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Endocrinology, doi:10.1210/en.2008-0063
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Endocrinology Vol. 149, No. 9 4658-4668
Copyright © 2008 by The Endocrine Society

Crucial Role of Estrogen Receptor-{alpha} Interaction with Transcription Coregulators in Follicle-Stimulating Hormone and Transforming Growth Factor β1 Up-Regulation of Steroidogenesis in Rat Ovarian Granulosa Cells

Yun-Ju Chen, Ming-Ting Lee, Hsiao-Chun Yao, Pei-Wen Hsiao, Ferng-Chun Ke and Jiuan-Jiuan Hwang

Institute of Physiology, School of Medicine (Y.-J.C., H.-C.Y., J.-J.H.), National Yang-Ming University, Taipei 112, Taiwan; Institutes of Biological Chemistry (M.-T.L.) and BioAgricultural Sciences (P.-W.H.), Academia Sinica, Taipei 115, Taiwan; and Institute of Molecular and Cellular Biology (F.-C.K.), School of Life Science, National Taiwan University, Taipei 106, Taiwan

Address all correspondence and requests for reprints to: Jiuan-Jiuan Hwang, Institute of Physiology, School of Medicine, National Yang-Ming University, 155 Linong Street, Section 2, Taipei 112, Taiwan. E-mail: jiuanh{at}ym.edu.tw; or Ferng-Chun Ke, Institute of Molecular and Cellular Biology, School of Life Science, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan. E-mail: fck{at}ccms.ntu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study was to explore estrogen receptor (ER) involvement in FSH and TGFβ1-stimulated steroidogenesis in rat ovarian granulosa cells. We first determined the specific involvement of ER{alpha} and ERβ in the process, and then investigated the molecular interaction of ER{alpha} and transcription coregulators in FSH and TGFβ1 up-regulation of steroidogenic gene expression. Primary culture of ovarian granulosa cells from antral follicles of gonadotropin-primed immature rats was used. Interestingly, a selective ER{alpha} antagonist methyl-piperidino-pyrazole (MPP) [like ER antagonist ICI-182,780 (ICI)] decreased FSH ± TGFβ1-stimulated progesterone production, whereas an androgen receptor antagonist hydroxyflutamide and particularly a selective ERβ antagonist 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol had no significant effect. Consistent with this, a selective ERβ agonist diarylpropionitrile (unlike 17β-estradiol) also had no effect on FSH ± TGFβ1-stimulated progesterone production. Furthermore, a selective ER{alpha} agonist 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (like 17β-estradiol) enhanced FSH-stimulated progesterone production, and this was abolished by pretreatment with MPP. Immunoblotting and chromatin immunoprecipitation analyses indicate that MPP/ICI suppression of FSH ± TGFβ1 action is partly attributed to the reduced ER{alpha}-mediated expression of Hsd3b and Cyp11a1 genes, but not steroidogenic acute regulatory protein. Furthermore, FSH ± TGFβ1 increased ER{alpha} association with histone acetylases (CBP and SRC-1) and coactivator of peroxisome proliferator-activated receptor {gamma} (PGC-1{alpha}), and MPP/ICI dramatically reduced these interactions. In addition, FSH ± TGFβ1 increased CBP, SRC-1, and PGC-1{alpha} binding to Hsd3b and Cyp11a1 genes. Together, we demonstrate for the first time that ER{alpha} interaction with transcription coregulators, histone acetylases (CBP/SRC-1), and PGC-1{alpha} is crucial to FSH and TGFβ1-up-regulated expression of Hsd3b and Cyp11a1, and, thus, progesterone production in rat ovarian granulosa cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVARIAN GRANULOSA cell differentiation is critically regulated by pituitary hormones, FSH during follicle growth to preovulatory follicles, and LH during ovulation and luteinization (1, 2). In addition, an intraovarian factor, TGFβ1, in vitro facilitates FSH-induced granulosa cell differentiation with increased production of progesterone and estrogen, and increased expression of LH receptor, inhibin, and steroidogenic proteins [including steroidogenic acute regulatory (StAR) protein, cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and aromatase] (3, 4, 5, 6, 7, 8, 9), as well as increased gap junction formation (8). StAR protein, P450scc enzyme, and 3β-HSD enzyme are products of Star, Cyp11a1, and Hsd3b genes, respectively. The significance of TGFβ1 in female ovarian function in vivo has been implicated by TGFβ1-null female mice that display disrupted ovarian function (reduced serum progesterone level, ovulation rate, and the number of corpus luteum) and reduced fertility with early embryo arrest (10).

