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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 1 297-301
Copyright © 2000 by The Endocrine Society

Original Studies

Progestin-Epidermal Growth Factor Regulation of Tissue Factor Expression during Decidualization of Human Endometrial Stromal Cells1

Charles J. Lockwood, Graciela Krikun, Radmila Runic, Lisa B. Schwartz, Augusto F. Mesia and Frederick Schatz

Department of Obstetrics and Gynecology (C.J.L., G.K., R.R., L.S., F.S.)and Department of Pathology (A.M.), New York University School of Medicine, New York, New York 10016

Address correspondence and requests for reprints to: Charles J. Lockwood, M.D., Professor and Chairman, Department of Obstetrics and Gynecology, New York University School of Medicine, New York, New York 10016.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Biochemical assays
 Results
 Discussion
 References
 
Perivascular decidualized human endometrial stromal cells (HESCs) are ideally positioned to prevent peri-implantational hemorrhage during endovascular trophoblast invasion by expressing tissue factor (TF), the primary cellular mediator of hemostasis. Earlier in vivo and in vitro studies have demonstrated enhanced TF expression in estradiol (E2)-primed HESCs during progestin-induced decidualization. However, the absence of estrogen or progesterone response elements from the TF gene promoter suggests that paracrine factor(s) may mediate these effects. We now demonstrate that significant elevation of TF messenger RNA and protein levels in the cultured HESCs require incubation with both epidermal growth factor (EGF) and the progestin medroxyprogesterone acetate (MPA) added, with or without E2. By contrast, no effects were elicited by adding EGF with E2, or by the separate additions of EGF, MPA, or E2 plus MPA. Our finding, that transforming growth factor-alpha, but not transforming growth factor-beta or interleukin 1-beta mimics these EGF effects, indicates that progestin-enhanced TF expression in cultured HESCs requires activation of the EGF receptor (EGFR). Western blot analysis indicated that MPA increased EGFR levels 2- to 3-fold in cultured HESCs. The current results suggest that the progestin up-regulation of TF levels in decidualized HESCs is mediated by enhanced EGFR expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Biochemical assays
 Results
 Discussion
 References
 
DECIDUALIZATION of human endometrium involves programmed changes in growth, as well as biochemical and morphologic differentiation (1). In the midluteal phase of the menstrual cycle, progesterone stimulates estradiol (E2)-primed human endometrial stromal cells (HESCs) to decidualize around blood vessels. Such perivascular decidualized HESCs are ideally positioned to prevent peri-implantational hemorrhage during endovascular trophoblast invasion by expressing tissue factor (TF), the primary cellular mediator of hemostasis (2). The participation of decidual cells in placental hemostasis is indicated by the close correlation between the extent of trophoblast invasion and decidualization in species with a hemochorial placenta (1, 3). Moreover, hemorrhage generally complicates placenta accreta and ectopic pregnancy, conditions in which the decidua is deficient.

Earlier work from our laboratory indicated that decidualized HESCs in sections of luteal phase and pregnant endometrium displayed enhanced expression of TF messenger RNA (mRNA) and protein (4, 5, 6). In monolayers of HESCs, others have shown that progestins control the expression of decidualization markers such as PRL (7, 8) and IGF binding protein-1 (IGFBP-1) (9, 10), as well as laminin and fibronectin (7, 11). In this well-characterized in vitro decidualization model, progestins increased TF mRNA and protein levels (5). Moreover, antiprogestins reversed these effects (12). Despite a lack of response to E2, greater effects were observed in HESCs incubated with E2 plus progestin than with progestin alone (13). These differential steroid effects are consistent with E2 priming of the endometrium for the decidualizing actions of progesterone. The absence of estrogen and progesterone response elements from the published sequence of the TF gene promoter suggests that autocrine and/or paracrine factors may mediate steroid effects on TF expression.

