Discussion A single 6-catenin mRNA transcript of 3.3 kb was detected in all of the total RNA extracts prepared from the cultured endometrial stromal cells. This 6-catenin mRNA transcript has been previously detected in stomach, colon and breast carcinoma cells (19-21). In view of the direct correlation between catenin expression levels and the metastatic potential of carcinoma cells in vivo and in vi tro (22,23), previous studies have focused on the hormonal regulation of catenin mRNA and protein expression levels in epithelial cells. Fujimoto et al. (24) reported that P4 was not capable of increasing the levels of the mRNA transcripts encoding a-, and 6-catenin in Ishikawa cells, whereas E2 decreased the levels of these two mRNA transcripts in this endometrial adenocarcinoma cell line. The effects of E2 on a-, 131 and p-catenin mRNA levels were reversed by the addition of P4 to the culture medium. In contrast, P4 increased a-catenin mRNA levels in T47D breast carcinoma cells (25). Finally, retinoids, which increased p-catenin protein stability in SKBR3 breast carcinoma cells, had no significant effect on p-catenin mRNA levels in this cell line (20). In our studies, we have determined that P4 is capable of regulating p-catenin mRNA levels in primary cultures of endometrial stromal cells. The stimulatory effects of P4 on stromal p-catenin mRNA levels appears to be specific for this gonadal steroid as E2 or DHT had no significant effect on the levels of the p-catenin mRNA transcript present in these cell cultures. To our knowledge, this is the f i r s t demonstration that gonadal steroids are capable of regulating stromal p-catenin mRNA levels. Collectively, these observations suggest that the regulation of p-catenin mRNA levels is complex and that other factors, alone or in combination with the gonadal steroids may be involved in differentially regulating p-catenin mRNA levels in human epithelial and stromal cells. The P4-mediated increase in p-catenin mRNA levels in cultured human endometrial stromal cells correlates with the ability of this gonadal steroid to regulate cad-11 mRNA and protein expression levels in these primary cell cultures (18). Taken together, these studies suggest that p-catenin and cad-11 mRNA levels are coordinately regulated in human endometrial stromal cells in vitro. In view of these observations, it i s tempting to speculate that steroids exert their morphogenetic effects on the endometrium, at least in part, by virtue of their ability to 132 regulate 6-catenin and cad-11 expression. However, in contrast to the P4-mediated increase in stromal cad-11 mRNA levels, the stimulatory effects of P4 on the levels of the B-catenin mRNA transcript could not be potentiated by the addition of E2 to the culture medium. These observations suggest that P4 increases B-catenin and cad-11 mRNA levels in human endometrial stromal cells by different molecular mechanisms. To date, the mechanism(s) by which gonadal steroids regulate B-catenin and cad-11 mRNA and protein expression levels have not been defined. The biological role(s) of the cad-11/B-catenin complex in cellular differentiation remain poorly understood. Co-localisation of cad-11 and B-catenin mRNA transcripts in signet cell carcinoma cells has led to the proposal that this cadherin/catenin complex may be involved i n modulating the invasive capacity of these cells (26). In addition, cad-11 has been shown to be co-expressed with B-catenin during the terminal differentiation of human osteoblasts in vitro (27). In the human endometrium, 6-catenin mRNA levels have been shown to increase during the secretory phase of the menstrual cycle when P4 i s the predominant steroid (16). Similarly, cad-11 is first detected in areas of early decidualisation in the secretory endometrium (14,15). The spatiotemporal expression of cad-11 and B-catenin in the human endometrium during the secretory phase of the menstrual cycle suggests that this cadherin/catenin complex may play a central role in the P4-mediated terminal differentiation of endometrial stromal cells into decidual cells. The ability of P4 to regulate B-catenin and cad-11 mRNA levels in human endometrial stromal cells in vitro suggests that 133 these cells may provide a useful model system to define the role(s) of the cad-11/6-catenin complex in cellular differentiation. In summary, we have demonstrated that P4, but not E2 or DHT, is capable of regulating 6-catenin mRNA levels in cultured human endometrial stromal cells. These studies not only add to our understanding of the cell biology of 6-catenin but give us useful insight into the mechanism(s) by which gonadal steroids regulate the cyclic remodeling processes which occur in the human endometrium