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Concerted Regulation of the Porcine Steroidogenic Acute Regulatory Protein Gene Promoter Activity by Follicle-Stimulating Hormone and Insulin-Like Growth Factor I in Granulosa Cells Involves GATA-4 and CCAAT/Enhancer Binding Protein ß

Department of Cell and Developmental Biology and Anatomy, University of South Carolina School of Medicine, Columbia, South Carolina 29208

Address all correspondence and requests for reprints to: Dr. Holly A. LaVoie, Department of Cell and Developmental Biology and Anatomy, Building 1, University of South Carolina School of Medicine, Columbia, South Carolina 29208. E-mail: hlavoie{at}med.sc.edu.

	Abstract

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We previously demonstrated that FSH alone or in combination with IGF-I activated the porcine steroidogenic acute regulatory protein gene promoter in a concerted manner in primary cultures of granulosa cells. Studies were undertaken to further delineate cis- and trans-acting elements of the porcine promoter and mechanisms mediating FSH stimulation and its augmentation by IGF-I. Mutation of several putative regulatory elements localized hormone-stimulated activity to the highly conserved GATA site and identified novel nucleotides downstream as a functional CCAAT/enhancer binding protein (C/EBP)ß site. In granulosa cell nuclear extracts, GATA-4 and C/EBPß formed a high-molecular-weight complex with an oligonucleotide spanning –76 to –32 bp of the porcine promoter. The intensity of this high-molecular-weight complex was increased in granulosa cell nuclear extracts by treatment with FSH alone and was enhanced with the combination of FSH and IGF-I at 2–3 h of treatment. GATA-4 and C/EBPß proteins were uniformly expressed with all treatments at time points associated with increased DNA binding. Treatment (2 h) with FSH alone or FSH + IGF-I increased phosphorylation of GATA-4 on a protein kinase A consensus site. The 38-kDa isoform of C/EBPß exhibited greater phosphorylation with FSH + IGF-I treatment. Porcine luteal cell nuclear extracts also demonstrated GATA-4 and C/EBPß binding to the –76 to –32 bp region of the promoter providing evidence for their cooperation in vivo. These data indicate that GATA-4 and C/EBPß are both required for FSH ± IGF-I stimulation of the porcine steroidogenic acute regulatory protein gene promoter in homologous granulosa cell cultures.

	Introduction

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IN 1994, STEROIDOGENIC ACUTE regulatory protein (StAR) was cloned from MA-10 Leydig tumor cells and characterized as the cycloheximide-sensitive protein responsible in part for the movement of cholesterol to the inner mitochondrial membrane requisite for steroidogenesis (1). Subsequent cotransfection studies using the full-length StAR cDNA with other cDNAs required for steroid biosynthesis-induced steroidogenesis in nonsteroidogenic COS cells (2). These reports led to the discovery that inactivating mutations in the human StAR gene were responsible for congenital lipoid adrenal hyperplasia, a condition associated with complete inability to produce gonadal and adrenal steroids (3). Because acute steroidogenesis in gonads and the adrenals is stimulated by peptide hormones, which activate the cAMP signaling cascade, numerous subsequent studies have focused on StAR gene transcription by this signaling pathway (reviewed in Refs.4, 5, 6).

In the ovary, StAR message and protein are dramatically up-regulated during the postovulatory luteinization of follicular cells into the mature corpus luteum, a period that directly correlates with increased progesterone synthesis (7, 8). Although StAR mRNA is undetectable in granulosa cells from antral follicles (8, 9), it is expressed in the granulosa cell layer in vivo after an experimental gonadotropin [pregnant mare serum gonadotropin (PMSG), human chorionic gonadotropin (hCG)] surge (10, 11). A PMSG (predominantly FSH-like) stimulus produces a short transient stimulation, whereas hCG (LH) results in a more prolonged stimulation of StAR mRNA (10). In culture, both gonadotropins (FSH, LH) can induce StAR in granulosa cells at specific stages of differentiation (9, 12, 13). In some species, IGF-I can augment gonadotropin-induced progesterone production, and in porcine granulosa cells this IGF-I effect includes enhancement of StAR mRNA and protein accumulation and transcriptional activation (12, 14, 15). Similar effects are observed when the cells are cocultured with gonadotropins and insulin (9, 13, 15). In addition, treatment of porcine granulosa cells with cAMP analogs yields responses similar to gonadotropins and can also cooperate with IGF-I to enhance StAR transcription (12, 13, 14, 15). IGF-I enhancement of gonadotropin-induced steroidogenic end points has been proposed to provide a selective advantage to follicles (16).