Progestin-epidermal growth factor (EGF) interactions are implicated in regulating cell growth, PRL, laminin, and fibronectin expression in cultured HESCs (14). The cellular effects of EGF are mediated by binding to its receptor (EGFR), a cell surface-sequestered structural homologue of the c-Erb b oncogene protein product (15). Ligand binding induces the EGFR to homodimerize or to form heterodimers with other members of the EGF/ErbB receptor family, thereby triggering complex pathways of intracellular gene-activating phosphorylation (16, 17).

The current study reveals that EGFR ligands are involved in mediating progestin-enhanced TF expression during in vitro decidualization of HESCs. In addition, expression of EGFR was found to be progestin-enhanced during in vitro decidualization of HESCs. These progestin-EGF interactions should play an integral role in mechanisms regulating TF expression and consequent hemostasis in peri-implantational and pregnant decidua. These mechanisms are also expected to play a crucial role in determining the balance between the physiological expression of TF and its pathological expression in conditions in which pregnancy loss or maternal hemorrhage follows decidual hemorrhage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Biochemical assays
 Results
 Discussion
 References
 
Tissues

After obtaining informed consent in compliance with local institutional review board guidelines, endometrial specimens were collected from patients undergoing hysterectomies for myomas and transported on ice to a sterile laminar flow hood. Small portions were formalin-fixed for dating by the histologic criteria of Noyes et al. (18), and others were extracted in preparation for Western blotting as described below.

Cell isolation and culture

Stromal cells were isolated from the remainder of each specimen of cycling endometrium as previously described (19), with more than 99% purity confirmed by immunohistochemical staining for cytokeratin and vimentin (5). Primary HESCs were grown to confluence (3–4 x 104 cells/cm2) in a 37C, 95% air: 5% CO2 incubator in basal medium (BMS), which consists of (BM), a phenol red-free 1:1, v:v mix of Dulbecco’s MEM (Life Technologies, Inc., Grand Island, NY) and Ham’s F-12 (Flow Laboratories, Rockville, MD), with 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL fungizone, supplemented with 10% charcoal-stripped calf serum (S), renewing the medium every 4–5 days.

Experimental culture conditions

Confluent HESCs were incubated in parallel groups in BMS + 0.1% ethanol (vehicle control) or 10-8 mol/L E2, 10-7 mol/L medroxyprogesterone acetate (MPA), or E2 plus MPA. After 4 days in the incubator, the conditioned medium was removed, and the cultures were washed twice with HBSS to remove residual serum elements. The medium was exchanged for a defined medium (DM) [BM plus ITS+ (Collaborative Research, Waltham, MA), 5 µM FeSO4, 50 µM ZnSO4, 1 nM CuSO4, 20 nM Na2SeO3, trace elements (Life Technologies, Inc.), and 50 µg/mL ascorbic acid (Sigma, St Louis, MO), containing corresponding vehicle control or steroid(s) added with or without EGF, transforming growth factor-{alpha} (TGF{alpha} ), TGFß , or interleukin-1ß (IL-1ß ) (Collaborative Research). At the end of the treatment period in DM, the medium was removed, and the monolayers were washed with HBSS. The cells were harvested, centrifuged, and the pellets stored at -70C. The RNA was extracted from HESC monolayers in parallel experiments. In other experiments, confluent HESCs were incubated for 4 days in BMS containing E2, or E2 plus MPA. The cells were then harvested for Western blotting as described below.


   Biochemical assays
Top
 Abstract
 Introduction
 Materials and Methods
 Biochemical assays
Results
 Discussion
 References
 
Immunoreactive TF in the sonicated cell pellets was measured with a sensitive enzyme-linked immunoabsorbent assay (ELISA) (American Diagnostica, Greenwich, CT). Protein and DNA content were measured in aliquots of the cell pellets as described (5).