during each menstrual cycle. Future studies will define the role(s) of the cad-11/6-catenin complex in the decidualisation of human endometrial stromal cells in vitro. Acknowledgments The authors thank Dr. S.W. Byers, Department of Anatomy and Cell Biology, Georgetown University, Washington D.C. for kindly providing the human 6-catenin cDNA probe and Dr. M.D. Stephenson, Department of Obstetrics and Gynaecology, University of British Columbia for providing the endometrial biopsy specimens used in these studies. S.G. is the recipient of a Doctoral Research Award from the Medical Research Council of Canada* 134 Fig. 1: Autoradiograms of a Northern blot containing total RNA extracted from endometrial stromal cells cultured in the presence of vehicle (0.1% ethanol). The cells were harvested 0, 6, 12, 24, 48, 72, or 96 h after treatment (lanes a-g, respectively). The blot was probed for p-catenin (top panel) or 18S rRNA (bottom panel). The autoradiograms were scanned using a laser densitometer. The absorbance values obtained for the p-catenin mRNA transcript were then normalised to the values obtained for the 18S rRNA. The results derived from this analysis, as well as from two other studies (autoradiograms not shown) were standardised to the 0 h control and are represented (mean + SEM; n=3) in the bar graph (*p <0.05). 135 136 Fig. 2: Autoradiograms of a Northern blot containing total RNA extracted from endometrial stromal cells cultured in the presence of 1 uM P4. The cells were harvested 0, 6, 12, 24, 48, 72, or 96 h after treatment (lanes a-g, respectively). See Fig. 1 for further methodological details. 137 1 3 8 Fig 3: Autoradiograms of a Northern blot containing total RNA extracted from endometrial stromal cells cultured in the presence of vehicle or 0.1, 0.5, 1 and 5 uM P4 (lanes a-e, respectively) for 96h. See Fig. 1 for further methodological details. The results for this and two other studies were standardised to the vehicle control and are represented (mean + SEM; n=3) in the bar graph (*p <0.05). 139 140 Fig 4: Autoradiograms of a Northern blot containing total RNA extracted from endometrial stromal cells cultured in the presence of vehicle, 30 nM E2, 1 uM P4, or 1 /iM P4 plus 0.5, 1,5, 10, 30, or 100 nM E2 for 96h (lanes a-i, respectively). See Fig. 1 for further methodological details. 141 142 References 1. Takeichi, M. (1991). Science. 251, 1451-1455. 2. Takeichi, M. (1995). Curr Opin Cell Biol. 7, 619-627. 3. Suzuki, ST. (1996). J Cell Biochem. 61, 531-542. 4. Larue, L., Antos, C, Butz, S., Huber, 0., Delmas, V., Dominis, M., and Kemler, R. (1996). Development. 122, 3185-3194. 5. Ozawa, M., Baribault, H., and Kemler, R. (1989). EMBO J. 8, 1711-1717. 6. Knudsen, K.A., and Wheelock, M. J. (1992). J Cell Biol. 118, 671 -679. 7. Kemler, R. (1993). Trends Genet. 9, 317-321. 8. Butz, S., and Kemler, R. (1994). 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Anal Biochem. 162, 1 56-159. 34. MacCalman, CD., and Blaschuk, O.W. (1994). Endocrine J. 2, 1 57-163. 35. MacCalman, CD., Bardeesy, N., Holland, P.C and Blaschuk, O.W. (1992). Dev Dyn. 195, 127-132. 145 CHAPTER IV: GENERAL DISCUSSION In order to fully comprehend the mechanism(s) by which P4 regulates cad-11 expression, it will be necessary to characterise the regulatory elements present in the promoter region of the gene encoding this CAM. To date, there is no information available regarding the DNA sequence of the 5' promoter region of the human cad-11 gene. To date, the genomic organisation of chicken E-cad and mouse E-, P- and N-cad have been examined in detail. However, as these genes although differing in size, exhibit the same genomic organisation (Ringwald et al., 1991; Mityani et al., 1992; Faraldo and Cano, 1993) these studies may give us useful insight into the structure of human cad-11 gene. In particular, each gene contains 16 exons of similar size *and has conserved exon-intron boundaries (Ringwald et al., 1991). This conserved genomic organisation has been linked to the formation of the functional domains characteristic of the cadherins (Miyatani et al., 1992). The relationship between the genomic and biochemical structure of the cadherins requires further clarif ication. In addition, the 5' regions of the mouse E- and P-cad genes exhibit structural similarity (Ringwald et al., 1991; Mityani et al., 1992; Farlado and Cano, 1993). For example, the transcription initiation site of both genes is in a GC rich region with no TATA box. Both genes also contain conserved DNA sequences encoding several putative binding sites for trans acting regulatory proteins. Furthermore, the putative PR binding sites identified in the E-cad gene of the mouse (Ringwald et 146 al., 1991) is further evidence to