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Endocrinology Vol. 138, No. 10 4308-4315
Copyright © 1997 by The Endocrine Society

ARTICLES

Progesterone Regulates Osteopontin Expression in Human Trophoblasts: A Model of Paracrine Control in the Placenta?1

Akinyinka Omigbodun, Piotr Ziolkiewicz, Cheryl Tessler, John R. Hoyer and Christos Coutifaris

Departments of Obstetrics and Gynecology and Pediatrics (J.R.H.) and the Center for Research in Reproduction and Women’s Health, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Christos Coutifaris, M.D., Ph.D., Department of Obstetrics and Gynecology, 106 Dulles Pavilion, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, Pennsylvania 19104. E-mail: ccoutifaris{at}obgyn.upenn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Osteopontin (OPN), a matrix glycosylated phosphoprotein, has been proposed to play a role(s) in basic cellular processes, such as neovascularization and tissue remodeling, which are essential to placental morphogenesis and embryo implantation. We have shown OPN to be expressed by cytotrophoblasts of the chorionic villus, and a putative progesterone regulatory element in the OPN promoter suggests hormonal regulatory control. This led us to test the hypothesis that progesterone regulates OPN expression in human cytotrophoblasts. Cytotrophoblasts isolated from human placentas were treated with combinations of progesterone, RU486, and/or aminoglutethimide, and their expression of OPN was assessed by Northern hybridization and immunocytochemistry. The expression of OPN messenger RNA (mRNA) declined as trophoblasts aggregated, but rebounded at later times when syncytia and mononuclear cytotrophoblasts coexisted in culture. Progesterone increased OPN mRNA expression by aggregating mononuclear cytotrophoblasts. Aminoglutethimide suppression of endogenous steroidogenesis by syncytiotrophoblasts inhibited OPN expression, whereas the addition of exogenous progesterone to cells treated with aminoglutethimide reversed this inhibitory effect. These observations were confirmed at the protein level by immunocytochemistry. Treatment of cytotrophoblasts with both progesterone and RU486 inhibited the up-regulatory effect on OPN mRNA associated with exposure to progesterone alone, further confirming a direct effect of progesterone. We conclude that progesterone up-regulates OPN expression in human cytotrophoblasts, and we propose that in vivo, progesterone secretion by syncytiotrophoblasts regulates the expression of OPN by the underlying cytotrophoblasts. As the receptors for OPN, {alpha}v integrins, are expressed by syncytiotrophoblasts, we postulate that these paracrine regulatory mechanisms contribute to the adhesive and/or signaling events between the two trophoblast cell types of the chorionic villus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOPONTIN (OPN) is a secreted glycosylated phosphoprotein that was originally isolated from bone (1, 2) and has since been demonstrated to be expressed in a variety of human tissues, including the kidneys, thyroid, gastrointestinal tract, breast, testis, endometrium, uterine decidual cells, and placenta (3, 4, 5, 6). It is also present in secretions such as milk (7), bile (4), and urine (5). There is evidence that OPN plays an important role in cell physiology at these sites, including cell adhesion and signaling, regulation of intracellular calcium levels, and modulation of the immune response to infections and neoplasia (8). Evidence is also emerging that it may play a role in angiogenesis and tissue remodeling (9, 10).

We recently demonstrated that OPN is expressed by human cytotrophoblasts in a differentiation-dependent manner both in vivo and in vitro (6). These observations provided the opportunity for study of the regulation of OPN expression in a human cell model. In our previous study (6), OPN messenger RNA (mRNA) expression was shown to decline as trophoblasts aggregated in culture. We were prompted to perform additional experiments in which the cells were cultured for longer periods due to an observed upward trend in mRNA abundance at the later stages of the in vitro differentiation of the cells. This was done to search for the factors that may be triggering the resurgence in OPN mRNA expression. The major hormones produced by trophoblasts, and specifically syncytiotrophoblasts, in the later stages of their differentiation in vitro are hCG and progesterone (11, 12). The pattern of OPN expression in the endometrium and decidua of pregnancy (3) and in chorionic villi (6) strongly suggests a susceptibility to progesterone regulation. Moreover, previous studies have demonstrated the presence of a putative progesterone regulatory element in the 5'-flanking region of the murine OPN gene (13), raising the possibility that a similar mechanism may exist in the regulation of expression of the human OPN gene. These observations led us to test the hypothesis that progesterone regulates the expression of OPN by human cytotrophoblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture materials and reagents
All reagents were of analytical grade and were obtained from Sigma Chemical Co. (St. Louis, MO), unless otherwise stated. Hanks’ Balanced Salt Solution, DMEM, FBS, and gentamicin were all obtained from Life Technologies (Grand Island, NY). Mifepristone (RU486) was generously provided by Dr. S. S. Koide (The Population Council, New York, NY). Culture dishes were obtained from Nunclon Delta (Copenhagen, Denmark).