Northern blot analysis

Total RNA was extracted from cultured HESCs by the guanidinium thiocyanate-chloroform method, as previously described (5). Total RNA (25 µg) from each of the experimental cultures and molecular weight standards (Roche Molecular Biochemicals, Indianapolis, IN) were separated on a 1% agarose gel containing 2.2 mol/L formaldehyde, then transferred to a Zeta-Probe nylon membrane (BioRad, Hercules, CA). As previously described (5), the TF probe was labeled with [32P]deoxy-CTP to high specific activity by random priming with a Random Primed DNA Labeling kit, and hybridization was performed by standard methods. The washed membranes were exposed to Kodak XAR film (Eastman Kodak Co., Rochester, NY) with signal intensity assessed by densitometry. Total RNA loads were standardized by re-examining stripped membranes with a 32P-labeled probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Western blot analysis

Cultured HESCs harvested by scraping with a spatula, or approximately 0.5 g endometrium, were each disrupted with a Dounce (Kimble/Kontes Glass, Vineland, NJ) homogenizer in ice-cold 10 mM Tris, 250 mM sucrose, 1 mM EDTA at pH 7.4 containing a protease inhibitor cocktail (5). The homogenate was centrifuged at 600 g for 10 min, and then 12,000 g for 30 min. After adding sodium chloride (100 mM) and magnesium sulfate (0.2 mM), the supernatant was centrifuged at 40,000 g for 40 min (19). The pellet was solubilized in 25 mM TRIS, 150 mM NaCl, 2 mM EDTA, pH 7.6 containing 0.5% NP-40, and resolved by SDS-PAGE under denaturing conditions. Western blotting was conducted as described (5). The blots were incubated in 1:100 dilution of rabbit polyclonal EGFR antibody (Oncogene Sciences, Cambridge, MA). Immunodetection was performed using ECL Western blotting protocols (Amersham Pharmacia Biotech, Arlington Heights, IL). Densitometric analysis of the blots was carried out with Kodak Digital Science 1D Image Analysis Software (Rochester, NY).

Statistical analysis

Statistical analysis was performed by the Mann-Whitney rank sum test, with P < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Biochemical assays
 Results
Discussion
 References
 
In vitro effects of steroids on EGFR expression in HESCs

The Western blot shown in Fig. 1Go, A was carried out to determine whether EGFR expression is progestin-regulated in cultured HESCs. It demonstrates the presence of the EGFR in cultured HESCs after parallel incubations in E2, which was used as the control condition to simulate priming of HESCs for the decidualizing effects of progesterone in vivo, vs. E2 plus MPA. As indicated, the HESCs contain a doublet at 170 kDa, the magnitude of which increased 2- to 3-fold in incubations with E2 plus MPA compared with E2 alone. We next confirmed that decidualization was associated with increased immunoreactive EGFR levels in vivo. The Western blot shown in Fig. 1Go, B is consistent with a previous report (20) that EGFR levels are much higher in secretory phase than in proliferative phase endometrial extracts.



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  		Figure 1. EGFR levels in cultured HESCs and endometrial biopsies. A, Confluent cultures were maintained in BMS with either 10-8 mol/L E2, or 10-8 mol/L E2 + 10-7 mol/L MPA (P) for 4 days. The medium was removed, the cells were washed with HBSS, harvested, and analyzed by Western blotting for the presence of EGFR (see Materials and Methods). Densitometric comparisons in cultures derived from three specimens indicated an increase of 253% ± 94 (mean ± SEM) in HESCs from three specimens treated with E2 plus MPA vs. E2. B, Western blotting for the EGFR in extracts from proliferative phase (Pro) and day 23 secretory phase (d23) endometrium (see Materials and Methods).