suggesting that gonadal steroids may be capable of directly regulating cadherin gene expression. Progesterone has been shown to play an important role in the growth, differentiation and function of a wide spectrum of tissues other than the endometrium. These include the testis, ovary, placenta, bone, and central nervous system (Graham and Clarke 1997). In the human, cad-11 mRNA transcripts and/or protein expression has been detected in al l of these tissues. Furthermore, cad-11 mRNA and protein expression levels are tightly regulated during the luteinisation of human granulosa cells (MacCalman et al., 1997), the terminal differentiation and fusion of human cyotrophoblasts isolated from the first trimester and term placenta (Getsios et al. 1998; MacCalman et al. 1996b), the formation of multinucleated osteoblasts (Cheng et al. 1998) and the formation of neurons (Kimura et al. 1996) in vitro. Consistent with the expression pattern observed during the decidualisation of cultured endometrial stromal cells, there is a marked increase in cad-11 expression as these various cell types undergo terminal differentiation (Kimura e t a l . 1995). Furthermore, as P4 is capable of coordinately regulating cad-11 and -catenin in human endometrial stromal cells, steroid-mediated catenin expression may provide an alternative, but related, mechanisms by which gonadal steroids regulate cellular differentiation and the subsequent formation of tissues. Alterations in steroid levels have been shown to have profound effects on the formation and organisation of tissues, including the central 147 nervous system (MacLusky and Naftolin 1981), testis (Stillman et al., 1982; Gaytan et al. 1986), ovary and endometrium (Stillman et al. 1982; Kaufman et al. 1986). For example, exposure to high levels of gonadal steroids during development have been shown to disrupt the formation and organisation of the seminiferous epithelium in the murine and human testes (Stillman et al., 1982; Gaytan et al. 1986). Similarly, women exposed to diethylstibestrol (DES) have a higher incidence of structural and functional reproductive tract disorders (Stillman et al. 1982; Kaufman et al. 1986). In addition, it has been reported that DES may predispose these individuals to in fer t i l i t y through alterations in endometrial function (Kaufaman et al. 1986; Castelbaum eta l . 1995). Antisteroidal compounds also have a profound effect on the development and function of these hormonally sensitive tissues. For example, antiestrogens have been shown to inhibit uterine growth and development in prepubertal rats (Branham et al. 1996) and modulate endometrial development and/or function in the primate (Dukes et al. 1992,1993). Antiprogestins have been shown to have a direct effect on the integrity of the human endometrium (Swahn et al. 1982; Schindler e ta l . 1985). As the cadherins are directly involved in the ability of cells to form functional collectives in a highly specific manner and several cadherin subtypes, including cad-11, E-cad and N-cad, are hormonally regulated (MacCalman et al., 1994a,b,c), it is tempting to speculate that some of the morphogenetic effects of gonadal steroids, are exerted, at least in part by their ability to regulate this family of CAMs. 148 Cellular invasion is the major cause of therapy failure in cancer treatment (Sporn 1997). Approximately 50% of all cancer deaths is caused by local cellular invasion with or without the involvement of the local lymph nodes; the other half being caused by metastases and cellular invasion in distant organs. Invasion is therefore a major challenge for therapy. The ability of cells to undergo transformation from a differentiated epithelial cell to an invasive carcinoma cell which can disseminate and invade into the underlying tissues requires the disruption and/or dysregulation of the complement of CAMs expressed on the cell surface. Cad-11 mRNA transcripts have been detected in prostate (Bussemakers et al. 1996; Morton et al. 1997), renal (Shimazui et al. 1996), stomach (Shibata et al. 1996) and breast (Byers et al. 1998), cancer cell lines, the majority of which have a poorly differentiated and invasive phenotype (Shimazui e ta l . 1996; and Shibata et al. 1996). In addition, reduced IS-catenin expression levels have been reported in malignant carcinoma cells (Oyama et al. 1994). In view of these observations, it is imperative that the factors capable of regulating cad-11 and B-catenin expression are identified. The ability of steroids to stimulate cad-11 and/or G-catenin expression may alter the metastatic and invasive capacity of carcinoma cells. Cell-cell interactions mediated by cad-11 may reduce the metastatic and/or invasive capacity of these cells by promoting interactions between the carcinoma cells and the underlying stroma, thereby preventing the release of carcinoma cells from the site of the primary tumor. 