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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 5 2312-2318
Copyright © 2004 by The Endocrine Society

Estrogen Receptor-Mediated Down-Regulation of Corticotropin-Releasing Hormone Gene Expression Is Dependent on a Cyclic Adenosine 3',5'-Monophosphate Regulatory Element in Human Placental Syncytiotrophoblast Cells

Xin Ni, Yue Hou, Bruce R. King, Xiaolu Tang, Mark A. Read, Roger Smith and Richard C. Nicholson

Department of Physiology (X.N., Y.H., X.T.), Second Military Medical University, Shanghai 200433, People’s Republic of China; and Mothers and Babies Research Center (B.R.K., M.A.R., R.S., R.C.N.), Hunter Medical Research Institute, University of Newcastle, New South Wales 2310, Australia

Address all correspondence and requests for reprints to: Dr. Richard C. Nicholson, Mothers and Babies Research Center, Endocrine Unit, John Hunter Hospital, Locked Bag 1, Hunter Region Mail Center, New South Wales 2310, Australia. E-mail: rick.nicholson{at}hunter.health.nsw.gov.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Placental CRH plays a major role in the mechanisms controlling human pregnancy and parturition. Understanding how placental CRH production is regulated is therefore of importance. Previously we have shown that placental expression of CRH peptide and mRNA are inhibited by estrogens, in contrast to the stimulatory effects of estrogen on hypothalamic CRH production. Our current study found that in placental cells cotransfected with a CRH promoter construct and an estrogen receptor-{alpha} expression vector results in a differential regulation whereby 17ß-estradiol (E2) decreased and the putative pure estrogen antagonist, ICI 182780, increased CRH promoter activity. Sequential deletion of the CRH promoter indicated that the region between –248 and –213 bp was essential for the effect of both E2 and ICI 182780. This region contains a consensus cAMP regulatory element (CRE) that is a requirement for E2- and ICI 182780-mediated activity because the CRE motif can confer E2 inhibition on a heterologous promoter such as rabbit ß-globin. Mutation of the CRE resulted in a complete reversal of E2 and ICI 182780 regulatory effects. In summary, our results demonstrate that a consensus CRE is required for the action of estrogen receptor ligands in human placental syncytiotrophoblast cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRH (1), A 41-AMINO ACID neuroendocrine peptide, is synthesized in the paraventricular nucleus of the hypothalamus and secreted into the hypophyseal-portal system in response to stress (1). CRH is the principal mediator of the hypothalamic-pituitary-adrenal response to stress, stimulating proopiomelanocortin gene expression and ACTH release in the anterior pituitary, which in turn, stimulates release of glucocorticoids from the adrenal cortex (2). CRH is also found in cortex and other sites in the central and peripheral nervous system and is widely expressed in the gastrointestinal tract (3), placenta (4), skin (5), ovaries (6, 7), and vasculature (8). During pregnancy the placenta becomes the major source of CRH production (4, 9). Placental CRH is structurally and functionally identical with that produced in the hypothalamus, but a number of differences in the regulation of placental and hypothalamic CRH production are apparent.

Hypothalamic production of CRH is ubiquitous in mammals, in contrast to placental expression of CRH, which appears to be limited to primates (10, 11). During human pregnancy placental syncytiotrophoblast cells secrete large amounts of CRH into the maternal and, to a lesser degree, the fetal circulations (12). Maternal plasma CRH concentrations rise exponentially from midgestation, consistent with increases in placental synthesis (13). Abnormally elevated levels of CRH during pregnancy are associated with preterm delivery, whereas unusually low levels correlate with longer-than-normal gestations. Consequently these findings have led to the theory that CRH plays a key role in the timing of birth (14, 15). Critical to understanding how this timing mechanism may operate is an appreciation of the mechanisms controlling CRH production in placental syncytiotrophoblast cells.