In recent years there has been much progress regarding the transcriptional regulation of the StAR gene in multiple species. Several transcription factors have been implicated in the stimulation of this gene, and most binding sites have been localized to the proximal region of the promoter. These factors include steroidogenic factor 1 (SF-1), GATA-4/-6, CCAAT/enhancer-binding protein (C/EBP) and -ß, sterol-regulatory element binding protein 1 (SREBP-1), cAMP response element modulator (CREM)/activating transcription factor (ATF)-1, and specificity protein 1 (Sp1) (17, 18, 19, 20, 21, 22, 23, 24, 25). A few inhibitory factors have been shown to mediate repression including DAX-1, Yin-Yang 1, c-fos, and fra-1 (26, 27, 28, 29). However, several of the studies have been performed by overexpressing transcription factors with reporter genes using clonal cells that do not normally express StAR (25) and may not be representative of primary cells (27). We hypothesize that regulation of the StAR gene in the adrenals, testes, and ovaries may use some common transcriptional elements but that there is some tissue selectivity to hormonal control of the StAR gene. Of the implicated transcription factors, GATA-4, C/EBPß, and SF-1 have been shown to mediate cAMP induction of StAR in ovarian cells (18, 21, 22, 30). The GATA site is highly conserved among all mammalian promoter sequences studied; however, the pertinent C/EBPß and SF-1 sites tend to vary in location (4, 5). Some studies have failed to show SF-1 involvement or binding to the StAR promoter (22). Silverman et al. (22), working with rat granulosa cells, were the first to implicate GATA-4 and C/EBPß in the FSH stimulation of StAR gene promoter constructs. Whether FSH stimulates increased transcription factor binding to DNA elements has not been investigated. Although numerous studies have looked at cAMP analog regulation of the StAR gene promoter in steroidogenic cells, few studies have addressed the actions of hormones in primary ovarian cells (14, 15, 22, 31). Moreover, regulation of StAR transcription by more than one trophic factor has been minimally explored. Because transcription in situ is likely the coordinated effort of multiple hormones, our studies aimed to begin to address the multihormonal regulation of the StAR gene.

We have previously shown that FSH and LH transactivation of the porcine StAR gene promoter constructs in porcine granulosa cells is localized within the –139-bp region upstream of the transcriptional start site in porcine granulosa cells (14, 15). In addition, IGF-I augmented gonadotropin-stimulated promoter activities, although it had no significant transactivation ability alone. The ability of IGF-I to augment gonadotropin-stimulated transactivation was accompanied by IGF-I-enhancement of gonadotropin-stimulated cAMP accumulation (14, 15). Furthermore, other events downstream of cAMP accumulation can be modified as well because IGF-I enhanced protein kinase A (PKA)-catalytic subunit activation of StAR reporter gene constructs in cotransfection studies. Thus, the ability of IGF-I to augment gonadotropin actions appears to require a cAMP signal, although this pathway is not necessarily the only pathway mediating IGF-I effects. We hypothesized that these signaling pathways converge at the level of the trans-acting factors. It is therefore our goal to identify the transcription factors and their cis-elements that are responsible for gonadotropin action and its augmentation by IGF-I.

In this study we identified the cis- and trans-acting elements responsible for the FSH ± IGF-I transactivation of the StAR gene in primary cultures of porcine granulosa cells and implicated GATA-4 and C/EBPß. We identified a highly conserved element downstream of the GATA site that binds C/EBPß. In addition, we evaluated the ability of hormonal stimulation to modify the DNA binding capacity of trans-acting factors. Furthermore, in light of emerging data regarding regulation of these transcription factors by phosphorylation, we present evidence that phosphorylation events are temporally associated with enhanced DNA binding. In addition, we analyzed the DNA binding ability of luteal nuclear extracts to determine whether these DNA complexes potentially exist in vivo.