Cell preparation and culture
Utilization of human tissue, including placentas, for our ongoing studies has been approved by the institutional review board of our university. All human tissue used in this study was "residual" or excess tissue that was not required for histopathological diagnosis. Cytotrophoblasts were prepared from human placentas as previously described (14). Briefly, chorionic villi were minced and then digested with trypsin and deoxyribonuclease in Hanks’ Balanced Salt Solution that was free of phenol red, calcium, and magnesium ions. This was followed by centrifugation on a 5–70% Percoll gradient. Cells banding at a density of 1048–1065 g/ml represent a highly enriched (up to 95% pure) and viable preparation of cytotrophoblasts. These cells were collected and cultured in DMEM containing 25 mM glucose, 25 mM HEPES, and 50 µg/ml gentamicin at 37 C in an atmosphere containing 5% carbon dioxide. The culture medium was free of phenol red, and it was supplemented with 10% heat-inactivated FCS that had been stripped of steroids with activated charcoal. Some of the cells were cultured in the presence of progesterone (1 µM), the progesterone antagonist RU486 (1 µM), the steroid hormone synthesis inhibitor aminoglutethimide (1 µM), or a combination of these agents. Preliminary experiments performed with different concentrations of progesterone (10 nM to 1 mM) showed optimal response at 1 µM, which led to the use of this concentration for subsequent experiments. The cells remained in primary culture until there was a mixture of mononuclear and syncytial trophoblasts in culture (~48–72 h). The cells were thereafter harvested for total RNA isolation.

In some experiments, cells were seeded onto glass coverslips (2 x 2 cm) and placed in six-well plates at a density of 1.0 x 105 cells/cm2 for indirect immunofluorescence studies. For total RNA preparation, cells were cultured in 100-mm culture dishes using a concentration of 20 million cells/culture dish. For the experiments presented in Fig. 1Go, cells were plated at a concentration of 30 million cells/culture dish.



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  		Figure 1. Northern blot analysis for OPN during differentiation of human trophoblasts in vitro. Mononuclear cytotrophoblasts isolated from human placentas were placed in culture (A; arrowheads), and over a period of 48–72 h some formed multinucleated syncytia (B; arrows) that coexisted with nonfused mononuclear cells (arrowheads in B; bar = 50 µm). Total RNA extracted at the time of trophoblast isolation (0 h) and at time points up to 72 h was blotted as described in Materials and Methods and hybridized with cDNAs for OPN and 28S (C). Quantitative analysis of the OPN/28S densitometric ratio from three different experiments (mean ± SD) is shown in D. Note the decline in OPN mRNA as the cells aggregated (12–24 h) and the rebound when both syncytia and single cells were present at later times in culture, i.e. 48–72 h. *, P < 0.05 vs. 0, 4, 48, and 72 h.

 
RNA isolation and Northern analysis
Total RNA was extracted from the cytotrophoblast cells after Percoll gradient isolation and after the specified period in primary cell culture, using an acid guanidine thiocyanate-phenol-chloroform method (15). The RNA samples were stored at -70 C until use.