FSH stimulates synthesis of estrogen in granulosa cells, and TGFβ1 enhances FSH action (3). In addition, estrogen exerts local feedback regulation to augment FSH-induced granulosa cell differentiation with increased gap junction formation, progesterone synthesis, and LH receptor expression and LH responsiveness (11). Estrogen is currently known to act mainly through two members of the nuclear receptor superfamily of hormone-regulated transcription factors, namely estrogen receptor (ER) {alpha} and ERβ (12). Both ER forms are present in granulosa cells of the rodent ovary, and ERβ is suggested to be the predominant form (13). ERβ is essential for granulosa cell proliferation, whereas ER{alpha} plays specific roles in ovulation, corpus luteum formation, and interstitial glandular cell development (12, 13, 14). Although ER{alpha} and ERβ display certain functional redundancy, they are different in the ligand activation and transcriptional properties (12, 13). ER regulation of target gene expression is mediated through direct binding to estrogen response element (ERE), and/or indirect interaction with coregulators and other transcription factors [e.g. activator protein-1 (AP-1)] resulting in recruitment of RNA polymerase II-containing transcription initiation complex that enhances target gene transcription with a coordinated and timely cycling manner (15, 16, 17, 18, 19). Presently, a few interacting partners of ER{alpha} have been demonstrated. ER{alpha} could recruit histone acetylases (CBP, SRC-1, and RAC-3) that facilitate chromatin remodeling and, therefore, enhance transcription of ER-regulated genes (20, 21). Interestingly, the coactivator of peroxisome proliferator-activated receptor {gamma} (PGC-1{alpha}) also serves as a transcription coactivator of ER{alpha} in both a ligand-independent and ligand-dependent manner (22). In addition, our most recent study demonstrates that FSH increased phosphorylation of fork-head box protein (Fox) O1 and FoxO3a in rat ovarian granulosa cells (9). It has been reported that phosphorylation of FoxOs leads to their nuclear exit and, thus, the release of their suppression of target gene transcription (23, 24). In addition, FoxO1 interaction with ER{alpha} augmented ligand-dependent ER{alpha} transactivation (25). The transcriptional regulation activity of ER{alpha} could be modulated by coregulators in a cell context-specific manner (16, 26, 27).

ER{alpha} and ERβ play differential roles in follicle growth and differentiation during rodent ovarian cycle (12, 13, 14). During luteinization, ERβ is down-regulated (28, 29) in coincidence with up-regulation of ER{alpha} (30, 31). This inspires us to explore the role of ER{alpha} in FSH and TGFβ1-promoted differentiation of rat ovarian granulosa cells. There were two specific aims. The first was to determine the specific role of ER{alpha} and ERβ in FSH and TGFβ1-stimulated progesterone production and the associated key steroidogenic proteins. The second aim was to investigate the molecular interaction of ER{alpha} and transcription coregulators in FSH and TGFβ1 up-regulation of steroidogenic gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Ovine FSH (oFSH-19-SIAFP) and equine chorionic gonadotropin were purchased from the National Institute of Diabetes and Digestive and Kidney Diseases’s National Hormone & Peptide Program and Dr. A. F. Parlow (Harbor-UCLA Medical Center, Torrance, CA). Recombinant human TGFβ1 was obtained from R&D System, Inc. (Minneapolis, MN). Penicillin and streptomycin were from GIBCO Invitrogen Corp. (Carlsbad, CA). ICI-182,780 (ICI), diarylpropionitrile (DPN), 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol (PHTPP), and 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) were purchased from Tocris (Bristol, UK). Hydroxyflutamide (HF) was kindly provided by Schering-Plough Pharmaceutical (Kenilworth, NJ). Antisera against P450scc enzyme were a generous gift from Dr. Bon-Chu Chung (Academia Sinica, Taipei, Taiwan). Antibodies against ER{alpha}, CBP, PGC-1{alpha}, RAC-3, β-catenin, phosphorylated Smad2 and phosphorylated Smad3, and Protein-A/G plus agarose bead were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against SRC-1, c-Jun, FoxO1, and FoxO3a were from Cell Signaling Technology, Inc. (Beverly, MA). Antibody against phosphorylated cAMP response element-binding protein (CREB) was from Upstate Biotechnology Inc. (Lake Placid, NY). All other chemicals used were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated.

Animals
Immature Sprague Dawley rats (25–27 d) were obtained from the Animal Center at National Yang-Ming University (Taipei, Taiwan). Rats were maintained under controlled temperature (20–23 C) and light conditions (14-h light, 10-h darkness). Food (LabDiet; PMI Feeds, St. Louis, MO) and water were available ad libitum. This study was conducted in accordance with the U.S. National Research Council’s Guide for the Care and Use of Laboratory Animals and the institutional guidelines.

Cell culture and treatment
Isolation and culture of ovarian granulosa cells from antral follicles of equine chorionic gonadotropin-treated immature rats were performed as previously described (7, 8, 9). In brief, granulosa cells (~5 x 105) were inoculated into 24-well plates coated with matrigel (derived from Engelbreth-Holm-Swarm sarcoma tumors; Sigma) in DMEM/F12 medium containing 2 µg/ml bovine insulin, 0.1% fatty acid-free BSA, 100 U/ml penicillin, and 100 µg/ml streptomycin, and allowed to attach for 24 h at 37 C, 5% CO2-95% air. Cultured cells were then incubated in DMEM/F12 medium containing 0.1% lactalbumin hydrolysate, 100 U/ml penicillin, and 100 µg/ml streptomycin for 24 h before the beginning of treatment. Cells were pretreated for 1 h with ethanol vehicle or various doses of ER antagonist ICI (32), ER{alpha} antagonist methyl-piperidino-pyrazole (MPP) (33), androgen receptor (AR) antagonist HF (34), ERβ antagonist PHTPP (35), or ERβ agonist DPN (28), and then treated with FSH (10 ng/ml) and/or TGFβ1 (5 ng/ml) for an additional 48 h. To determine the involvement of ER{alpha} in estradiol augmentation of FSH-stimulated progesterone production, granulosa cells were pretreated for 1 h with ethanol vehicle or ER{alpha} antagonist MPP, and then treated with FSH and/or a selective ER{alpha} agonist PPT for an additional 48 h. All doses of drugs used throughout the study had no obvious cytotoxic effect. At the end of incubation, conditioned media were collected, cleared by centrifugation, and stored at –70 C until assayed for progesterone content by ELISA. Cell number was determined using the crystal violet assay as previously described (36).