A variety of endogenous factors is known to regulate placental CRH production, including glucocorticoids, catecholamines, acetylcholine, IL-1, and vasopressin (16, 17, 18). Estrogen is also implicated in the regulation of CRH production. The human placental syncytiotrophoblast synthesizes large amounts of estrogen, from fetal adrenal dehydroepiandrosterone sulfate precursors, and there is a correlation between maternal plasma estrogens and CRH levels in humans (19, 20). Superficially this suggests that estrogen may stimulate CRH gene expression, as has been demonstrated in the hypothalamus (21, 22). Recent observations by Giussani et al. (23) indicating that maternal treatment with 17ß-estradiol (E2) in the rhesus monkey stimulates placental CRH production supports this hypothesis. However, we have recently shown that this is not the case, and estrogens actually have an inhibitory effect on placental expression of CRH peptide and mRNA (24). The mechanisms by which estrogen inhibits placental CRH production remain to be characterized, although our earlier study examining estrogen regulation of CRH production indicated that estrogen acts through the CRH gene promoter via an estrogen receptor (ER)-dependent mechanism (24).

In this report we used primary cultures of human syncytiotrophoblast cells to identify the regulatory elements involved in estrogen inhibition of human CRH gene expression in placenta. We show that E2 functions through the {alpha}-subtype of ER to down-regulate CRH gene promoter activity via a mechanism that is specifically directed through a cAMP response element (CRE) located in the CRH gene promoter region.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

E2, trypsin, and deoxyribnuclease-1 were obtained from SigmaAldrich (Sydney, Australia). ICI 182780 was purchased from Tocris Cookson (Bristol, UK). pGL3-luciferase reporter plasmid, pRL-TK vector, and the dual-luciferase reporter assay system were obtained from Promega Corp. (Madison, WI). 8-Bromo-cAMP was obtained from Calbiochem (La Jolla, CA). DMEM was obtained from Life Technologies, Inc., (Grand Island, NY). Fetal Calf Serum (FCS) was obtained from CSL (Melbourne, Australia). Percoll was obtained from Amersham Biosciences (Buckinghamshire, UK).

Plasmids

PGL3-luciferase reporter plasmids were used for transient transfections. The 5.5-kb CRH 5'-flanking DNA was isolated from a human CRH genomic clone, CRH 1001+ (a kind gift from Dr. J. Majzoub, Harvard University, Boston, MA), the deletions and mutants of the CRH promoter, and the CRE-globin constructs were described previously (25, 26). The expression vectors for ER{alpha} (pCMV-ER{alpha}) and ERß (pcDNA-ERß) were generously provided, respectively, by Dr. B. S. Katzenellenbogen (University of Illinois, Urbana, IL) and Dr. P. Fuller (Prince Henry’s Institute of Medical Research, Melbourne, Australia).

Cell culture

Human placentae were obtained from normal pregnant women at term after spontaneous vaginal delivery or elective cesarean section. Collections of placentae were performed with the approval of Shanghai Hospital ethics and the Hunter Area Health Service and the University of Newcastle human ethics committees. Cytotrophoblasts were cultured as described previously (27) using a method modified from that described by Kliman et al. (28). Briefly, cotyledons removed from the maternal side were dispersed with trypsin and deoxyribonuclease I, and a purified fraction of cytotrophoblasts was obtained by Percoll gradient centrifugation and then plated and grown in phenol red-free DMEM with 10% FCS.

Transfections

Transient transfections were performed using a calcium-phosphate method as described previously (24, 29, 30). Typically, 1 d before transfection, 1 x 105 cells/well were seeded and fed with phenol red-free DMEM containing 10% charcoal stripped FCS in 12-well plates and incubated in an atmosphere of 5% CO2 at 37 C. Each well was transfected with 0.15 ml HEPES buffered saline-CaCl2 solution containing 6.5 µg DNA and 0.2 µg control DNA (pRL-TK vector, Promega Corp.) and incubated in 5% CO2 at 37 C. Eight hours later, culture media were changed to DMEM without serum and treated with various agents as indicated. E2, ICI 182780, or 8-bromo-cAMP were added to the treatment media as stock solutions in absolute ethanol or dimethylsulfoxide. Control media contained the same concentration of vehicle (<=0.01% vol/vol). Luciferase assays were carried out 3–48 h later using the dual-luciferase assay kit. Relative luciferase activity is presented as firefly luciferase values normalized to renilla luciferase activity.