Denatured total RNA (20 µg in each well) was electrophoretically separated by size in a formaldehyde agarose-denaturing gel, transferred to a nylon membrane (Nytran, Schleicher and Schuell, Keene, NH) by capillary action using 20 x SSC (standard saline citrate) solution, and cross-linked to the membrane by UV radiation (Stratalinker 1800, Stratagene, La Jolla, CA) using 12 x 105 µJ radiation. The membrane was prehybridized with 50% (vol/vol) formamide, standard saline phosphate, and ethylenediamine acetate buffer (SSPE), 1 x Denhardt’s solution, and 0.1% SDS at 42 C for 2 h. A 1493-bp complementary DNA (cDNA) encoding the entire protein region of human OPN (a gift from M. Young, NIH) served as the template for synthesis of a labeled DNA using random priming (12) with [32P]deoxy-CTP (New England Nuclear Corp., Boston, MA), a mixture of the other deoxynucleoside triphosphates, and Klenow enzyme (Random Primed DNA Labeling Kit, Boehringer Mannheim Biochemicals, Indianapolis, IN) to a specific activity of 1 x 106 to 1 x 107 cpm/ng probe. Hybridization was performed with 2 x 106 cpm denatured probe/ml hybridization solution for 18 h at 42 C. The blots were washed by four 15-min incubations, two at 42 C and another two at 55 C, with 1 x SSC (containing 0.1% SDS). Autoradiography was performed using Kodak XAR film (Eastman Kodak, Rochester, NY) for 1–72 h at -70 C until the desired exposure was obtained. We also used a 1.2-kilobase cDNA encoding the 28S ribosomal RNA (rRNA) subunit or a 300-bp cDNA encoding the 18S rRNA to prepare probes for normalization of the blots.

The intensity of the autoradiographic images of OPN mRNA relative to those of 28S or 18S rRNA were determined by scanning densitometry with a desk-top scanner using the ImageQuant computer program (Molecular Dynamics, Kemsing, UK).

Indirect immunofluorescence
Trophoblast cells on coverslips were fixed for 10 min in Bouin’s solution after a period of 24 h in culture. These coverslips were used for indirect immunofluorescence detection of OPN with a rat monoclonal antibody to human OPN. This antibody has been previously characterized (5). It was used at a concentration of 12 µg/ml, and a fluorescein-conjugated goat antirat IgG (Jackson ImmunoResearch, West Grove, PA) at a concentration of 20 µg/ml served as the secondary antibody. Briefly, the coverslips were incubated with 10% goat serum for 60 min to block nonspecific binding before incubation with the primary antibody for 60 min. This was followed by rinsing three times with PBS, a 30-min incubation with the secondary antibody, and rinsing three times with PBS before mounting. Controls consisted of sections incubated without primary antibody. Staining was evaluated with a Nikon Microphot FXA fluorescent microscope (Nikon Corp., Tokyo, Japan).

Statistical analysis
The densitometric results are expressed as the mean ± SD. Differences were determined using one-way ANOVA. The Bonferroni t test was used for post-hoc multiple comparisons to determine differences between individual groups. The level of statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Changes in OPN mRNA expression in human trophoblasts during their differentiation in vitro
When freshly isolated mononuclear cytotrophoblasts are kept in monolayer culture, they aggregate and eventually fuse to form multinucleated syncytia. This morphogenetic process recapitulates the in vivo differentiation of trophoblasts from cytotrophoblasts to syncytiotrophoblasts. The time course of this process is cell density dependent. As we had previously shown by Northern analysis (6), freshly isolated trophoblasts abundantly expressed OPN mRNA, but this expression declines rapidly as the cells aggregate in culture. When cells were plated at a higher density and the time course of the experiment was extended, expression of OPN mRNA rebounded upward, reaching the levels observed in freshly isolated cells (Fig. 1Go). These differences were statistically significant (F = 17.78; P = 0.000008). Immunocytochemistry indicated that the nonfused mononuclear cytotrophoblasts were responsible for this dramatic increase in OPN expression and not the syncytiotrophoblasts that had formed in culture (data not shown). This observation led us to further investigate the possibility of an endogenously produced, syncytiotrophoblast-derived factor (progesterone)regulating OPN expression in mononuclear cytotro-phoblasts.

Exogenous progesterone increases OPN mRNA expression in trophoblasts
Exposure of aggregating trophoblasts to exogenous progesterone over 48 h increased OPN mRNA expression compared with that in controls (Fig. 2Go). By 72 h of culture, as a larger proportion of the trophoblasts fused into progesterone-secreting syncytia, an increase in OPN mRNA expression was observed in untreated trophoblasts comparable to the level of expression seen in progesterone-treated trophoblasts at 48 h (Fig. 2Go). These differences were statistically significant (F = 21.1; P = 0.000045). Post-hoc analysis demonstrated a significant drop in OPN mRNA expression from 0 to 48 h (P = 0.0001) and up-regulation by progesterone at 48 h (P = 0.047). There was no significant difference between 48 h plus progesterone and 72 h. Thus, progesterone accelerated the rebound in OPN mRNA expression by cytotrophoblasts in vitro.