ELISA for progesterone
Progesterone level in conditioned media was measured by ELISA as previously described (7, 8, 9). Antisera against progesterone (37) were kindly provided by Dr. O. David Sherwood (University of Illinois, Urbana, IL).

Immunoblotting
Granulosa cells (~6 x 106) were cultured in Matrigel-coated 60-mm culture dishes, pretreated with ethanol vehicle, or various doses of MPP or ICI for 1 h, and then treated with FSH (10 ng/ml) and/or TGFβ1 (5 ng/ml) for an additional 48 h to determine their effects on the protein levels of StAR (38), P450scc (39), and 3β-HSD enzymes (40). Cell lysates were prepared, and immunoblotting was performed as previously described (7, 8, 9). Relative quantification of chemiluminescent signals on x-ray film was analyzed using a two-dimensional laser scanning densitometer (Molecular Dynamics, Sunnyvale, CA).

Coimmunoprecipitation
Granulosa cells (~15 x 106) were cultured in Matrigel-coated 100-mm culture dishes, pretreated with ethanol vehicle, MPP, or ICI for 1 h, and then treated with FSH (10 ng/ml) and/or TGFβ1 (5 ng/ml) for 30 min to determine their effects on the ER{alpha} interaction with transcription coregulators. Cell lysates (500 µg proteins) were precleared with 20 µl protein A/G plus agarose, incubated for 2 h at 4 C. After centrifugation at 15,000 x g for 15 min, the supernatant was incubated with 1 µg ER{alpha} antibody for 6 h at 4 C, followed by addition of 30 µl protein A/G plus agarose and rotated overnight at 4 C. The immune precipitated products were separated by centrifugation, and analyzed by immunoblotting using antibodies of ER{alpha}, histone acetylases (CBP, SRC-1, and RAC-3), PGC-1{alpha}, and transcription factors (c-Jun, β-catenin, phospho-Smad2, phospho-Smad3, phospho-CREB, FoxO1, and FoxO3a).

Chromatin immunoprecipitation (ChIP) assay
Granulosa cells (~15 x 106) were cultured in Matrigel-coated 100-mm culture dishes, pretreated with ethanol vehicle, MPP, or ICI for 1 h, and then treated with FSH (10 ng/ml) and/or TGFβ1 (5 ng/ml) for 3 h to determine their effects on the binding of ER{alpha}, CBP, SRC-1, and PGC-1{alpha} to DNA using ChIP assay as previously described (41, 42). In brief, cells were first fixed in 1% formaldehyde in PBS (10 min, 37 C) to cross-link DNA and proteins, and then 125 mM glycine was added (5 min, 37 C) to stop the reaction. Cells were then lysed in Nonidet P-40 lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40] by incubating on ice for 10 min, and centrifuged at 1500 x g for 5 min. The nuclear pellet was further sonicated in lysis buffer [10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 2% Triton X-100], and centrifuged at 15,000 x g for 20 min. The DNA fragment sizes were at the range of approximately 400-1000 bp. Small aliquots of the supernatants were kept and served as input to normalize PCR results. Supernatants were then precleared by protein A/G-agarose followed by centrifugation, and the supernatants were immune precipitated as described previously in the Coimmunoprecipitation section. Normal IgG in place of antibody of interest was used as the negative control. To remove RNAs and proteins, the immune complexes were incubated sequentially in 200 mM NaCl containing 2 µg/ml ribonuclease A for 2 h at 65 C, and then with 300 µg/ml proteinase K for an additional 2 h at 65 C. DNA was then extracted by phenol/chloroform (1:1, vol/vol), followed by isopropanol precipitation, and then dissolved in TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA]. PCR was performed using PerkinElmer GeneAmp PCR system 2400 (PerkinElmer Life And Analytical Sciences, Inc., Waltham, MA). For Star, the DNA was amplified for 30 cycles (denaturation: 95 C, 1 min; annealing: 64 C, 1 min; elongation: 72 C, 1 min) using antisense and sense primers (5'-CATCCAGCAAGGAGAGGAAG-3' and 5'-CGTGAGTTTGGTCTTTGAGG-3') (–853 to –358, accession no. NC005115). For Cyp11a1, the DNA was amplified for 25 cycles (denaturation: 95 C, 1 min; annealing: 45 C, 1 min; elongation: 72 C, 1 min) using antisense and sense primers (5'-ATCACAGAGATGCTGGCAGGA-3' and 5'-GCACGTTGATGAGGAAGATGG-3') (–801 to –321, accession no. NC005107). For Hsd3b, the DNA was amplified for 25 cycles (denaturation: 95 C, 1 min; annealing: 58 C, 70 sec; elongation: 72 C, 70 sec) using antisense and sense primers (5'-ACTGGCAAATTCTCCATAG-3' and 5'-TTCCTCCCAGCTGACAAGTGG-3') (–552 to –151, accession no. L17138). All of these regions contain ERE half-site. The primer pairs used correspond to the rat nucleotide sequences (43, 44). The PCR cycle number chosen for each gene was at linearity range of amplification. PCR products were separated on 2% agarose gel and visualized by ethidium bromide staining.