 
Paracrine and/or steroids effects on HESC-expressed TF

Previously, we showed that progestins elevated HESC-expressed TF in both a serum-containing (BMS) and a serum-free (DM) medium, and that E2 augmented these responses despite a lack of response to E2 alone (12, 13). Moreover, our studies revealed a lag period of 24–48 h between exposure of the HESCs to progestin and detection of enhanced TF levels (13). Figure 2Go displays the effects of EGF and E2 plus MPA, added separately or together, on TF expression in HESCs. In the absence of E2 plus MPA, 50 ng/mL of EGF did not alter TF levels, whereas without EGF, E2 plus MPA failed to affect TF levels. However, the combination of EGF and E2 plus MPA produced much greater than additive increases of TF levels (7-fold, P < 0.0001) (2A). As expected from our earlier studies (12, 13), TF protein levels were elevated when EGF was added with MPA alone, but not with E2 alone (results not shown). The dose-response relationships for EGF shown in Fig. 2Go, B indicate that, when added with E2 plus MPA, threshold increases of TF expression were evident at 0.5ng/mL EGF, with half-maximal and maximal increases occurring at about 4 ng/mL EGF and 50ng/mL EGF, respectively.



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  		Figure 2. Effects of EGF and steroids on immunoreactive (ir) TF levels. A, Effects of EGF and steroids on ir TF levels in cultured HESCs. HESCs were incubated for 4 days in BMS containing either vehicle control, or 10-8 mol/L E2 (E) +10-7 mol/L MPA (P), and then exposed to the corresponding control or E+P, with or without 50 ng/mL EGF for 36 h. Cell-associated TF was measured by ELISA and normalized to cell protein in 10 separate experiments. *, control (±EGF) vs. E+P+EGF, (P < 0.0001). B, Dose-response effects of EGF on ir TF levels in cultured HESCs incubated for 4 days in BMS with either vehicle control or 10-8 mol/L E2 +10-7 mol/L MPA, and then exposed to control or E2+ MPA with 0, 0.5, 5, and 50 ng/mL EGF for 36 h (see Materials and Methods). Cell-associated TF was determined by ELISA, normalized to cell protein, and presented as percent of values in cultures without EGF.

 
Consistent with these changes in TF protein expression, the Northern blot of Fig. 3Go indicates that TF mRNA expression was refractory to the separate addition of E2 plus MPA, or EGF, or TGF{alpha} , which also binds to and activates the EGFR (16). However, when adjusted for differences in RNA loading, simultaneous exposure of the HESCs to E2 plus MPA with either EGF or TGF{alpha} increased TF mRNA levels about 3-fold. Figure 4Go further supports the involvement of the EGFR in mediating progestin-enhanced TF expression in HESCs by comparing the effects of EGF with TGFß and IL-1ß. In contrast with the marked enhancement of TF levels elicited by the combination of EGF and E2 plus MPA, no elevation in TF levels was observed with either TGFß (2 ng/mL) or IL-1ß (1.0 U/mL) added in combination with E2 plus MPA. Similar results were obtained in an additional experiment in which TGFß was added at 4 ng/mL, and IL-1ß was added at 2.0 U/mL. The concentrations used generally exceeded those shown to markedly enhance DNA, IL-8, integrin, and matrix metalloproteinase expression in cultured HESCs, while inhibiting prolactin expression (21, 22, 23, 24, 25, 26). Similar results were obtained when normalizing TF content to total culture DNA, indicating that these EGF-progestin effects on TF expression were not a consequence of altered cell growth in these confluent HESC monolayers.



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  		Figure 3. Northern analysis of growth factor and steroid effects on TF mRNA levels in cultured HESCs. After the incubation protocol described in Fig. 3AGo, total cell RNA was extracted and subjected to Northern blot analysis as described in Materials and Methods. RNA loads were normalized for steady state levels of GAPDH mRNA; lane 1 = control, lane 2 = 50 ng/mL EGF, lane 3 = 50 ng/mL TGF{alpha} , lane 4 = 100 ng/mL TGF{alpha} , lane 5 = 10-8 mol/L E2 + 10-7 mol/L MPA, lane 6 = E2+MPA+50 ng/mL EGF, lane 7 = E2+MPA + 50 ng/mL TGF{alpha} , lane 8 = E2+MPA+100 ng/mL TGF{alpha} .

