Molecular Human Reproduction, Vol. 5, No. 9, 789-796,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular endocrinology |
The decidualizing effect of progesterone may involve direct transcriptional activation of corticotrophin-releasing hormone from human endometrial stromal cells
1 Departments of Pharmacology and 2 Clinical Chemistry, School of Medicine, University of Crete, Heraklion, Greece and 3 Developmental Endocrinology Branch, National Institutes of Health, Bethesda, MD, USA
Abstract
The hypothalamic neuropeptide corticotrophin-releasing hormone (CRH) is also produced by human endometrial cells and is directly involved in the decidualization process as a paracrine inducer. The aim of the present work was to examine the effect of progesterone, the main decidualizing factor, on endometrial CRH, in primary cultures of human endometrial stromal cells. The effect of progesterone was examined by measuring the effects of medroxyprogesterone acetate (MPA) on (i) the concentration of immunoreactive CRH in isolated human endometrial stromal cells and (ii) the activity of the CRH promoter in human endometrial stromal cells transfected with a 0.9 kb fragment of the 5'-flanking region of the human CRH gene coupled to luciferase reporter. The data show that MPA increased the production and secretion of immunoreactive CRH from stromal cells and induced the activity of the CRH promoter, both in a dose-dependent manner. These effects were partially reversed by a molar excess of the antiprogestin RU 486 and were completely abolished in the presence of 100 nmol/l of the cAMP inhibitor, Rp-cAMP. The effect of progesterone on the CRH promoter requires the existence of an intact CRH sequence since experiments carried out with a deleted palindromic cAMP response element (CRE: 5'-TGACGTCA) at –224 bp of the CRH promoter resulted in a complete loss of MPA effect. In conclusion, these data provide evidence that progesterone induces the transcription of CRH gene in human endometrial stroma. This effect coupled with the decidualizing properties of progesterone and CRH may indicate that progesterone and CRH form a decidualizing local pathway within the human endometrium.
CRF/corticotrophin-releasing hormone/decidualization/endometrium/progesterone
Introduction
Progesterone is the principal decidualizing effector of human endometrium (Psychoyos et al., 1995
). Recently, the hypothalamic peptide corticotrophin-releasing hormone (CRH) has been shown to be produced by the epithelial and stromal cells of human endometrium (Markigiannakis et al., 1995a; Mastorakos et al., 1996; DiBlazio et al., 1997) and to participate in the decidualization process (Ferrari et al., 1995) by acting locally on the CRH-R1 receptors present in stromal cells (DiBlazio et al., 1997). The present work is based on the hypothesis that the decidualizing effect of progesterone may be partially mediated by locally produced CRH. It is possible that the following sequence of events takes place in the endometrium during decidualization: among other effects, progesterone also induces the production of endometrial CRH which in turn stimulates the production of local decidualizing factors which initiate or modify the decidualizing process itself. To test this hypothesis, the in-vitro effect of synthetic progesterone agonists and antagonists on the production of CRH by human endometrial stromal cells in primary culture and on the activity of the CRH promoter in purified human stromal cells, transiently transfected with a 0.9 kb fragment of the 5'-flanking region of the human CRH gene coupled to luciferase reporter, was examined. Since cAMP plays an important role in the regulation of the activity of the CRH promoter (Seasholtz et al., 1988), the effect of progestins on the cAMP-mediated production of CRH was also examined. Thus, human endometrial stromal cells were transfected with a CRH promoter deprived of its cAMP sensitive sequence. Both types of transfectants were exposed to the synthetic progestin medroxyprogesterone acetate (MPA), in the absence or presence of cAMP inhibitor, Rp-cAMP. Subsequently, the activity of luciferase reporter was determined in cell lysates. The results provide strong evidence that progestins induce the production of CRH in human endometrial stroma, through direct transcriptional activation of the CRH gene, suggesting that CRH may mediate the progesterone-induced decidualization.