Statistics
Quantitative data were analyzed by ANOVA and Duncan’s multiple range tests at a significance level of 0.05 using the general linear model of the SAS program (SAS Institute Inc., Cary, NC). In addition, the Student’s t test was used to identify significant differences between the two treatment groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Specific involvement of ER{alpha} in FSH and TGFβ1-stimulated steroidogenesis
We first determine the specific involvement of ER in FSH and TGFβ1-stimulated steroidogenesis in rat ovarian granulosa cells. ER antagonist ICI dose dependently (10–10 to 10–6 M) decreased FSH plus TGFβ1-induced progesterone production, whereas AR antagonist HF (10–6 and 10–5 M) had no effect (Fig. 1Go). Therefore, because both ER{alpha} and ERβ are present in granulosa cells, we further investigated their specific involvement by using selective modulators. Interestingly, a selective ER{alpha} antagonist MPP, like ICI, dose dependently decreased FSH ± TGFβ1-stimulated progesterone production, and the inhibitory effect of MPP at the dose of 5 x 10–6 M was similar to that of ICI at 10–6 M (Fig. 2Go). Whereas a selective ERβ antagonist PHTPP had no obvious effect on FSH ± TGFβ1-stimulated progesterone production in rat granulosa cells (Fig. 3Go). In addition, 17β-estradiol (E2) is known to enhance FSH-induced progesterone secretion in granulosa cells (45). Here, we show that MPP and ICI reduced this action of estradiol, whereas PHTPP had no effect (Fig. 3Go). Also unlike estradiol, a selective ERβ agonist DPN exerted no significant effect on FSH ± TGFβ1-induced progesterone production (Fig. 3AGo). In addition, a selective ER{alpha} agonist PPT (like E2) augmented FSH-stimulated progesterone production, and pretreatment with ER{alpha} antagonist MPP abolished this action of PPT (Fig. 3BGo). Together, these results implicate for the first time the crucial role of ER{alpha} mediation of FSH and TGFβ1-stimulated progesterone production in rat ovarian granulosa cells.


Figure 1
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  		FIG. 1. Effect of ER and AR antagonists on FSH and TGFβ1-induced progesterone production in rat ovarian granulosa cells. Cells were pretreated with ethanol vehicle or various doses of an ER antagonist ICI-182780 and an AR antagonist HF for 1 h, and then treated with vehicle control (C), 10 ng/ml FSH, and/or 5 ng/ml TGFβ1 for an additional 48 h. Conditioned media were collected and analyzed for progesterone content by ELISA. Each bar represents the mean (±SE) progesterone production (n = 9). Different lowercase letters indicate significant differences among treatment groups in the absence of antagonists (P < 0.05). Asterisk indicates a significant difference compared with the FSH plus TGFβ1-treated group (P < 0.05).

 

Figure 2
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  		FIG. 2. Effect of selective ER{alpha} antagonist on FSH and TGFβ1-induced progesterone production in rat ovarian granulosa cells. Cells were pretreated with ethanol vehicle, or various doses of ICI or an ER{alpha} antagonist MPP for 1 h, and then treated with FSH and/or TGFβ1 for an additional 48 h. Each bar represents the mean (±SE) progesterone production (n = 9). Different lowercase letters indicate significant differences among treatment groups in the absence of antagonists (P < 0.05). Asterisk indicates a significant difference compared with the respective control (C) without antagonists (P < 0.05).

 

Figure 3
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  		FIG. 3. Effect of selective ERβ modulators and ER{alpha} agonist on FSH and TGFβ1-induced progesterone production in rat ovarian granulosa cells. A, Cells were pretreated with vehicle, various doses of an ERβ antagonist PHTPP or agonist DPN for 1 h, and then treated with FSH and/or TGFβ1, or 10–9 M E2 for an additional 48 h. Pretreatment with 5 x 10–6 M MPP and 10–6 M ICI was included as reference. B, Cells were pretreated with vehicle or an ER{alpha} antagonist MPP (5 x 10–6 M) for 1 h, and then treated with FSH in the absence or presence of an ER{alpha} agonist PPT (10–8 and 10–7 M) for an additional 48 h. Each bar represents the mean (±SE) progesterone production (n = 6). Different lowercase letters indicate significant differences among treatment groups in the absence of antagonists (P < 0.05). Asterisk indicates a significant difference compared with the respective control without antagonists (P < 0.05).