 


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  		Figure 4. Growth factor, cytokine, and steroid effects on TF levels in cultured HESCs. HESCs were exposed for 4 days in BMS containing 0.1% ETOH vehicle control (C), or primed with 10-8 mol/L E2 (E) + 10-7 mol/L MPA (P). The cultures were then incubated for 36 h in DM containing corresponding vehicle or E+P, with and without 50ng/mL EGF, or 2ng/mL TGFß or 1.0 U/mL IL-1ß . Cultures were analyzed for ir TF by ELISA and normalized to total culture protein. Means ± SEM of four separate experiments are presented. Only comparisons of control vs. E2 + MPA + EGF are significant (P < 0.03).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Biochemical assays
 Results
 Discussion
References
 
TF is the transmembrane cell receptor for coagulation factor VII and its activated form (VIIa). Under physiological conditions, TF is constitutively expressed by some perivascular cells, but not by endothelial cells. As a result of vascular disruption, perivascular cell-bound TF is exposed to blood. These events are rapidly followed by formation of a complex between TF and plasma-derived factors VII or VIIa. The ensuing cascade enables TF to act as the physiological initiator of hemostasis via the activation of factor X, which, in turn, converts prothrombin to thrombin (27, 28).

Previously, we showed that ovarian steroid-regulated decidualization of HESCs was associated with enhanced TF expression in vivo and in vitro (4, 5, 6, 12, 13). For the latter, progestins elevated HESC-expressed TF and E2 augmented these effects despite a lack of response to E2 alone. These differential effects applied to functionally active TF, as measured by a clotting assay, and to immunogenic TF as measured by ELISA and validated by Western blot analysis. Parallel effects on TF mRNA levels were observed by Northern analysis (5, 12, 13). Transient transfections with TF-reporter constructs indicated that progestins augmented TF mRNA levels via enhanced transcription and not by altered mRNA half-life (29). The absence of estrogen and progesterone response elements from the TF promoter suggests that paracrine intermediaries modulated these steroid effects. Reports that EGF-steroid interactions control growth and differentiation of the murine uterus (30, 31, 32) as well as decidualization-related endpoints in HESCs (14) prompted us to evaluate the separate and interactive effects of EGF and steroids on TF expression in cultured HESCs. Cytokines, growth factors, and serum transiently (1–4 h) induced TF mRNA and protein in cultured cells from diverse tissues (33, 34, 35, 36). By contrast, MPA-enhanced TF expression was only detected in HESCs after a lag of 24–48 h, with E2 plus MPA maintaining TF at elevated levels for up to 3 weeks (13). Operating within the constraints of this unique time course, we evaluated progestin-EGF interactions on TF expression in HESCs after a 36-h test period in a serum-free medium. Dose-response studies revealed that maximal effects were elicited by adding EGF at a concentration of 50 ng/mL. Given the heterogeneity of the available endometrial samples in terms of patient age, ethnicity, and time of the menstrual cycle, EGF was added at 50 ng/mL to ensure maximal enhancement of TF expression.

Results presented in this study indicated that, consistent with our previous studies of TF expression (4, 5, 6), increased EGFR expression was also tightly coupled to the progestin-regulated decidualization reaction. These effects on EGFR expression were consistent with previous reports linking enhanced EGFR expression with decidualization in humans (37) and baboons (38). Among members of the EGF/ErbB receptor family, only EGFR was activated by EGF and its structural homologues, TGF{alpha} , heparin-binding EGF, and amphiregulin. Ligand binding induced the EGFR to form homodimers or to heterodimerize with ErbB family members such as ErbB2 and ErbB3. Receptor dimerization initiated autophosphorylation of EGFR and transphosphorylation of ErbB2 or ErbB3 at tyrosine residues in the carboxyl terminus (17). These phosphorylated tyrosine residues served as docking sites for proteins bearing src homology 2 (SH2) domains, which concentrated substrates for the tyrosine kinase at its active site. Phosphorylation of these substrates by the activated EGFR tyrosine kinase established a common link to multiple intracellular signal transduction pathways (39).