	Materials and Methods

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Materials
General chemicals and protease inhibitors were obtained from Sigma (St. Louis, MO) and Fisher Scientific (Fairlawn, NJ). Cell culture reagents and all synthesized oligonucleotides were purchased from Life Technologies, Inc./Invitrogen (Carlsbad, CA). NIDDK o-FSH-20 was obtained from the National Hormone and Pituitary Program (National Institutes of Health, Bethesda, MD). Recombinant human IGF-I was purchased from Bachem (Torrance, CA). Reagents for PCR were obtained from Clontech (Palo Alto, CA). Isotope was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Abattoir ovaries from prepubertal gilts were purchased from Greenwood Packing Plant, Inc. (Greenwood, SC). Adult ovaries were obtained from Caughman Meat Packing (Lexington, SC).

Plasmids
The porcine –1423-bp StAR promoter firefly luciferase construct (p-1423StAR/luc) and the porcine GATA-4 and GATA-6 expression vectors have been previously described (14, 32). The rat C/EBPß cDNA vector (33) was a generous gift of Dr. S. L. McKnight (University of Texas Southwestern, Dallas, TX).

The porcine SF-1 cDNA, which codes for the full-length SF-1 protein, was amplified by PCR using primers corresponding to nt 841–860 and nt 2258–2239 derived from GenBank accession no. U84399. PCR was performed using cDNA synthesized with the Marathon cDNA amplification kit (Clontech) using polyA RNA isolated from pooled porcine corpora lutea. The PCR product was ligated into pCRII for sequence confirmation and then subcloned into the expression vector pcDNA3.1 and orientation confirmed by sequencing (University of Maine DNA sequencing facility, Orono, ME).

Culture and transfection of porcine granulosa cells
Granulosa cells were isolated from 1- to 5-mm ovarian follicles of prepubertal gilts by needle aspiration as previously described (14). Cells were plated in MEM containing antibiotics plus 3% fetal calf serum at a density of 1.6–1.8 x 10⁶ live cells/well in 24-well culture plates (Falcon, Franklin Lakes, NJ). After 39–43 h of culture, cells were rinsed with serum-free medium and transfected with 1 µg total plasmid DNA using 6 µl LipofectAMINE reagent in MEM for 5 h. Plasmids included the wild-type –1423-bp pStAR/luc vector (14) and similar constructs harboring point mutations of putative transcriptional elements (0.975 µg/well). The renilla luciferase vector with minimal thymidine kinase promoter, ptkRL/luc (Promega Corp., Madison, WI), was cotransfected as an internal control (25 ng/well). After transfection, medium was replaced with fresh MEM containing hormone treatments (5–25 ng/ml o-FSH-20; 100 ng/ml IGF-I, Bachem) or appropriate vehicle and incubated 0–8 h (time-course studies) or 4 h (found to be optimal for FSH + IGF-I interactions). Cells were lysed using 1 x passive lysis buffer, and the lysates were assayed with the Dual Luciferase Assay kit (Promega) using a Turner 20E luminometer (Turner Designs, Sunnyvale, CA).

For transfections experiments in which the GATA-4 and C/EBPß cDNA plasmids were overexpressed, the cells were transfected for 5 h as above and then returned to MEM/antibiotics/3% fetal calf serum for 13 h to allow serum to drive the viral promoters. Cells were then switched to serum-free medium containing vehicle or 8-bromoadenosine-cAMP (8-Br-cAMP) (1 mM) for the last 4 h of culture. Optimal concentrations of GATA-4 and C/EBPß cDNA-containing plasmids were determined in preliminary experiments. For this series of transfections, 10 ng porcine GATA-4 and 100 ng rat C/EBPß and/or pcDNA3.1 plasmids were cotransfected with 0.98 µg –1423-bp pStAR/luc vector (total 1 µg DNA/well). Cells were lysed using 1 x passive lysis buffer, assayed for firefly luciferase activity, and activity normalized for protein in the lysate as previously described (34).