 
We next investigated the role of ER{alpha} in FSH and TGFβ1 regulation of the three key players in progesterone production (StAR protein, P450scc, and 3β-HSD enzymes) at the level of protein and gene expression, respectively, using immunoblotting and ChIP assays, respectively. Consistent with our recent studies (7, 8, 9), FSH alone at the dose of 10 ng/ml increased StAR protein and 3β-HSD enzyme levels and had no significant effect on P450scc enzyme level, and the combined treatment with TGFβ1 further increased the levels of all three key molecules (Fig. 4Go). TGFβ1 alone did not affect the content of all three players (data not shown). Here, we demonstrate that MPP and ICI reduced the protein levels of FSH-increased 3β-HSD enzyme and FSH plus TGFβ1-increased 3β-HSD and P450scc, but not the StAR protein level (Fig. 4Go). ChIP assays further support that ER{alpha} exerts a critical role in FSH and TGFβ1-induced gene expression of Hsd3b and Cyp11a1, but not the Star. FSH significantly increased ER{alpha} binding (directly and/or indirectly) to Hsd3b gene, and FSH plus TGFβ1 further increased ER{alpha} binding to Hsd3b and Cyp11a1 genes (Fig. 5Go). Furthermore, MPP and ICI significantly reduced these interactions. In addition, we did not detect any significant association of ER{alpha} with Star gene in all groups (Fig. 5Go). These results indicate ER{alpha} crucial mediation of FSH and TGFβ1-stimulated progesterone production in rat ovarian granulosa cells is attributed at least partly through up-regulation of the expression of Hsd3b and Cyp11a1 genes.


Figure 4
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  		FIG. 4. Effects of selective ER{alpha} antagonists on FSH and TGFβ1-regulated key players of steroidogenesis in rat ovarian granulosa cells. Cells were treated as described in Fig. 2Go. Cell lysates were analyzed by immunoblotting for StAR protein, P450scc and 3β-HSD enzymes with β-actin used as an internal control (C). Quantitative analysis of StAR, P450scc, and 3β-HSD in reference to β-actin was performed using two-dimensional scanning densitometry. Relative density ratios were calculated using the FSH-treated group value or control group value as one. Each bar represents the mean (± SE) relative density (n = 3). Different lowercase letters indicate significant differences among treatment groups in the absence of inhibitors (P < 0.05). Asterisk indicates a significant difference compared with the respective control without inhibitors (P < 0.05).

 

Figure 5
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  		FIG. 5. Effect of selective ER{alpha} antagonists on FSH and TGFβ1-induced ER{alpha} binding to gene of key players of steroidogenesis in rat ovarian granulosa cells. Cells were pretreated with ethanol vehicle, 5 x 10–6 M MPP or 10–6M ICI for 1 h, and then treated with vehicle control (C), 10 ng/ml FSH and/or 5 ng/ml TGFβ1 for an additional 3 h. Nuclear extracts were analyzed by ChIP assay with ER{alpha} antibody or normal IgG as a negative control (–), and input DNA served as an internal control. Ethidium bromide-stained intensity of the PCR products was determined and normalized to the input DNA. Relative density ratios were calculated using the control group value as one. Each bar represents the mean (±SE) relative density (n = 3). Different lowercase letters indicate significant differences among treatment groups in the absence of inhibitors (P < 0.05). Asterisk indicates a significant difference compared with the respective control without inhibitors (P < 0.05). FT, FSH plus TGFβ1.

 
Molecular interaction of ER{alpha} with transcription coregulators in FSH and TGFβ1 up-regulation of steroidogenic gene expression
Stimulation of target gene expression in response to ER is mediated by its direct binding to ERE and/or indirect interaction with other transcription coregulators (15). Therefore, we further explored ER{alpha} interaction with transcription coregulators in FSH and TGFβ1-stimulated expression of Hsd3b and Cyp11a1 genes in rat ovarian granulosa cells using coimmunoprecipitation with ER{alpha} antibody. Interestingly, FSH increased ER{alpha} interaction with histone acetylases CBP and SRC-1, but not RAC-3, and TGFβ1 significantly enhanced FSH-induced ER{alpha} interaction with CBP (Fig. 6Go). Surprisingly, TGFβ1 in the presence of FSH increased ER{alpha} association with PGC-1{alpha}, whereas FSH alone had no effect. In addition, TGFβ1 alone had no significant effect on ER{alpha} association with CBP, SRC-1, and PGC-1{alpha} (Fig. 6Go). Furthermore, MPP and ICI dramatically suppressed the FSH ± TGFβ1-induced ER{alpha} interaction with CBP and SRC-1, as well as PGC-1{alpha}. Both FSH and TGFβ1 had no obvious effect on the ER{alpha} association with transcription factors c-Jun and β-catenin, and this is not affected by pretreatment with either MPP or ICI (Fig. 6Go). In addition, FSH and TGFβ1 had no obvious effect on the ER{alpha} association with TGFβ1 signaling transcription factors, phosphorylated Smad2 and Smad3. Our most recent work shows that FSH increased the phosphorylation of CREB and FoxOs (FoxO1 and FoxO3a) in rat granulosa cells (9). Here, we detected no clear association of ER{alpha} with phosphorylated CREB, as well as FoxO1 and FoxO3a in all groups (data not shown).