Materials and methods
Isolation and purification of endometrial stromal cells
Late proliferative endometrial specimens were obtained from patients undergoing biopsy for fertility evaluation or hysterectomy. The tissue samples were trimmed and minced under a laminar flow hood in minimum essential medium (MEM) containing 1% of an antibiotic–antimycotic mixture (Grand Island Biological Co., Grand Island, NY, USA). Cell dispersion and isolation was carried out in MEM supplemented with 10 µg/ml porcine insulin (Nordisk-USA, Bethesda, MD, USA), 1% antibiotic–antimycotic mixture (Gibco, Grand Island, NY, USA) and 10% fetal calf serum (MEM + 10% FCS). As discussed in more detail elsewhere (Makrigiannakis et al., 1995b) it involves: (i) digestion of the minced tissue for 90 min at 37°C using 0.25% of Type I collagenase, (ii) separation of glands and stroma by filtration through a 45 µm stainless steel sieve, (iii) backwashing the glands from the sieve, followed by pelleting by centrifugation and separation of epithelial from stromal cells in the filtrate by taking advantage of the more rapid adhesion of the stromal cells to tissue culture plastic at 37°C.
Measurement of immunoreactive CRH
Stromal cells were cultured in the presence of various agents, then cells were collected and homogenized in 0.1 N HCl. Immunoreactive CRH in cell homogenates and culture media was concentrated by C-18 reverse phase columns (Sep-Pak; Waters Associates, Milford, MA, USA) following acidification in 2 volumes of 0.1 N HCl and centrifugation at 10,000 g for 10 min. The supernatants were extracted by activated Sep-Pak cartridges, washed with 20 ml 0.1 N HCl, eluted with 3 ml acetonitrile 80–0.01% HCI, then dried under vacuum (Speed-Vac). CRH was assayed by radioimmunoassay using a rabbit antiserum purchased from Neosystem Laboratoire (Strasbourg, France). The CRH antiserum was raised against human CRH and exhibits 100% cross-reactivity to rat CRH, and no cross-reactivity to ovine CRH, human adrenocorticotrophic hormone (ACTH), human ß-endorphin, luteinizing hormone-releasing hormone (LHRH), or AVP. The sensitivity of the assay was 0.1 pg/tube. The intra-assay coefficient of variation was 4.4% and the inter-assay 6.6%. Results are expressed as pg of CRH per mg of total cellular protein, determined on whole cellular homogenates, by the Bradford method using bovine serum albumin as standard (Bradford, 1976).
Description of the human (hCRH) constructs used and the transfection protocol
Figure 1 shows the structure of the hCRH promoter construct used in these experiments. It extends from the HindIII site at –918 bp to the +38 bp of exon 1 of the human CRH gene (Vamvakopoulos and Chrousos, 1993). It is subcloned in a sense orientation into the promoterless luciferase plasmid pA3luc (Dorin et al., 1993). The pA3luc luciferase expression vector has negligible non-specific luciferase activity. Upstream of the hCRH promoter insertion site, a cassette containing a trimerized SV40 poly(A) termination signal was introduced which virtually eliminated luciferase activity contributing to cryptic plasmid transcription, thus allowing a sensitive measurement of the activity of the promoter to be tested. Endometrial stromal cells transfected with the CRH-promoterless pA3luc construct were found to have negligible background luciferase activity, even in transfectants exposed to steroids (data not shown).
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The hCRH promoter with the deletion of the cAMP response element (CRE) sequence at –224 bp was kindly provided to us by Drs S.Malkoski and R.Dorin (Albuquerque, NM, USA). Deletion of the palindromic CRE (5'-TGACGTCA-3') from the CRH promoter was achieved by digestion with the enzyme AatII, blunting with the Klenow fragment of DNA polymerase, and ligation of synthetic XhoI linker to create the plasmid hCRH(-918)[
CRE]luc (Makloski et al., 1997). This construct contains a single 8 bp XhoI linker in the place of the deleted internal 4 bp of the CRE at –224 bp and re-ligation following Klenow blunting. This sequence has been confirmed by dideoxynucleotide sequencing using the Sequenase 2.0 kit (United States Biochemical, Cleveland, OH, USA).