Western blot analysis

Cells were scraped off dishes in the presence of lysis buffer consisting of 60 mM Tris-HCl, 2% sodium dodecyl sulfate, 10% sucrose, 2 mM phenylmethylsulfonyl fluoride (Merck, Darmstadt, Germany), 1 mM sodium orthovanadate (Sigma-Aldrich), and 10 µg/ml aprotinin (Bayer, Leverkusen, Germany). Lysates were quickly sonified, heated 5 min at 95 C, and stored at –80 C until used. Protein concentrations were measured using a modified Bradford assay and samples were diluted in sample buffer [250 mM Tris-HCl (pH 6.8), containing 4% sodium dodecyl sulfate, 10% glycerol, 2% ß-mercaptoethanol, and 0.002% bromophenol blue] and heated at 95 C for a further 5 min. Aliquots of proteins were separated by SDS-PAGE (10%) and subsequently transferred to nitrocellulose membranes by electroblotting. Membranes were blocked in 5% nonfat milk in TBS/Tween 20 (0.5%) for 30 min and incubated in the presence of the ER{alpha} antibody (sc-543, Santa Cruz Biotechnology, Inc., Santa Cruz, CA.) or ERß antibody (sc-6820, Santa Cruz Biotechnology) overnight at 4 C. Proteins were detected using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ) after incubation of blots for 2 h at room temperature with corresponding horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology).

Statistical analyses

Statistical multiple comparison analyses were carried out by one-way ANOVA. The values are expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ER ligand-mediated regulation of CRH promoter activity

Primary cultures of human placental cells were transfected with a 5500-bp human CRH promoter-luciferase construct (pCRH 5500-GL3) and treated with E2 (10–7 M) for periods up to 48 h. CRH promoter activity was significantly decreased after 12 h with no further changes observed thereafter (Fig. 1AGo). Concentrations of E2 were examined from 10–9 to 10–6 M, and the effects of E2 were maximal at 10–7 M (Fig. 1BGo). This concentration (10–7 M) was subsequently used for all further experiments. 10–9 to 10–7 M is within the range of free E2 in plasma of women in the third trimester of pregnancy (24).



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  		FIG. 1. A, Luciferase activity in placental syncytiotrophoblast cells in culture transfected with pCRH (5500)-GL3 plasmid DNA and exposed to 10–7 M E2 for periods up to 48 h. Activity is expressed relative to vehicle controls. Values represent the mean ± SEM of experiments in triplicate with cells obtained from three placentae. *, P < 0.01, compared with vehicle controls. B, Luciferase activity in placental syncytiotrophoblast cells in culture transfected with pCRH (5500)-GL3 plasmid DNA and exposed to either vehicle (0) or E2 (10–9 to 10–6 M) for 24 h. Promoter activity is shown relative to vehicle control. Values represent the mean ± SEM of experiments in triplicate with cells obtained from four placentae. *, P < 0.01, compared with vehicle controls.

 
ICI 182780, a putative pure estrogen antagonist, increased CRH promoter activity by approximately 40% at 24 h. ICI 182780 was also able to completely overcome the inhibitory effects of E2 because E2 and ICI 182783 in combination resulted in an approximate 25% increase in CRH promoter activity above control values (Fig. 2Go).



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  		FIG. 2. Luciferase activity in placental syncytiotrophoblast cells treated with E2 and ICI 182780. Cells were transfected with pCRH(5500)-GL3 reporter gene and exposed to 10–7 M E2 or 10–6 M ICI 182780 alone or in combination for 24 h. Promoter activity is shown relative to vehicle control. Values represent the mean ± SEM of experiments in triplicate with cells obtained from three placentae. *, P < 0.01, compared with vehicle controls; #, P < 0.01, compared with E2.