Figure 6
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  		FIG. 6. Effect of selective ER{alpha} antagonists on FSH (F) and TGFβ1 (T)-regulated ER{alpha} interaction with transcription coregulators in rat ovarian granulosa cells. Cells were pretreated with ethanol vehicle, 5 x 10–6M MPP or 10–6M ICI for 1 h, and then treated with vehicle control (C), 10 ng/ml FSH, and/or 5 ng/ml TGFβ1 for an additional 30 min. Cell lysates were immunoprecipitated with ER{alpha} antibody, and the immune complexes were analyzed by immunoblotting for ER{alpha} and transcription coregulators (CBP, SRC-1, RAC-3, PGC-1{alpha}, c-Jun, β-catenin, phospho-Smad2 and Smad3). Quantitative analysis was performed in reference to ER{alpha} using two-dimensional scanning densitometry. Relative density ratios were calculated using the control group value as one. Each bar represents the mean (±SE) relative density (n = 3). Different lowercase letters indicate significant differences among treatment groups in the absence of antagonists (P < 0.05). Asterisk indicates a significant difference compared with the respective control without antagonists (P < 0.05). FT, FSH plus TGFβ1.

 
To study further the critical association of ER{alpha} and transcription regulator complex in FSH and TGFβ1 induction of steroidogenic gene expression in rat ovarian granulosa cells, ChIP assays with antibodies of CBP, SRC-1, and PGC-1{alpha} were used. Our results clearly demonstrate CBP, SRC-1, and PGC-1{alpha} binding to Hsd3b and Cyp11a1 genes, and CBP could also bind to Star gene (Fig. 7Go). FSH increased CBP, SRC-1, and PGC-1{alpha} binding to Hsd3b gene, and had no significant effect on Cyp11a1 gene. TGFβ1 plus FSH further increased the binding of these coregulators to Hsd3b and Cyp11a1 genes (Fig. 7Go). Furthermore, MPP and ICI dramatically suppressed the FSH and TGFβ1-induced CBP, SRC-1, and PGC-1{alpha} binding to Hsd3b and Cyp11a1 genes. We also noticed that FSH, in the absence or presence of TGFβ1, similarly increased CBP binding to Star gene, and this was not affected by the pretreatment of MPP or ICI (Fig. 7AGo).


Figure 7
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  		FIG. 7. Effect of selective ER{alpha} antagonists on FSH and TGFβ1-induced binding of histone acetylases and PGC-1{alpha} to key steroidogenic genes in rat ovarian granulosa cells. Cells were treated, and nuclear extracts were analyzed by ChIP assay as described in Fig. 5Go. Nuclear extracts were immunoprecipitated with antibodies for CBP (A), SRC-1 (B), and PGC-1{alpha} (C), or normal IgG as negative control (–). Ethidium bromide-stained intensity of the PCR products was determined and normalized to the input DNA. Relative density ratios were calculated using the control group value as one. Each bar represents the mean (±SE) relative density (n = 3). Different lowercase letters indicate significant differences among treatment groups in the absence of antagonists (P < 0.05). Asterisk indicates a significant difference compared with the respective control without antagonists (P < 0.05). C, Control; FT, FSH plus TGFβ1.

 
Together, these results suggest that FSH-induced expression of Hsd3b gene in rat granulosa cells is partly through recruitment of ER{alpha} and histone acetylases (CBP/SRC-1). Furthermore, TGFβ1 enhancement of Hsd3b and Cyp11a1 expression in the presence of FSH is partly through recruitment of ER{alpha} together with CBP/SRC-1 and PGC-1{alpha}. In addition, FSH and TGFβ1 act in an ER{alpha}-independent manner to increase Star expression in rat granulosa cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Specific involvement of ER{alpha} in FSH and TGFβ1-stimulated steroidogenesis
The ovarian endocrine is very complicated and interesting. In our laboratory we are devoted to study the mechanism(s) of pituitary FSH and intraovarian TGFβ1-induced granulosa cell differentiation characterized by increased steroidogenic activity, production of both progesterone and estrogen. Our recent studies show that TGFβ1 augmentation of FSH-induced increase in progesterone production in rat granulosa cells of antral follicles is partly attributed to increased protein levels of StAR protein, and P450scc and 3β-HSD enzymes (7, 8, 9). This study further implicates for the first time that ER{alpha} is a critical mediator in FSH and TGFβ1-induced progesterone production, partly acting through modulating the expression of Hsd3b and Cyp11a1 genes, but not Star gene in rat ovarian granulosa cells, and interestingly ERβ appears not to be significantly involved in the process as evidenced by the following. A selective ER{alpha} antagonist MPP, like ER antagonist ICI, suppressed the FSH ± TGFβ1-stimulated progesterone production in rat granulosa cells, whereas AR antagonist HF as well as selective ERβ antagonist PHTPP and agonist DPN all had no significant effect (Figs. 1–3GoGoGo). This is partly attributed to the suppressive effects of MPP and ICI on the FSH ± TGFβ1-induced expression of Hsd3b and Cyp11a1, but not the Star, as indicated by the results of immunoblotting and ChIP analyses (Figs. 4Go and 5Go). Furthermore, a selective ER{alpha} agonist PPT (like E2) enhanced FSH-stimulated progesterone, and this was blocked by pretreatment with ER{alpha} antagonist MPP (Fig. 3BGo). Therefore, because FSH and TGFβ1 increased ER{alpha} binding directly and/or indirectly to Hsd3b and Cyp11a1 genes (Fig. 5Go), we explored the molecular mechanism of ER{alpha} mediation of FSH and TGFβ1-regulated expression of Hsd3b and Cyp11a1 in rat ovarian granulosa cells.