149 Gonadal steroids appear to play a critical role in the development and progression of cancer. Evidence for steroid involvement in the neoplastic transformation is most convincing for cancers of the breast (Blankenstein et al. 1992) reproductive system (Pasquilini and Sumida 1986) and endometrium (Yamamoto et al. 1993). Of these, the depth of information is greatest for carcinomas of the breast. This tissue system has therefore been taken as an illustrative example by which to evaluate the potential significance of the interactions that may exist between steroids, cadherins and cancer. Among breast cancer cases, there is a direct correlation between tumor differentiation and the presence of ER and PR which, in turn is reflected in the prognosis (Murphy et al. 1998). In general, carcinoma cells expressing ER and PR are significantly less invasive than their steroid-independent counterparts (Murphy 1998). In these cases, and in normal tissues, steroids regulate cell proliferation and differentiation. Although a direct correlation between ER/PR and cadherin expression levels in breast carcinoma cells has not been determined, studies by many laboratories indicate that poorly differentiated human carcinomas contain either reduced or undetectable levels of the hormonally regulated cadherin subtype, E-cad (Shiozaki et al. 1996). Similarly, i t is tempting to speculate that cad-11 and ER/PR expression levels w i l l be coordinated regulated during the neoplastic transformation of these cells. Further evidence to suggest a direct link between cad-11 expression, P4, and the neoplastic transformation of cells is the empirical treatment of endometrial cancer with this gonadal steroid (Yamamoto et al. 1993). 150 Trophoblast invasion mimics many of the events associated w i th tumour cell metastasis (Lala and Hamilton 1996). However, unlike tumour cell invasion, the migration of trophoblast cells in the endometrium is a highly regulated process. For example, the transplantation of murine blastocysts in extrauterine tissues such as the kidney (Kirby 1960), spleen (Kirby 1963b) and testis (Kirby 1963a), consistently increases the invasive capacity of trophoblast cells. These studies suggest that trophoblasts possess an intrinsic capacity for invasiveness and/or that the decidua is capable of limiting this invasion. The accumulation of cad-11 in the endometrial stroma correlates with the early events of implantation in the human. As cad-11 has been localised to the syncytial trophoblast and extravillous cytotrophoblast columns of the human placenta, the two cell types that form intimate interactions with the decidualised cells of the endometrium, it is tempting to speculate that cad-11 mediates trophoblast-decidual cell interactions in a homophilic manner. This cellular interaction may anchor the trophoblast cells to the decidua and arrest their invasion. In support of this hypothesis, cad-11 is not expressed in extravillous cytotrophoblasts propagated from f i r s t trimester placental explants. These findings are not surprising as the isolated extravillous cytotrophoblasts did not appear to form extensive cellular interactions with one another. Treatment of extravillous cytotrophoblasts with TGFli-1, which has been shown to increase cellular differentiation and reduce the invasive capacity of isolated extravillous cytotrophoblasts, was associated with an increase in cad-11 mRNA and protein expression levels. Large cellular aggregates, some of which contained multinucleated cells, were also observed in 151 these treated cultures. As intense immunostaining for cad-11 was detected in all of the cell aggregates, these observations are further evidence to suggest that cad-11 plays a central role in regulating the invasive capacity of cells. The maternal environment plays a critical role in both the establishment and maintenance of a pregnancy. Recently, it has been postulated that an underlying deficiency in the endometrium may provide an environment that allows for the early stages of implantation to occur but is subsequently unable to support a viable fetal-placental unit (Aplin et al. 1996). If such an abnormality were persistent, repeated pregnancy failure may occur. To date, molecular markers which identify a competent endometrium (i.e. one which is able to maintain a pregnancy), as opposed to a receptive endometrium remain to be identified. The development of such cellular markers would allow us to go beyond the morphological and descriptive cr i ter ia presently used to assess the endometrium. Clearly, cellular