 
ER{alpha} overexpression enhances selective ER modulator responsiveness, whereas ERß expression results in loss of E2 effects on CRH promoter

Placental cytotrophoblasts express both ER{alpha} and ERß. Transfection of human ER{alpha} and ERß expression vectors into placental cells resulted in an increase in the corresponding ER{alpha} (Fig. 3AGo) or ERß (Fig. 3BGo) protein levels.



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  		FIG. 3. Effect of overexpressing ER{alpha} and ERß on CRH promoter activity in placental cells treated with E2 and ICI. A and B, Western blot analysis of ERs in placental cells. Total cellular protein was isolated from primary placental cells 36 h after transfection with 0.5 µg of an ER{alpha}- or ERß-expressing plasmid and then separated and immunoblotted with ER{alpha} or ERß antibody as described in Materials and Methods. 1, Basal expression of ER; 2, overexpression of ER. C, Luciferase activity in placental syncytiotrophoblast cells overexpressing ER{alpha}. Cells were transfected with pCRH(5500)-GL3 reporter gene and exposed to 10–7 M E2 or 10–6 M ICI 182780 without (control) or with cotransfection of pCMV-ER{alpha} or pCMV5 plasmid. Relative promoter activities are shown as percentage of (vehicle-treated) pCRH(5500)-GL3-transfected control cells. Values represent the mean ± SEM from three independent experiments. *, P < 0.01, compared with vehicle controls. D, Luciferase activity in placental syncytiotrophoblast cells overexpressing ERß. Cells were transfected with pCRH(5500)-GL3 reporter gene and exposed to 10–7 M E2 or 10–6 M ICI 182780 without (control) or with cotransfection of pcDNA-ERß or pcDNA plasmid. Relative promoter activities are shown as percentage of (vehicle-treated) pCRH(5500)-GL3 transfected control cells. Values represent the mean ± SEM from three independent experiments. *, P < 0.01, compared with vehicle controls.

 
Cotransfection of the CRH-promoter-reporter plasmid along with an ER{alpha} expression plasmid, or pCMV5 empty vector as a control, resulted in significant enhancement of the responsiveness to E2 and ICI182780 in the placental cells overexpressing ER{alpha} (Fig. 3CGo). In contrast, overexpression of ERß resulted in a loss of response to either E2 or ICI 182780 (Fig. 3DGo). No noticeable changes occurred in basal CRH promoter activity in either the ER{alpha} or ERß overexpressing cells, and cotransfection of control plasmids had no effect on CRH expression or the E2 and ICI 182780 responses.

Localization of estrogen response region in CRH 5'-flanking region

To identify the regulatory sequences required for E2- and ICI 182780-mediated effects on CRH gene expression, CRH reporter plasmids containing progressively shorter sections of the CRH gene were transfected into placental cells. Cells transfected with CRH promoter sequences from –5500 to –248 bp displayed similar results in response to E2 or ICI 182780 treatment. However, when the CRH promoter was truncated to 213 bp, both the estrogen inhibition and ICI 182780 stimulation were lost (Fig. 4Go). This indicates that the region of the CRH promoter between –248 and –213 bp is essential for ER ligand-mediated CRH gene expression in placental cells.



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  		FIG. 4. Luciferase activity in placental syncytiotrophoblast cells transfected with constructs containing different lengths of the CRH gene promoter from –5500 to –99 bp and treated with 10–7 M E2 or 10–6 M ICI 182780 for 24 h. Promoter activity is shown relative to vehicle control. Values represent the mean ± SEM of experiments in triplicate with cells obtained from three placentae. *, P < 0.01, compared with vehicle controls.

 
E2 inhibits cAMP-simulated CRH promoter activity

Treatment of placental syncytiotrophoblast cells with 5 x 10–4 M 8-bromo-cAMP increased CRH promoter activity (Fig. 5Go). The inhibitory effects of E2 on CRH promoter activity in cAMP stimulated cells was markedly greater than seen in nonstimulated cells. However, there was only a modest increase in CRH promoter activity observed with ICI 182780 in cAMP-treated cells (Fig. 5Go). Deletion of the CRH promoter region between –248 and –213 bp resulted in loss of any responsiveness to E2, ICI 182780, or cAMP, either alone or in combination. The amount of cAMP stimulation observed in these experiments is much lower than the 5- to 8-fold induction reported previously for CRH promoter activity in placental cells (25). These large responses are typically observed when the experiments are carried out in cells cultured with 10% charcoal-stripped serum containing media. More modest responses are observed when experiments are conducted in serum-free media, as was done in the experiments presented in the current study.