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  		Figure 2. Effect of progesterone on OPN mRNA expression in trophoblasts. Total RNA was extracted for Northern analysis from freshly isolated trophoblasts (0h), at 48 h in the absence (48h) or presence (48h +P) of 1 µM progesterone, and at 72 h (72h). Autoradiographs of the resulting Northern blot after probing for OPN and 28S RNA are shown in A. The densitometric ratios of OPN/28S from four different experiments (mean ± SD) are shown in B. OPN mRNA expression decreased with cell differentiation, demonstrating a significant drop from 0 to 48 h (P < 0.05) and was up-regulated by exogenous progesterone at 48 h (P < 0.05). There was no significant difference between 48h+P and 72h, indicating that exogenous progesterone accelerated OPN mRNA up-regulation.

 
Inhibition of endogenous steroidogenesis inhibits OPN mRNA expression
When aminoglutethimide (an inhibitor of the P450 side-chain cleavage enzyme complex that is the rate-determining enzyme in steroid hormone biosynthesis) was included in the culture medium, there was a marked inhibition of OPN mRNA production by the trophoblasts (Fig. 3AGo). When exogenous progesterone was introduced into the culture medium of the cells exposed to aminoglutethimide, the inhibitory effect of aminoglutethimide on OPN mRNA expression was reversed (Fig. 3BGo). Densitometric analysis demonstrated these differences to be significant (F = 40.44; P = 0.0067). Exposure of the cells to estradiol (up to 1 µM) or dexamethasone (up to 1 µM) over the same period of time was not associated with increases in OPN mRNA expression (data not shown).



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  		Figure 3. Effects of the inhibition of endogenous steroidogenesis on OPN mRNA expression in trophoblasts. Cytotrophoblasts were isolated and cultured in the presence or absence of aminoglutethimide (AG) and/or progesterone (P). Results from two different experiments are presented in A and B. Hybridization with a probe for 18S rRNA was used to control for RNA loading. Note that the addition of AG, which inhibits steroid biosynthesis, led to a sharp drop in OPN mRNA expression. The addition of exogenous progesterone reversed this inhibitory AG effect.

 
Transcriptional regulation by progesterone is reflected in OPN protein production
OPN immunocytochemistry of trophoblasts differentiating in vitro revealed that the regulatory effects of progesterone and aminoglutethimide on OPN mRNA expression are reflected at the translational level. Cells treated with progesterone showed an increase in fluorescence after staining for OPN compared with controls (Fig. 4Go). In addition, there was a marked reduction in fluorescence in cells treated with aminoglutethimide, whereas the addition of exogenous progesterone reversed this inhibitory effect.



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  		Figure 4. Immunocytochemical localization of OPN in trophoblasts. Cytotrophoblasts isolated from placenta were cultured under standard conditions (A) or in the presence of 1 µM aminoglutethimide (B), 1 µM progesterone (C), or a combination of both aminoglutethimide and progesterone (D). The cells were fixed after 24 h in culture and stained with a specific antibody to OPN, as described in Materials and Methods. Note the reduced staining observed when aminoglutethimide was added to the cells (B), the increase in fluorescence when progesterone was added to the cells (C), and the overriding of the aminoglutethimide effect by exogenously added progesterone (D). Bar = 10 µm.

 
RU-486 blocks the progesterone-induced increase in OPN mRNA expression
To confirm that the observed effect of progesterone on OPN mRNA expression was directly attributable to this steroid hormone, cultured trophoblasts were exposed to both exogenous progesterone and the progesterone antagonist RU486. Progesterone alone up-regulated OPN mRNA at 24 and 48 h, whereas the addition of RU486 inhibited the up-regulatory effect of progesterone alone on OPN mRNA expression at both time points (Fig. 5Go), proving that the observed increase was a direct progesterone effect. The differences observed in OPN mRNA expression were statistically significant at 24 h (F = 33.96; P = 0.000067) and 48 h (F = 17.43; P = 0.0007). Addition of RU486 alone to the trophoblasts at 24 h when endogenous progesterone levels were very low resulted, paradoxically, in an increase in OPN mRNA expression (P = 0.025), although not to the extent observed with progesterone (Fig. 5Go). This seemingly contradictory effect has been observed in other cell culture systems, especially where there is a high cAMP concentration (see Discussion). This paradoxical effect was not observed at 48 h, presumably due to the presence of higher endogenous progesterone levels.