Molecular interaction of ER{alpha} with transcription coregulators in FSH and TGFβ1 up-regulation of steroidogenic gene expression
The molecular mechanisms of transcription regulation of steroidogenic genes Hsd3b, Cyp11a1, and Star remain largely unclear. There are regulators reported to be involved in transcription regulation of human Cyp11a1 and mouse Star genes, including CREB, CBP, and AP-1 (heterodimer of c-Jun and c-fos) (46). According to nucleotide sequence analysis, promoters of rat Hsd3b, Cyp11a1, and Star genes all lack a consensus cAMP response element site (TGACGTCA) (47, 48), and all have consensus AP-1 site (GTCGTCA) (47, 49). In addition, all three rat gene promoters only have ERE half-site (AGGTCA) and not full palindrome ERE (50, 51). Up to date, no report has clearly documented ER{alpha} involvement in transcription regulation of Hsd3b, Cyp11a1, and Star genes. Several coregulators have been reported to activate ER{alpha} transcription activity, including histone acetylases (CBP, SRC-1, and RAC-3), and transcription factors (Smad3 and FoxO1) in cancer cells (21, 25, 52). Histone acetylases facilitate histone acetylation, leading to chromatin remodeling and transcription initiation by formation of stable RNA polymerase II complex. Moreover, the peroxisome proliferator-activated receptor (PPAR) {gamma} PGC-1{alpha} and β-catenin have also acted as coactivators of ER{alpha} that enhanced SRC-1 and CBP binding to target genes and increased ER{alpha} transcription activity (53, 54, 55, 56). In addition, ER{alpha} interaction with histone acetylases enhanced AP-1 transcription activity (19).

Our present study indicates for the first time that FSH-increased expression of Hsd3b gene in rat granulosa cells is partly through recruitment of ER{alpha} and histone acetylases (CBP/SRC-1), as indicated by results of coimmunoprecipitation (Fig. 6Go) and ChIP assays (Figs. 5Go and 7Go). Moreover, this study implicates for the first time that TGFβ1 enhancement of FSH-induced expression of Hsd3b and Cyp11a1 genes in rat granulosa cells is through recruitment of ER{alpha} together with histone acetylases (CBP/SRC-1) and PGC-1{alpha}, as indicated by results of coimmunoprecipitation (Fig. 6Go) and ChIP assays (Figs. 5Go and 7Go). All these FSH and TGFβ1-induced effects were suppressed by MPP and ICI (Figs. 5–7GoGoGo). ER{alpha}, in the presence of antagonists, may fail to establish a proper conformation for recruitment of coregulators to activate transcription. The PGC-1{alpha} involvement in stimulation of steroidogenesis is partially supported by a previous study reporting that PPAR{gamma} activator increased estradiol and progesterone production in rat granulosa cells (57). Because PGC-1{alpha} is known to play a critical role in mitochondria biogenesis (58), we think TGFβ1 may enhance FSH effect via increasing the capacity of steroidogenesis that takes place inside the mitochondria. Therefore, we suspect that PGC-1{alpha} involvement in FSH and TGFβ1-stimulated steroidogenesis may manifest the coordinated transcription program between enzymes for steroidogenesis and mitochondria biogenesis. In addition, TGFβ1 involvement in enhancing FSH-induced Hsd3b gene expression is partially supported by a recent study reporting a reduction of Hsd3b mRNA level in the ovary of gonadotropin-primed TGFβ1-null prepubertal female mice (10).

ER transcription regulation could also act indirectly through non-ERE (such as AP-1-responsive elements) (19). The promoters of Hsd3b, Cyp11a1, and Star gene all have a consensus AP-1 site (47, 50). And our results demonstrate ER{alpha} association with c-Jun in rat granulosa cells; however, this interaction was not altered by the presence of FSH and/or TGFβ1 (Fig. 6Go). In addition, β-catenin plays a pivotal role in the Wnt-signaling pathway and cell adherens junction formation (56). β-Catenin has also been reported to interact with many nuclear receptors, including AR, ER, glucocorticoid receptor and thyroid hormone receptor, and PPAR. In prostate cancer cells, β-catenin specifically interacted with AR, but not ER{alpha}, progesterone receptor β and glucocorticoid receptor (59). The present study demonstrates ER{alpha} association with β-catenin in rat granulosa cells; however, like c-Jun-ER{alpha}, this interaction was not altered by the presence of FSH and/or TGFβ1 (Fig. 6Go).