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  		FIG. 5. Luciferase activity in placental syncytiotrophoblast cells transfected with constructs containing different lengths of the CRH gene promoter from –663 to –213 bp and treated with 10–7 M E2 or 10–6 M ICI 182780 for 24 h under basal conditions or after stimulation of CRH promoter activity with 0.5 mM 8-bromo-cAMP. Promoter activity is shown relative to vehicle controls. Values represent the mean ± SEM of experiments in triplicate with cells obtained from three placentae. *, P < 0.01, compared with vehicle controls; #, P < 0.01, compared with cAMP.

 
ER ligands regulate the CRH promoter through the cAMP regulatory element

There is a consensus CRE in the region between –248 and –213 bp of the human CRH gene promoter (25, 26, 30, 31). Transfection of a mutant CRH-reporter construct into placental syncytiotroblast cells, in which the CRE sequence was specifically mutated, resulted in a complete reversal of E2- and ICI 182780-mediated activity in comparison with wild-type control (Fig. 6AGo).



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  		FIG. 6. A, Luciferase activity in placental syncytiotrophoblast cells transfected with wild-type or mutant pCRH(663)-GL3 plasmid constructs and treated with 10–7 M E2 or 10–6 M ICI 182780 for 24 h. B, Luciferase activity in placental syncytiotrophoblast cells transfected with pGLOB-GL3 or pCRE-GLOB-GL3 constructs and treated with 10–7 M E2 or 10–6 M ICI 182780 for 24 h. Promoter activity is shown relative to vehicle control. Values represent the mean ± SEM of experiments in triplicate with cells obtained from three placentae. *, P < 0.01, compared with vehicle controls.

 
Confirmation that the CRE is a sufficient and adequate element to confer estrogen responsiveness in the CRH promoter was shown by using a chimeric construct containing the human CRH promoter CRE sequence linked to the basal rabbit ß-globin promoter. E2 and ICI 182780 normally have no effect on rabbit ß-globin promoter activity; however, when the rabbit ß-globin promoter is linked to the CRE, as found in the CRH promoter, both E2 and ICI 182780 modulate promoter activity of the globin promoter in a similar fashion to that of the wild-type CRH promoter (Fig. 6BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the traditional model of estrogen action, ERs, with bound ligand, function as either homodimers or heterodimers to bind to a specific DNA sequence, the estrogen response element (ERE), in the promoters of many estrogen-responsive genes. Although there are no full palindromic ERE sequence elements in the human CRH gene promoter there is a half-ERE site at position –312 bp. Vamvakopoulos and Chrousos (21) suggested that this site was a potential ER binding site in CV-1 cells, although this has been disputed by Dibbs et al. (22). We have now demonstrated that ER modulation of gene regulation maps to a region between –248 and –213 bp of the CRH gene promoter. This region of the CRH promoter does not contain an ERE but does contain a consensus CRE.

Insertion of a short CRH promoter fragment containing a functional CRE to drive a basal rabbit ß-globin promoter conferred E2 and ICI 182780 responsiveness, similar to that seen with the intact CRH promoter. This confirms the role of the CRE in mediating down-regulation of the CRH promoter in these cells. However, mutation of the CRE within the CRH promoter context resulted in complete reversal of E2 and ICI 182780 responsiveness. In the 663-bp CRH promoter constructs containing a mutant CRE, E2 stimulated levels of CRH promoter activity by approximately a 3-fold greater amount than in E2-treated wild-type controls and 1.5-fold greater amount than untreated controls. In contrast, there was no difference in the effect of ICI 182780 with the mutant CRE construct in comparison with nontreated controls. It is possible that following mutation of the CRE the inhibitory effects of E2 mediated through this site are lost, unmasking stimulatory actions of E2 elsewhere on the CRH promoter.