For protein isolation studies in which the cells were not transfected, 2 x 10⁷ cells were plated in 60-mm dishes (Falcon) and cultured identically for the first 39–43 h and then changed to serum-free MEM for 5 h (equivalent to the period of transfection) before treatment in serum-free MEM with vehicle or hormones for the indicated times.

Site-directed mutagenesis
Mutations of the –1423-bp pStAR/Luc construct were performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). PAGE-purified sense and antisense strand oligonucleotides corresponding to the porcine StAR promoter nt –133 to –95, –115 to –75, and –76 to –32 with the appropriate mutations were used for mutagenesis. Mutants were identified by DNA sequencing. The sense strands of the oligonucleotides used for site-directed mutagenesis (with mutations indicated in lower-case letters) were: MUT-A, 5'-GGTTTTTTTAagTCCAGATGATGAAACACAACCTTCAGCTGGAGG-3'; MUT-B, 5'-GGTTTTTTTATCTCCAGAacATGAAACACAACCTTCAGCTGGAGG-3'; MUT-C, 5'-GGTTTTTTTATCTCCAGATGAaaAAACACAACCTTCAGCTGGAGG-3'; MUT-D, 5'-GGTTTTTTTATCTCCAGATGATGAAACACAAaaTTCAGCTGGAGG-3'; MUT-E, 5'-GGTTTTTTTATCTCCAGATGATGAAACACAACCTTttGCTGGAGG-3'; MUT-AB, 5'-GGTTTTTTTAagTCCAGAacATGAAACACAACCTTCAGCTGGAGG-3'; MUT-AC, 5'-GGTTTTTTTAagTCCAGATGAaaAAACACAACCTTCAGCTGGAGG-3'; MUT-ABC, 5'-GGTTTTTTTAagTCCAGAacAaaAAACACAACCTTCAGCTGGAGG-3'; MUT-F, 5'-GGCTGCTTGTGAtttAATCCCTCTATCCTTGGCCCCTTCC-3'; MUT-G, 5'-GGCTGCTTGTGAGGCAATCCCTCTAaagTTGGCCCCTTCC-3'; and MUT-H, 5'-CCTCTATCCTTGGCCCCTTCCTTTGCAAGGTaaAGTGATG-3'.

Nuclear protein isolation and EMSA
Crude nuclear proteins were isolated from treated cells at the selected time (as determined in preliminary studies). Cells were harvested and swollen in hypotonic buffer [50 mM HEPES (pH 7.5), 10 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 5 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml soybean trypsin inhibitor, 0.1 mM sodium orthovanadate, 1 mM sodium fluoride, and 1 mM tetrasodium pyrophosphate] on ice for 10 min followed by the addition of Triton X-100 (0.8%), and then nuclei were pelleted. Nuclei were resuspended in the above buffer containing 400 mM KCl and proteins extracted for 30 min on ice. Protein concentrations were determined on an aliquot of the extract and the remainder was snap frozen and stored at –70 C until later use. Protein concentrations were determined using a protein dye reagent (Bio-Rad Laboratories, Hercules, CA). Nuclear extracts from corpora lutea were isolated as above after homogenization of tissue as previously described (32). Corpora lutea assessed to be between d 5 and d 8 of the estrus cycle based on color, diameter, and vascularity were used (35). This stage of the pig estrus cycle corresponds to the period of increasing StAR mRNA accumulation in corpora lutea (7).