The purified endometrial stromal cells were transiently transfected with either the hCRH(-918)pA3luc or the hCRH(-918)[CRE]luc constructs employing the lipofectin method (Stamatatos et al., 1988). They were maintained in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Flow Labs, Irvine, UK), containing 10% charcoal-stripped FCS, in 60 mm Petri dishes at 37°C under 95% air–5% CO2 until 70% confluency. Before transfection, 105 cells were washed with DMEM/F12 medium without serum, then they were incubated with the DNA–lipofectin complex, prepared as described by the supplier of lipofectin (Boerhinger Mannheim, Munich, Germany). The DNA transfected was hCRH(-918)pA3luc (5 µg), or hCRH(-918)[CRE]luc (5 µg), and pRSV-ßgal (1 µg) per plate. The latter was used as control to normalize luciferase values which were divided by their corresponding galactosidase values. A total of 6 µg of DNA per transfection was used. The DNA was dissolved in 100 µl of HBSS (HEPES 20 mmol/l, NaCI 150 mmol/l, pH 7.4) and mixed with an equal volume of lipofectin diluted 1:1 in HBSS. The 200 µl transfection mixture was delivered to properly prepared cells, over 6 h, then culture medium was changed and transfectants remained for at least another 24 h in culture. Cell growth was resumed in DMEM/F12 medium without serum containing different agents, diluted in ethanol. For comparison, ethanol was added in the control media at a final concentration of 0.1%. Transfectants were harvested 24 h after incubation with the compounds under testing or placebo ethanol and luciferase and ß-galactosidase activities were determined in cell lysates.
Measurement of luciferase and ß-galactosidase activity
Luciferase activity was determined in cell lysates as relative fluorescence units using the Promega luciferase assay (Promega Corp., Madison, WI, USA), (Schenborn and Goiffon, 1993). Transfectants were washed once with phosphate-buffered saline (PBS), then 300 µl of the lysis buffer, provided by Promega, was added. Lysed cells were centrifuged at 4°C for 2 min at 13,000 g and the supernatant was used to measure luciferase and ß-galactosidase activities. In brief, 20 µl of cell extract was added at 25°C in 100 µl of luciferase assay buffer containing 20 mmol/l tricine, 1.07 mmol/l (MgCO3)4Mg(OH)2•5H2O, 2.67 mmol/l MgSO4, 0.1 mmol/l EDTA, 33.3 mmol/l dithiothreitol, 270 µmol/l coenzyme A, 470 µmol/l luciferin and 530 µmol/l ATP, pH 7.8, then the light produced for a period of 10 s was counted in a Lumac/3M luminometer (Biocounter, Shoesberg, The Netherlands). For the measurement of ß-galactosidase activity in cell lysates the Promega assay was used as previously described (Rosenthal, 1987). In brief, 150 µl of cell extract was incubated at 37°C in a total volume of 300 µl of 0.1 mol/l sodium phosphate, pH 7.5, 0.67 mg/ml o-nitrophenyl-ß-D-galactopyranoside, 1 mmol/l MgCI2, and 50 mmol/l ß-mercaptoethanol. The reaction was stopped by adding 500 µl of 1 mol/l Na2CO3 and the optical density was determined at 420 nm.