markers of endometrial receptivity and/or competency should have known function(s), should be spatiotemporally expressed in the human endometrium, particularly at the time of implantation and failure to express this marker should be associated with suboptimal or failed implantation. The regulated expression of cad-11 during the decidualisation of the endometrial stroma suggest that this CAM could be used to monitor the development of the endometrium during the luteal phase of the menstrual cycle. Aberrant development of the endometrium during the luteal phase of the menstrual cycle is believed to be an underlying cause of infertility and spontaneous abortion 152 (Jordan et al. 1994). In contrast, the identification of proteins involved in the formation, organisation, and differentiation of the human endometrium, such as cadherin-11, may lead to the development of new cellular based therapeutic agents, including contraceptives, which would ultimately result in the improvement of women's health. Aberrant and/or alterations in cadherin expression may also play key roles in the development of other endometrial-associated pathologies. For example, retrograde flow of menstrual effluent and dissemination of endometrial tissue fragments into the peritoneal cavity is believed to be the major underlying cause of endometriosis (Sampson, 1940; Kruitwagen et al. 1991). Recent studies suggest that the establishment of endometriotic explants in the peritoneal cavity is dependent on the invasion of the ECM (Spuijbroek et al., 1992; Van der Linden et al., 1994). However the mechanism(s) which mediate the adhesion of the endometrial tissue to the peritoneum have not been characterised. In view of the central role that cadherins play in the cellular invasion and metastasis, it would seem likely that this family of CAMs are likely to be involved in the development of endometriosis. In particular, we speculate that aberrant expression of cad-11 in endometrial tissues present in the peritoneal cavity may promote cellular interactions between these two tissues and/or the surface epithelium of the ovary. The expression pattern of cad-11 in these endometriotic explants is likely to reflect those observed in normal endometrial tissues during the menstrual cycle. Consequently, reduced P4 levels during the late luteal phase of the menstrual cycle w i l l reduce cad-11 expression in these endometrial tissue explants. This in 153 turn is likely to result in the dissociating of endometrial cells into the peritoneal cavity and the development of new endometriotic lesions. The ability to regulate cad-11 expression levels may lead to development of new cellular based treatments for this debilitating disease. In view of these observations, P4 appears to be a key regulator of cad-11 mRNA and protein expression levels in human endometrial stromal cells. These findings are not only important to our understanding of the molecular and cellular biology of reproduction but are likely to find relevance in the fields of development and cancer cell biology. Future studies will characterise the promoter region of the human cad-11 gene and define the function of this CAM in the cyclic remodeling processes which occur in the human endometrium during each menstrual cycle. 154 SUMMARY AND CONCLUSIONS These studies are the first demonstration that gonadal steroids are key regulators of cad-11 and its associated intracellular protein, fc-catenin in cultured human endometrial stromal. In particular, P4, but not E2 or DHT, was capable of regulating cad-11 mRNA and protein expression levels in human endometrial stromal cells in vitro. Similarly, P4 increased the mRNA levels of the cadherin-associated cytoplasmic protein, 6-catenin, in these primary cell cultures. Furthermore, E2 was capable of potentiating the stimulatory effects of progesterone on stromal cadherin-11 expression. Maximum levels of cadherin-11 were detected in endometrial stromal cells which had undergone decidualisation in response to long term culture in the presence of these two gonadal steroids. The regulated expression of cad-11 during the steroid-mediated differentiation of endometrial stromal cells suggests that this CAM may serve as a cellular marker for decidualisation in the human. Cad-11 expression in decidualised endometrial stromal cells was effectively reduced by the antiprogestin RU486, the antiestrogen ICI 182,780, and steroid withdrawal suggesting that the P4-mediated increase in stromal cad-11 expression is dependent on endogenous estrogens. 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A negative control in which primary antiserum was omitted is shown in panel C. 183 184 

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