Nuclear protein extracts used for EMSA were used undialyzed or dialyzed to concentrate as previously described (32) so that the final binding reaction consisted of 5% glycerol, 0.5 mM dithiothreitol, 100 mM KCl, 10 mM Tris-HCL (pH 7.5), 2 µg poly dI-dC (Sigma), 0.25 mM EDTA, 1.25 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 2.5 µg/ml leupeptin, 0.25 µg/ml pepstatin A, 0.25 µg/ml soybean trypsin inhibitor, 25 µM sodium orthovanadate, 0.25 mM sodium fluoride, and 0.25 mM tetrasodium pyrophosphate. Double-stranded oligonucleotides were end labeled with ³²P-ATP and T₄ polynucleotide kinase or ³²P-dATP and Klenow for oligos with overhangs. Nuclear extracts (8–18 µg) were incubated with high specific-activity ³²P-labeled probe, approximately 100,000 cpm in binding buffer for 30 min on ice. The reactions were run on native 4–5% polyacrylamide gels for 2.5–3 h at 150 V, dried, and visualized by autoradiography using Biomax MR film (Kodak, Rochester, NY). Supershift and competition assays were performed by incubating the binding buffer protein mixture with antisera (usually 4–8 µg) or excess cold competitor oligonucleotides for 30 min on ice before the addition of labeled oligonucleotide and then incubated an additional 30 min on ice. Antibodies used for supershift assays were: GATA-4 sc-1237X, GATA-6 sc-7245X, C/EBPß sc-150X, C/EBP sc-61X, C/EBP sc-636X, C/EBPß (–2) sc-746X (cross-reactive with C/EBP, -, and -), SREBP-1 sc-367X, c-Jun sc-45X, SF-1 sc-10976X, LRH-1 sc-5993X, CREM-1 sc-440X, ATF-1 sc-270X, USF-1 sc-229X, USF-2 sc-862X, Sp1 sc-420X (Santa Cruz Biotechnology, Santa Cruz, CA), and SF-1, 06–431 (Upstate Biotechnology, Lake Placid, NY). Consensus oligonucleotides for GATA (GATA-2X), cAMP response element-binding protein, and USF were obtained from Santa Cruz Biotechnology and Sp1 and activator protein-1 (AP-1) from Promega.

The oligonucleotides used for EMSA included annealed sense and antisense strands of the mutant primers listed above under the Site-directed mutagenesis section and the following (sense strand shown unless noted): –76 to –32, 5'-GGTTTTTTTATCTCCAGATGATGAAACACAACCTTCAGCTGGAGG-3'; –72 to –55, 5'-TTTTTATCTCCAGATGAT-3'; –66 to –37, 5'-TCTCCAGATGATGAAACACAACCTTCAGCT; C/EBP-2X, 5'-TGCAGATTGCGCAATCTGCATTGCGCAATCTTCG-3'; C/EBP-GATA oligonucleotide (36); sense, 5'-GATCCTAGATATCCCTGATTGCGCAATAGGCTCAAAGCTG-3'; and antisense, 5'-AATTCAGCTTTGAGCCTATTGCGCAATCAGGGATATCTAG-3'.

For the –72 to –55 and –66 to –37 regions, double-stranded oligonucleotides were prepared for labeling with Klenow (for higher specific activity) by omitting the last two nucleotides of the 3' end during oligonucleotide synthesis.

In vitro-translated proteins were generated using the TNT Rabbit Reticulocyte Lysate kit (Promega) and 500 ng of each plasmid, complete amino acid mixture, and T7 polymerase. Three microliters of each in vitro-translated reaction was incubated with 100,000 cpm wild-type or mutant oligonucleotide in the reaction buffer listed above with the addition of 50 ng denatured salmon testis DNA at room temperature for 30 min before separation on 5% native gels.

Total phosphoprotein isolation
Isolation of the phosphoprotein-enriched fraction was performed with the Phosphoprotein column isolation kit (Qiagen, Valencia, CA). This kit isolates total phosphoprotein from whole-cell extracts reflective of the phosphorylation profile in the cell at the time of lysis. Approximately 10 60-mm dishes of granulosa cells per treatment were incubated with hormones or vehicle as described above. Cell lysates were processed according to the manufacturer’s protocol. Eluted fractions containing protein were pooled and concentrated on Nanosep ultrafiltration columns before SDS-PAGE.

Immunoprecipitation
Antibodies (200 µg) to GATA-4 (sc-1237X) were conjugated to Aminolink Plus Coupling gel (200 µl) using the Seize Primary Mammalian Immunoprecipitation kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s directions. Nuclear protein (350 µg) isolated from treated cells was shaken gently overnight at 4 C with 50 µl packed beads. Beads were washed four times with Dulbecco’s PBS and eluted. The elution was separated by SDS-PAGE followed by immunoblot analysis. Immunoblots were first probed with anti-phospho-(Ser/Thr) PKA-substrate (Cell Signaling Technology, Beverly, MA) or pSER¹⁰⁵GATA-4 (Biosource International, Camarillo, CA) and subsequently stripped and reprobed for total GATA-4.