Statistical analysis
Immunoreactive CRH data were analysed by multiple comparison tests which included analysis of variance (ANOVA) and subsequently Fisher's significance test. Luciferase values were first corrected for transfection efficiency according to ß-galactosidase values, then normalized to the respective controls (transfected cells cultured in the absence of any compound). Results were expressed as mean ± SEM of at least six experiments. To evaluate the dose–response curve of each compound tested, normalized luciferase activity was compared with the concentration. Statistical analysis for these data was performed by two non-parametric statistical methods (NPS; Kruskal–Wallis and Mann–Whitney) since luciferase activity was subjected to double normalization, i.e. ß-galactosidase correction and normalization to the respective controls. The data were also analysed normalized only for the transfection efficiency (luciferase values divided by their corresponding ß-galactosidase values) by multiple comparison tests (MCT) which included ANOVA and subsequently Fisher's significance test.
Results
Progesterone stimulates the production of CRH from human endometrial stromal cells
It is well established that human endometrial stromal cells contain functional progesterone (PR) and glucocorticoid receptors (GR) (Press et al., 1988). Following their isolation, endometrial stromal cells were placed in 6 cm wells and were left to grow until 75% confluency. To test the hypothesis that MPA affects the production of endometrial CRH, cultured stromal cells were exposed to various concentrations of MPA, and the immunoreactive CRH in cell homogenates and culture media and the activity of the CRH promoter in the transfected cells were measured.
Stromal cell content and culture media concentration of immunoreactive CRH increased in a dose-dependent manner following exposure to MPA in concentrations ranging from 1 to 1000 nmol/l (Figure 2). The maximal effect was observed at 1000 nmol/l. Indeed, the concentration of immunoreactive CRH in the cell extract increased from 0.2 pg/mg protein in the absence of MPA to 3.79 ± 0.1 pg/mg protein in the presence of 1000 nmol/l MPA (Figure 2A). Identical effects were observed on the concentration of immunoreactive CRH in the culture media (Figure 2B). Similarly, the activity of the CRH promoter in stromal cells, transfected with the hCRH(-918)pA3luc construct, increased in a dose-dependent manner following exposure to the same range of MPA concentrations (Figure 3). The maximal effect was observed at 100 nmol/l and was 292 ± 1.2% of controls (n = 6, NPS: P < 0.02; MCT: P < 0.05). To test the hypothesis that MPA might exert its effect on CRH production through its binding to progesterone receptors, the above experiments were conducted in the absence or the presence of a molar excess of progesterone antagonist RU 486. The effect of MPA on immunoreactive CRH and promoter activity were partially, but significantly, blocked in the presence of a molar excess of the antiprogestin RU 486 (Figures 2 and 3). It should be noted that RU 486 did not have any effect by itself in doses ranging from 1 to 1000 nmol/l (Figure 3).
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The stimulatory effect of progesterone on endometrial stroma CRH does not appear to be mediated by glucocorticoid receptors
The possibility was explored that MPA might exert its effect on CRH through its binding to glucocorticoid receptors, acting as a glucocorticoid agonist since progesterone possesses a significant affinity towards the glucocorticoid receptor (Kontula et al., 1983
; Ojancon et al., 1989) and glucocorticoids are known modulators of CRH production and secretion in hypothalamus, placenta and adrenals (Jingami et al., 1985; Robinson et al., 1988; Venihaki et al., 1998). The effect of MPA on the activity of CRH promoter was measured in the absence or presence of the synthetic glucocorticoid, dexamethasone (DEX).
In the system used, the presence of DEX did not modify the stimulatory effect of MPA on endometrial CRH, neither did it have any effect by itself (Figure 3
). Specifically, stromal cells transfected with the hCRH(-918)pA3luc construct were incubated with a range of MPA concentrations (0.1–1000 nmol/l) for 1, 2, 4 and 6 days in the presence or absence of a molar excess of DEX (1000 nM) (Figure 4). DEX did not exert any modifying effect on the CRH dose–response curve to MPA, suggesting that the effect of progesterone is not mediated by glucocorticoid receptors. Interestingly, DEX alone did not have any effect on the activity of stromal cell CRH (Figure 4).