Our results clearly indicate that a functional CRE is necessary and adequate for E2 repression and ICI 182780 stimulation of CRH gene expression in placental syncytiotrophoblast cells. These result are also consistent with the findings of An et al. (32) in which E2-mediated repression of TNF{alpha} gene transcription was also shown to require an activator protein-1 (AP-1)/CRE-like sequence motif. In this case, the AP-1/CRE motif was also essential for ICI 182780 activation of TNF{alpha} gene expression. Several studies have shown that selective ER modulators can modulate gene expression by mechanisms that are independent of binding to a classical ERE (33, 34, 35). There is also considerable evidence indicating that ERs may also function by interacting with other transcription factors, including AP-1, specificity protein-1, and nuclear factor-{kappa}B, bound to their specific response elements in target genes that do not contain EREs (33, 36, 37). Moreover, it is evident that estrogen effects are not only dependent on the target gene but are also reliant on the cell context.

In addition to estrogen, there are many endogenous factors associated with pregnancy that are known to regulate placental CRH production, including glucocorticoids, prostaglandins, catecholamines, and IL-1 (16, 17, 38). In earlier studies we demonstrated that cAMP and glucocorticoids regulate the CRH gene promoter in placental cells through the CRE in the promoter region of the CRH gene (25, 26). Additionally we observed using EMSA that the protein complexes interacting with the CRE in the CRH gene promoter include the transcriptional activators CREB and the AP-1 component protein Jun (30, 31).

Previously we have shown that placental trophoblast cells express both ER{alpha} mRNA and ERß mRNA, but the ER{alpha} is predominant (24). Using Western blot analysis, we show here that these placental cells express the proteins of these two type of ERs, consistent with Bukovsky et al. (39). In placental cells overexpressing ER{alpha} and cotransfected with a CRH promoter-luciferase reporter, we observed greater E2 inhibition and ICI 182780 stimulation on CRH promoter activity, compared with control or pCMV transfected (i.e. non-ER{alpha} overexpressing cells). In contrast, when ERß was overexpressed, both E2 and ICI 182780 were without activity on promoter activity, compared with control or pcDNA-transfected cells. This suggests that ERß can act as an effective antagonist of ER{alpha} action in the CRH promoter. Similar results have been reported by Monroe (40) in osteoblastic cell lines. Collectively the evidence we have provided strongly indicates that estrogen-mediated expression of CRH gene expression in placental syncytiotrophoblast cells is mediated via ER{alpha}. Furthermore, given the low levels of ERß expression in placental cells, any functional significance of ERß-mediated effects on the CRH promoter is unlikely.

An important role for CRH produced by the placenta, and thereby placental CRH gene expression, in controlling the length of human pregnancy has been proposed by several groups (14, 41, 42). Hypothalamic CRH production represents the integration of many stress inputs, and placental CRH production may similarly represent the input of many signals from the mother and fetus. Our current studies indicate that E2, estrogen antagonists, and cAMP/protein kinase A activators all act through the CRE in the CRH promoter. The effects of E2 and ICI 182780 on the CRE are mediated via an ER{alpha}-dependent mechanism, and the CRE is a critical element involved in the regulation of the CRH gene promoter in placental syncytiotrophoblast cells. The rise in CRH expression in the presence of the ICI 182780 suggests that E2 has a tonic inhibitory effect on placental CRH gene expression. This effect of E2 on placental CRH production may have important physiological consequences in human pregnancy.


   Acknowledgments
 
The authors thank the nursing and medical staff of the delivery suites at Changhai Hospital in Shanghai and the John Hunter Hospital in Newcastle for their cooperation in obtaining placenta.


   Footnotes
 
This work was supported by the Natural Science Foundation of China Grants 39870300 and 30270511 (to X.N.) and the National Health and Medical Research Council of Australia (to R.S., R.C.N.).

Abbreviations: AP-1, Activator protein-1; CRE, cAMP response element; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; FCS, fetal calf serum.

Received June 2, 2003.

Accepted February 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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