Immunoblot analysis
Equivalent amounts of protein per treatment or equivalent elution volumes from immunoprecipitation were resolved by 10–12% SDS-PAGE and electrotransferred to PDVF membranes (Amersham). Membranes were blocked in 0.6 mg/ml gelatin in Tris-buffered saline containing 0.05% Tween 20 (TTBS) for antibodies recognizing phosphospecific substrates, anti-pSER¹⁰⁵GATA-4, and anti-phospho-(Ser/Thr)-PKA substrate. For other antibodies, membranes were blocked in 5% milk in TTBS (anti-GATA-4, sc-9053X; C/EBPß, sc-150X). Primary antibodies were incubated in either gelatin blocking solution or 1% milk-TTBS overnight at 4 C. After washing, membranes were incubated with horseradish peroxidase-coupled goat antirabbit secondary antibody (Zymed Laboratories, South San Francisco, CA) in gelatin or 5% milk-TTBS for 1 h. After extensive washing, immunoreactive bands were detected by enhanced chemiluminescence (Amersham). Membranes were stripped before reblocking and probing with other antisera.

Data analysis
For mutagenesis studies, the percentage of inhibition relative to the same treatment with the wild-type promoter construct was calculated. The percentage of inhibition of mutant constructs was compared by one-way ANOVA followed by Tukey’s multiple comparison test. P 0.05 was considered significant. For overexpression studies, raw data (luciferase activity per microgram protein) was first evaluated by repeated-measures ANOVA before Tukey’s test. For immunoprecipitation studies, densitometric analyses of immunoreactive bands were performed using Quantity One version 4.2.1 software (Bio-Rad). For each immunoprecipitation the phosphospecific GATA band was normalized to total GATA-4 on the membrane and expressed relative to the vehicle control in each experiment. The immunoprecipitation data were also analyzed by repeated-measures ANOVA and Tukey’s test. Statistical analyses were performed using GraphPad Prism version 3.0 for Windows (GraphPad Software, San Diego, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our previous studies determined the maximal and submaximal concentrations of FSH (o-20) needed to activate the porcine StAR promoter (14). Figure 1 shows the time course of responsiveness of the porcine –1423-bp StAR promoter construct to submaximal (5 ng/ml) and maximal (25 ng/ml) FSH alone or in the presence of IGF-I. The time-course studies revealed that IGF-I in the presence of FSH at either concentration reduces the time to achieve maximal stimulation from 6 h with FSH alone to 4 h. IGF-I also enhances the maximum stimulation observed with submaximal concentrations of FSH.

View larger version (12K): [in this window] [in a new window]   FIG. 1. Time course and concentration dependence of FSH activation and IGF-I enhancement of the porcine StAR promoter. Porcine granulosa cells were transiently cotransfected with the p-1423StAR/luc construct and ptkRL/luc control and subsequently treated with vehicle, IGF-I (100 ng/ml) alone or in combination with FSH for the indicated times. IGF-I shifts the time course and amplitude of submaximal FSH (5 ng/ml, left panel) and maximal FSH (right panel, 25 ng/ml) activation of the StAR promoter. Data represent one of three similar experiments. Data represent the mean and range of duplicate wells normalized for vehicle control at the same time point. FSH doses were plotted on separate graphs for purposes of clarity only.