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The stimulatory effect of progesterone on endometrial stroma CRH appears to be mediated by the cAMP response element on the CRH promoter
The hCRH promoter used does not contain a complete progesterone/glucocorticoid response element (PRE/GRE) but only the hexamer 5' TGTTCT or a half GRE site (1/2 GRE) at –598 bp (Vamvakopoulos and Chrousos, 1993
) (Figure 1) making it less likely that the effect of MPA is mediated by these response elements. An alternative site on the promoter may explain its effect. Indeed, it is possible that the effect of progesterone may involve cAMP response elements (CRE) which are known regulators of the CRH gene expression (Seasholtz et al., 1988; Adler et al., 1990). To test this hypothesis, i.e. that the effect of progesterone on the CRH promoter is carried out by the CRE motif, the following protocols were used: (i) transfected stromal cells were incubated with MPA (1–1000 nmol/l) in the absence or the presence of 100 nmol/l of Rp-cAMP, a specific cAMP inhibitor, and (ii) a CRH promoter was used with deleted palindromic cAMP response element (CRE: 5'-TGACGTCA) at –224 bp. This construct is identified as hCRH(-918)[CRE]luc. The transfection protocol was the same as that of the intact construct. To prove that the absence of CRE is not deleterious to the CRH promoter, the effect of interleukin-1 (IL-1) on stromal cells transfected with the defective construct hCRH(-918)[CRE]luc was compared to that of stromal cells transfected with the intact hCRH(-918)pA3luc construct. Interleukin-1 is a well-documented CRH inducer in hypothalamus, placenta and adrenals (Sapolsky et al., 1987; Petraglia et al., 1990; Navarra et al., 1991; Venihaki et al., 1998) and acts on gene expression through regulation of the NF-kB and the mitogen-activated protein (MAP) kinases pathways (Kishimoto et al., 1994). Indeed, the CRH promoter contains a functional NF-IL-6/EBP motif at position –299 bp (Stephanou et al., 1997).
The responsiveness of stromal cells, transfected with the hCRH(-918)pA3luc construct following exposure to 8-bromo-cAMP (Br-cAMP) for 24 h in the absence or presence of a cAMP-specific membrane permeable inhibitor, Rp-cAMP showed the following: 1 mM of Br-cAMP increased the activity of the CRH promoter by an impressive 1823 ± 59% with respect to controls (mean ± SEM, n = 6, P < 0.001, NPS, normalized data) (Figure 5A). These data were highly significant even in their non-normalized format by ANOVA and Fisher's MCT (P < 0.005). The effect of Br-cAMP was partially, but significantly, blocked by the cAMP inhibitor Rp-cAMP (Figure 5A). The presence of Rp-cAMP at 100 nmol/l blocked the expected stimulatory effect on the CRH promoter activity by 645 ± 72% (n = 6, NPS: P < 0.001; MCT: P < 0.005). These findings confirmed that the CRH promoter we have used contained a highly sensitive CRE motif.
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A second set of experiments was carried out using the CRH promoter with deleted palindromic cAMP response element (CRE: 5'-TGACGTCA) at –224 bp. The results obtained are as follows. (i) The absence of CRE is not deleterious to the CRH promoter since IL-1 induced the activity of the CRH promoter in both types of transfectants, i.e. independently of the absence or the presence of the CRE motif. Specifically, IL-1 at 100 nmol/l stimulated the activity of the CRH promoter by 258 ± 12% of controls (n = 6, NPS: P < 0.005; MCT: P < 0.05) in transfectants containing the CRE motif, and by 258 ± 23% of controls (n = 6, NPS: P < 0.005; MCT: P < 0.05) in transfectants deprived of the CRE motif (Figure 5A
). (ii) The induction of the CRH promoter was not significantly altered following co-incubation of IL-1 with 100 nM of the cAMP inhibitor Rp-cAMP (Figure 5A). (iii) Exposure of stromal cells transfected with the CRE-deleted construct hCRH(-918)[CRE]luc to a range of MPA (1–1000 nM) for 24 h resulted in the complete loss of the ability of MPA to induce the activity of CRH promoter, suggesting that this motif is important for this MPA effect (Figure 5B). (iv) Stromal cells transfected with the intact hCRH-pA3luc construct (i.e. containing the CRE motif) were incubated with MPA (1–1000 nM) in the presence or the absence of 100 nM of Rp-cAMP, a cAMP inhibitor. The presence of the cAMP inhibitor resulted in a complete inhibition of the MPA effect (Figure 5B).