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  		Figure 5. Effect of RU486 on OPN expression in human trophoblasts. Cytotrophoblasts were cultured in the presence of progesterone (P), RU486 (RU), or a combination of both (P/RU) for 24 h (24h) and 48 h (48h). Total RNA was extracted for Northern analysis as described in the text, and a representative autoradiograph is shown in A. A quantitation of OPN mRNA expression relative to 28S rRNA from three separate experiments is shown in B. Note that RU486 completely blocked the up-regulatory effect of P on OPN mRNA expression at both 24 and 48 h and the stimulatory effect of RU486 at 24h when endogenous P levels were low. *, P < 0.05 vs. C at the indicated times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings described in this study demonstrate that progesterone directly regulates the expression of OPN in human trophoblasts. The results presented further extend our understanding of how the expression of this secretory protein is regulated and provide insight into its potential role in the human placenta. We have previously demonstrated that it is the mononuclear cytotrophoblasts that are responsible for OPN production in the placenta (6, 16). In vivo, these cells exist in an endocrine milieu that is high in progesterone produced by the overlying syncytiotrophoblast layer of the chorionic villus, and we hypothesize that this contributes to the high levels of OPN mRNA expressed by freshly isolated cytotrophoblasts. The in vitro model used in the present study has been shown to recapitulate many critical events regulating the in vivo process of trophoblast differentiation and function (14, 17, 18). Both in vivo and in vitro, the morphological differentiation of these cells is accompanied by functional differentiation with respect to hormone production, including progesterone (12, 14, 18). Mononuclear cytotrophoblasts do not exhibit P450 side-chain cleavage enzyme activity to any appreciable extent, but as they differentiate into syncytia in culture, the activity of this enzyme increases markedly, leading to significant progesterone biosynthesis (19). We hypothesized that the observed coincident decline in cytotrophoblast OPN mRNA at early time points in culture may have been due to the absence of progesterone from the culture medium coupled with the inhibitory effect of increasing levels of intracellular cAMP on OPN expression during cell aggregation and fusion that we recently demonstrated (6). We further hypothesized that with the onset of syncytialization of the cells, increased secretion of endogenous progesterone leads to increased OPN mRNA and protein production by the remaining mononuclear cytotrophoblasts in culture. The data presented as well as the previously reported time course of endogenous progesterone secretion in this cell model of trophoblast differentiation (14, 17, 19) support these hypotheses.

Although progesterone is known to be important for successful embryo implantation, maintenance of pregnancy, and normal parturition, most of the work, to date, has focused on the actions of progesterone at the levels of the endometrium, myometrium, or breast. Nevertheless, as progesterone receptors have been shown to be present in cytotrophoblasts (20), an effect of progesterone at the level of the placenta should also be considered. The present investigation has discovered that one of the downstream molecular effects of binding of progesterone to these receptors appears to include regulation of the expression of OPN. The results clearly show that blocking endogenous steroid biosynthesis keeps OPN expression at a low level, and exogenous progesterone reverses this effect at both the mRNA and protein levels. Moreover, RU486, which antagonizes progesterone interaction with its receptor, also suppresses OPN mRNA expression in the presence of exogenous progesterone, further supporting this conclusion. It is noteworthy that adding RU486 alone to the trophoblast cultures had an up-regulatory effect on OPN mRNA expression, especially when endogenous progesterone levels are low, as opposed to its inhibitory effect when added together with progesterone. This paradoxical agonistic effect has also been reported in the endometrium of postmenopausal women (21) and in vitro when there is a high cAMP concentration in cell cultures (22). This conditional agonist activity has also been observed with other type II progesterone antagonists such as ZK112993 (23). The experimental evidence suggests that in the presence of high cAMP concentrations there is recruitment of a coactivator that mediates communication between the antagonist-receptor complexes and the basal transcription apparatus that, in turn, leads to agonist activity (24). Using the present in vitro model of trophoblast differentiation, we have previously shown that trophoblast aggregation is associated with an increase in intracellular cAMP (12), and thus, the observation of agonist activity when the cells are exposed to RU486 alone at a time of low endogenous progesterone (24h) agrees with results obtained using other experimental models.