A previous study reported that FoxO1 interaction with ER{alpha} results in increased ER{alpha} transactivation activity (25). Our most recent study demonstrated that FSH increases the phosphorylation of FoxO1 and FoxO3a in rat ovarian granulosa cells (9). Phosphorylation of FoxOs is known to promote their exit from the nucleus (23, 24), and consistent with this concept, we did not detect any significant ER{alpha} interaction with the phosphorylated form or nonphosphorylated form of FoxO1 and FoxO3a in rat granulosa cells, regardless of FSH and/or TGFβ1 treatment (data not shown). This suggests that ER{alpha} may act through FoxOs independent manner in mediation of FSH and TGFβ1-stimulated steroidogenesis in rat ovarian granulosa cells. In addition, TGFβ1 has enhanced ER{alpha}-mediated transcription activity through direct physical interactions between Smad2/Smad3 and ER{alpha}, resulting in translocation into the nucleus and recruitment of transcription coregulators such as CBP, c-Jun, and β-catenin (52, 60). Our current study demonstrates that TGFβ1 and/or FSH did not significantly affect ER{alpha} association with phosphorylated Smad2 and Smad3 (Fig. 6Go), suggesting that FSH and TGFβ1 up-regulation of Hsd3b and Cyp11a1 expression is not attributed to ER{alpha} interaction with Smad2/3 in rat granulosa cells.

Although the promoter of rat Star gene has an ERE half-site (AGGTCA) (49), we did not detect any association of ER{alpha} with Star gene in rat granulosa cells using ChIP assay (Fig. 5Go). And consistent with this, we also did not observe any significant effect of ER{alpha} antagonist MPP on FSH ± TGFβ1-increased StAR protein level (Fig. 4Go). Our study suggests that FSH and TGFβ1 up-regulation of Star gene expression in rat granulosa cells may be through an ER{alpha}-independent manner. In addition, the Star gene can be regulated by FSH-induced cAMP signaling (61, 62), yet it has no consensus sequence of cAMP response element (TGACGTCA) (48). Here, we show that FSH significantly enhanced CBP binding to Star gene in rat granulosa cells, and this was not altered by pretreatment with ER antagonists MPP and ICI (Fig. 7AGo), or by the presence of TGFβ1 (data not shown). Together, our previous (9) and present studies suggest that FSH up-regulates Star gene expression in rat granulosa cells through cAMP/CBP-dependent, and PKA-/PI3K-independent and ER{alpha}-independent pathway, and TGFβ1 augmentation of this FSH effect is through PKA-/PI3K-/rapamycin-dependent and ER{alpha}-independent pathway. In addition, several signaling pathways have played a permissive role in regulation of StAR protein, including protein kinase C, MAPK/ERKs, calcium messenger systems (63, 64). The molecular mechanism whereby FSH and TGFβ1 regulate Star gene expression awaits further study.

In conclusion, the present study demonstrates for the first time that ER{alpha}, but not ERβ, plays a critical role in FSH and TGFβ1-induced steroidogenesis in rat ovarian granulosa cells through up-regulation of gene expression of Hsd3b and Cyp11a1, but not Star. This is partly attributed to FSH and TGFβ1-induced recruitment of ER{alpha} together with histone acetylases (CBP/SRC-1) and PGC-1{alpha}. Our work furthers the understanding of the mechanism of FSH and TGFβ1 induction of ovarian granulosa cell differentiation that is critically linked to oocyte maturation function and, thus, vital to fertility control.


   Acknowledgments
 
We appreciate the generous gifts of antisera against progesterone, steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase enzymes, respectively, from Drs. O. David Sherwood (University of Illinois, Urbana, IL), Douglas M. Stocco (Texas Tech University Health Sciences Center, Lubbock, TX), Bon-Chu Chung (Academia Sinica, Taipei, Taiwan), and J. Ian Mason (Queen’s Medical Research Institute, Edinburgh University, Edinburgh, UK).


   Footnotes
 
This study was supported by grants from National Science Council of Taiwan NSC93-2320-B-010-002, NSC94-2320-B-010-030 (to J.-J.H.) and NSC94-2311-B-002-033 (to F.-C.K.), and a grant from the Ministry of Education, Aim for the Top University Plan.

Disclosure Summary: The authors have nothing to disclose.

First Published Online May 29, 2008

Abbreviations: AP-1, Activator protein-1; AR, androgen receptor; ChIP, chromatin immunoprecipitation; CREB, cAMP response element-binding protein; DPN, diarylpropionitrile; E2, 17β-estradiol; ER, estrogen receptor; ERE, estrogen response element; Fox, fork-head box protein; HF, hydroxyflutamide; 3β-HSD, 3β-hydroxysteroid dehydrogenase; ICI, ICI-182,780; MPP, methyl-piperidino-pyrazole; P450scc, cholesterol side-chain cleavage enzyme; PGC-1{alpha}, coactivator of PPAR{gamma}; PHTPP, 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol; PPAR, peroxisome proliferator-activated receptor; PPT, 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol; StAR, steroidogenic acute regulatory.

Received January 14, 2008.

Accepted for publication May 21, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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