Additional evidence for the involvement of the cAMP pathway in the effect of MPA was provided by studies on immunoreactive CRH. Indeed, exposure of endometrial stromal cells to various concentrations of MPA in the presence of 100 nmol/l of cAMP inhibitor, Rp-cAMP, resulted in the complete block of the effect of MPA on immunoreactive CRH in both cell extract and culture media (Figure 2).
Discussion
CRH is produced locally within the human endometrium by both epithelial and stromal cells (Makrigiannakis et al., 1995a; DiBlazio et al., 1997). Its physiological role is slowly becoming understood. Indeed, multiple lines of data suggest that the endometrial CRH may be involved in the decidualization process as well as the reception of the fertilized ovum (Makrigiannakis et al., 1995b). Since the steroid hormone progesterone is the dominant player in these processes, our experiments were designed to test the hypothesis that part of its effects on human endometrium are mediated by locally produced CRH. For this reason the effect of progesterone on CRH production by isolated human stromal cells in culture was examined. Their immunoreactive CRH content and secretion and the activity of a CRH promoter construct inserted into the same cells, i.e. into primary cultures of human endometrial stromal cells, were measured. The data suggest that progesterone exerts a stimulatory effect on CRH production and secretion from human stromal cells. This effect of MPA is dose-dependent, and is mediated by the progesterone and not the glucocorticoid receptor. Finally, stimulatory progesterone appears to need the cAMP response element on the CRH promoter.
The data in this study, showing that in normal endometrial stromal cells progesterone induces the transcription of CRH gene, are in contrast with recent reports describing an inhibitory effect of progesterone on CRH production in cultures of human fetal membranes and in the content of CRH transcript in hepatoma cells (Jones et al., 1989; Kontopoulos et al., 1998). These findings may suggest that the effect of progesterone on CRH gene may be cell-specific, i.e. depending on the presence of tissue-specific transcription factors, and that the molecular basis of the action of progestins on the CRH promoter is complex. Indeed, the hCRH promoter does not contain the complete PRE/GRE but only the hexamer 5' TGTTCT or 1/2 GRE at –598 bp (Vamvakopoulos and Chrousos, 1993). It can be postulated that the CRE may mediate the effect of progestins on the CRH gene since deletion of the cAMP response element at –224 bp from the CRH promoter resulted in the complete loss of MPA activity. This hypothesis is strengthened by Rp-cAMP, a cAMP inhibitor, which blocked completely the effect of MPA. Indeed, it is known that the CRE motif on the CRH promoter plays a crucial role (Seasholtz et al., 1988). It mediates the effect of a number of CRH regulators. In fact, the regulatory effects of glucocorticoids (inhibitory on hypothalamus and stimulatory on placenta) has been attributed to competition between the glucocorticoid–receptor complex and cAMP-related transcription factors, such as the cAMP response element binding protein (CREB), for binding on the CRE motif of the CRH promoter (Akerblom et al., 1989; Guardiola-Diaz et al., 1996). The activated PR may interact with the CREB protein via association with the binding protein of CREB (CBP), a transcription cofactor mediating the regulation of several steroid hormone-sensitive genes (Jenster et al., 1997). CBP binds to phosphorylated CREB enhancing its capacity for binding to CRE, recruiting the appropriate cofactors for the initiation of transcription (Arlas et al., 1994; Kwok et al., 1994). It has been shown that progesterone increases the phosphorylation of CREB in rat anteroventral periventricular nucleus (Gu et al., 1996). Taking this observation and combining it with the results of this study, it may be postulated that the effect of progesterone on the CRH promoter within the endometrial stroma involves activation of CREB and interaction with the CBP protein resulting in enhancement of transcription following the binding of CREB to the CRE motif on the CRH promoter. This hypothesis is further supported by recent reports describing `cross-talking' events between progesterone and the cAMP system in regulating gene expression of several cell types, including human breast cancer and porcine granulosa cells (Sirotkin and Nitray, 1993; Cho et al., 1994).