The human OPN gene has been cloned and mapped to chromosome 4q13 (3), and its 5'-upstream region has been recently characterized (25). There is a segment in this flanking region that has a sequence similar to the optimal progesterone recognition sequence (26). The murine gene, which is better characterized (8, 13), also has putative estrogen, glucocorticoid, and progesterone response elements that enable these steroids to exert transcriptional control on OPN expression in mouse tissues. Presumably, progesterone exerts its stimulatory effect on OPN expression by its action on the promoter region in the human gene. The observation of a partial agonistic activity of RU486 in the trophoblasts suggests binding of RU486-receptor complexes to the OPN promoter, which is additional evidence for the presence of a putative progesterone response element in the upstream region of the OPN gene.

Although the full functional significance of the presence of OPN in the chorionic villus remains to be determined, its actions in other cell systems suggest a potential role for this matrix protein in the regulation of important processes of the placenta. It has been shown that OPN can function as a cytokine and that it can exert a chemoattractant effect on macrophages and stimulate IgM and IgG antibody production by B cells (27). In addition, T cell activation by OPN has also been reported (28). Likewise, recent work indicates a role for OPN in nitric oxide synthesis (29), a process of potential significance in the course of normal placentation and placental function. Thus, abnormalities in the regulation of expression of OPN by human cytotrophoblasts and its action(s) on surrounding tissues may be related to the development of complications of pregnancy that are associated with altered nitric oxide synthase activity, such as preeclampsia. It has also been shown that OPN inhibits urinary calculi formation (5), thus making it conceivable that it may help to prevent calcifications of the placenta.

In addition, we propose that one of the critical roles of OPN in the placenta may be in the regulation of syncytiotrophoblast function. OPN’s main known receptors are members of the {alpha}v family of integrins (30), and the binding of OPN to its integrin receptors mediates several processes, including cell adhesion and signaling. It has been demonstrated that the {alpha}vß3 integrin is expressed by human syncytiotrophoblasts, but not by mononuclear cytotrophoblasts (31). Further, some of our recent findings demonstrate that human trophoblasts attach to OPN and that this adhesion is mediated at least in part by the {alpha}vß3 integrin (32). Preliminary results indicate that binding of OPN to syncytiotrophoblasts generates intracellular calcium oscillations, indicating a role for this molecule in trophoblast signaling (Coutifaris, C., unpublished observations). Thus, we propose that in vivo, the OPN synthesized and secreted by mononuclear cytotrophoblasts binds to {alpha}v integrins, i.e. {alpha}vß3, present in the overlying syncytiotrophoblast facilitating adhesion and communication between the two cell layers (see schematic representation in Fig. 6Go). We postulate that this adhesive and/or signaling event is vital for maintaining the structural integrity of the chorionic villus and has an as yet uncharacterized role in the normal function of the syncytiotrophoblast.



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  		Figure 6. Schematic representation of the epithelial cells of the chorionic villus and the postulated paracrine communication involving progesterone, OPN, and an {alpha}v integrin receptor. The findings from this study suggest that a regulatory feedback loop exists between the cytotrophoblast and syncytiotrophoblast layers of the villus in which an endocrine product of the latter (progesterone) regulates the expression of a paracrine product (OPN) by the former. In turn, OPN secreted by the cytotrophoblast layer is postulated to bind to one of its {alpha}v integrin receptors, {alpha}vß3, expressed by the overlying syncytium. We hypothesize that this ligand-receptor binding facilitates adhesion and communication between the two cell layers and may be vital for the structural integrity of the chorionic villus. For purposes of clarity, the space between the cytotrophoblast and syncytiotrophoblast layers has been exaggerated.

 
In this study we have demonstrated a novel regulatory feedback system between the trophoblast syncytium and the underlying cytotrophoblast cell layer: secretion of progesterone by the target cell (the syncytiotrophoblast) regulates the expression of OPN (the paracrine factor) by the underlying mononuclear cytotrophoblast. OPN, in turn, can then bind to its specific receptor present in the target cell. Work is underway to determine the precise molecular mechanism of the signaling events following the binding of OPN to its integrin receptor(s) on the syncytiotrophoblast and to uncover the regulatory role of OPN in trophoblast function.


   Acknowledgments
 
We thank Drs. Marian Young and Larry Fisher of the Bone Research Branch, National Institute of Dental Research (Bethesda, MD), for providing us with the OPN cDNA and for helpful discussion. We are grateful to Dr. S. S. Koide of The Population Council (New York, NY) for providing the RU486 used in this study.


   Footnotes
 
1 This work was supported by NIH Grants HD-06274 (to C.C.) and DK-33501 (to J.R.H.) and the Rockefeller Foundation. Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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