Consistent with our finding of an up-regulation of EGFR by progestins in cultured HESCs, Lange et al. (40) recently reported that progestins induced a 2- to 3-fold increase in EGFR as well as ErbB2 and ErbB3 in an epithelial breast cancer cell line. Increased expression of the c-EGFR/ErbB family of receptors was in turn associated with enhancement in a variety of phosphorylation-dependent intracellular signaling pathways (e.g. MAPK, STAT, JAK) in these cells (40). Recent studies from our laboratory indicated that progestin-effects on HESC TF mRNA transcription required binding to the SP-1 cis-acting element of the TF promoter (41). Because activation of SP1 cis-acting elements appears to require phosphorylation of the SP1 protein (42), we speculate that progestin-EGFR interactions may enhance SP-1 phosphorylation with resulting increases in TF mRNA transcription.


   Footnotes
 
1 This work was supported in part by Grant 5-RO1-HL33937-03 (to C.J.L.) from the National Institutes of Health. Back

Received May 3, 1999.

Revised September 27, 1999.

Accepted October 1, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Biochemical assays
 Results
 Discussion
 References
 

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C. J. Lockwood, G. Krikun, M. Hickey, S. J. Huang, and F. Schatz
Decidualized Human Endometrial Stromal Cells Mediate Hemostasis, Angiogenesis, and Abnormal Uterine Bleeding
Reproductive Sciences, February 1, 2009; 16(2): 162 - 170.
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 J. Clin. Endocrinol. Metab.Home page
K. Chobotova, N. Karpovich, J. Carver, S. Manek, W. J. Gullick, D. H. Barlow, and H. J. Mardon
Heparin-Binding Epidermal Growth Factor and Its Receptors Mediate Decidualization and Potentiate Survival of Human Endometrial Stromal Cells
J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 913 - 919.
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 J. Clin. Endocrinol. Metab.Home page
C.-S. Chou, C. D. MacCalman, and P. C. K. Leung
Differential Effects of Gonadotropin-Releasing Hormone I and II on the Urokinase-Type Plasminogen Activator/Plasminogen Activator Inhibitor System in Human Decidual Stromal Cells in Vitro
J. Clin. Endocrinol. Metab., August 1, 2003; 88(8): 3806 - 3815.
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 J. Clin. Endocrinol. Metab.Home page
C. J. Lockwood, G. Krikun, A. B. C. Koo, S. Kadner, and F. Schatz
Differential Effects of Thrombin and Hypoxia on Endometrial Stromal and Glandular Epithelial Cell Vascular Endothelial Growth Factor Expression
J. Clin. Endocrinol. Metab., September 1, 2002; 87(9): 4280 - 4286.
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G. Krikun, H. Critchley, F. Schatz, L. Wan, R. Caze, R. N. Baergen, and C. J. Lockwood
Abnormal Uterine Bleeding during Progestin-Only Contraception May Result from Free Radical-Induced Alterations in Angiopoietin Expression
Am. J. Pathol., September 1, 2002; 161(3): 979 - 986.
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T. Liu and T. F. Ogle
Signal Transducer and Activator of Transcription 3 Is Expressed in the Decidualized Mesometrium of Pregnancy and Associates with the Progesterone Receptor Through Protein-Protein Interactions
Biol Reprod, July 1, 2002; 67(1): 114 - 118.
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J. K. Richer, B. M. Jacobsen, N. G. Manning, M. G. Abel, D. M. Wolf, and K. B. Horwitz
Differential Gene Regulation by the Two Progesterone Receptor Isoforms in Human Breast Cancer Cells
J. Biol. Chem., February 8, 2002; 277(7): 5209 - 5218.
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 J. Clin. Endocrinol. Metab.Home page
R. Runic, F. Schatz, L. Wan, R. Demopoulos, G. Krikun, and C. J. Lockwood
Effects of Norplant on Endometrial Tissue Factor Expression and Blood Vessel Structure
J. Clin. Endocrinol. Metab., October 1, 2000; 85(10): 3853 - 3859.
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