Progesterone and MPA possess a significant affinity towards the GR (Kontula et al., 1983; Ojancon et al., 1989). Thus, the stimulatory effect of MPA on the CRH promoter may be mediated by the GR activated by pharmacological concentrations of MPA. This hypothesis appeared even more plausible since human endometrial stromal cells possess functional GR, in addition to their progesterone receptors (Press et al., 1988). Furthermore, glucocorticoids induce the transcription of the CRH gene in certain non-neuronal tissues including human placenta and adrenomedullary chromafin cells (Robinson et al., 1988; Venihaki et al., 1998). However, the data prescribed here exclude such a possibility since dexamethasone did not exert any significant effect on the activity of the CRH promoter. Furthermore, the dose–response curve of MPA on the CRH promoter was not altered by the simultaneous presence of a molar excess of dexamethasone. Surprisingly, dexamethasone did not exert any effect on the CRH promoter transfected into stromal cells. This lack of effect may indicate absence of transcription cofactors, necessary for an efficient regulation of the CRH promoter, and points to a differential regulation of endometrial CRH by various steroid hormones.
The decidualization process is considered to be physiologically induced by progesterone. However, recent in-vitro findings revealed that cAMP or other compounds generating cAMP, such as relaxin, prostaglandins and gonadotrophins, are primary inducers of differentiation of endometrial stromal cells (Telgmann and Gellersen, 1998). The involvement of cAMP in the effect of progesterone on endometrial CRH reported in the present work is in indirect agreement with recently published data. Indeed, it has been shown that progesterone-dependent decidualization is mediated by cAMP since the cAMP inhibitor Rp-Br-cAMP blocks the decidualizing effect of MPA in endometrial stromal cells in culture (Brar et al., 1997). Furthermore, the CREB mRNA and the encoding protein are present within stromal cells of human endometrium (Gellersen et al., 1997). However, comparison of undifferentiated and decidualized stromal cells revealed no difference in the level of expression of the CREB protein. Nevertheless, elevated concentrations of the phosphorylated (active) form of CREB during decidualization cannot be excluded, particularly under the stimulatory effect of progesterone, which may increase CREB phosphorylation (Gu et al., 1996). Meanwhile, it is worth mentioning that CRE does not only bind CREB: a variety of transcriptional activators or repressors of the CREB family (Gellersen et al., 1997) may bind on the CRE motif, which may also come into play in the presence of MPA.
In conclusion, the data presented provide evidence that progesterone induces the transcription of CRH gene in human endometrial stroma. This effect coupled with the decidualizing properties of progesterone and CRH may indicate that progesterone and CRH form a decidualizing local mechanism within the human endometrium.
Acknowledgments
We thank Drs R.Dorin and S.Malkoski (Arbouquerke, NM, USA) for providing the hCRH(-918)pA3luc and hCRH(-918)[CRE]luc constructs. This work was supported by a grant to A.G. from the General Secretariat of Research and Technology (PENED94).
Notes
4 To whom correspondence should be addressed at: Department of Pharmacology, Medical School, University of Crete, Heraklion GR-711 10, Greece 
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Submitted on March 3, 1999; accepted on June 8, 1999.
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