View larger version (32K): [in this window] [in a new window]   FIG. 2. FSH activation and its augmentation by IGF-I are localized to a GATA and a highly conserved C/EBP-like element. A, Sequence of the proximal region (nt –139 to –24) of the porcine StAR gene 5'-flanking region. Bold letters indicate the nucleotides mutated in this study. Nucleotides in italics represents a DNA element first noted by Reinhart et al. (17 ) to be conserved in all known mammalian StAR promoter sequences. For B and C, primary cultures of porcine granulosa cells were cultured and transfected with the wild-type or mutant p-1423StAR/luc construct and ptkRL/luc control. Posttransfection cells were treated with vehicle, FSH (5 or 25 ng/ml), IGF-I (100 ng/ml), or their combination for 4 h and promoter activity analyzed by dual luciferase assay. B, This graph represents the summary of mutations made within the –76- to –32-bp promoter region and functionally tested with a submaximal concentration of FSH (5 ng/ml). Black boxes represent mutation of elements as identified in wild-type promoter with open boxes. Data represent the mean of three individual experiments (performed with duplicate wells) normalized to the wild-type vector, FSH + IGF-I treatment ± SEM. The asterisks indicate that the treatment with the mutant exhibited significant inhibition, compared with the wild-type vector with the same treatment, *, P < 0.05; **, P < 0.01; ***, P < 0.001. C, Summary of three to seven individual experiments performed with a maximal FSH concentration (25 ng/ml). Data are presented as described in B.

We also mutated other conserved elements upstream between –129 and –76 bp (Fig. 2A) that have been shown to be functional in other species, depending on the cellular context. Evaluation of these single mutants (F, G, or H) with either concentration of FSH (5 ng/ml, n = 3; 25 ng/ml, n = 5) alone or with IGF-I resulted in no significant decrement in hormone-stimulated transactivation (data not shown).

Because the transactivation of FSH ± IGF-I was localized primarily to nucleotides in sites A–C, and to a lesser extent D, we wanted to carry out a more detailed analysis of the proteins binding to this regulated region. EMSAs were performed with oligonucleotides spanning the –76- to –32-bp region of the porcine promoter and regions therein. The –76- to –32-bp oligonucleotide, containing putative GATA, C/EBP, and SF-1 sites, was incubated with nuclear extracts from treated granulosa cell primary cultures. Preliminary studies showed similar binding with 5–25 ng/ml FSH alone or with IGF-I, so most subsequent experiments were performed with an intermediate concentration of FSH (15 ng/ml). FSH alone (15 ng/ml) and FSH + IGF-I (100 ng/ml) enhanced the intensity of a high-molecular-weight complex (Fig. 3A). This complex was greatest in the FSH + IGF-treated cells at 2–3 h and with FSH only-treated cells at 4 h treatment (not shown). To identify the proteins in this complex, supershift analyses were performed. Antibodies to C/EBPß, GATA-4, and to a lesser extent GATA-6 were able to shift this complex (Fig. 3B). These proteins could be shifted with all treatments (not shown).

View larger version (81K): [in this window] [in a new window]   FIG. 3. EMSA studies with the –76- to –32-bp oligonucleotide and treated (2–3 h) granulosa cell nuclear extracts. Granulosa cell nuclear extracts were isolated from cells that had been cultured according to the transfection protocol (but not transfected). A, Nuclear extracts from granulosa cells treated with vehicle (C), FSH (15 ng/ml, F); IGF-I (100 ng/ml, I), or their combination (F+I) were incubated with the radiolabeled probe and separated on a 5% native acrylamide gel. The arrow indicates a complex regulated by treatments with FSH alone or FSH plus IGF-I. B, Supershift analysis using F+I-treated nuclear protein and radiolabeled –76- to –32-bp oligonucleotide and the indicated antibodies for relevant C/EBP family, GATA family, and SF-1 family members (4% gel). Arrows (B and C) indicate the hormonally regulated complex. C, Competition EMSA performed with F+I nuclear protein using the –76- to –32-bp wild-type oligonucleotide. Cold competitor oligonucleotides were added at 50-fold molar excess 30 min before addition of labeled oligonucleotide (5% gels). Each experiment was repeated at least three times.

To confirm that the mutations used in Fig. 2 did indeed abolish the binding of proteins to their respective sites, in vitro-translated proteins for GATA-4, GATA-6, C/EBPß, and SF-1 were incubated with the –76- to –32-bp wild-type oligonucleotide or its respective mutants (Fig. 4). Each of the recombinant proteins was able to bind the wild-type oligonucleotide, and the appropriate mutants abolished their binding. These gel shift experiments also verified that sites D and E could actually bind SF-1, even though granulosa cell nuclear extracts did not exhibit SF-1 binding. Of interest was the observation that mutation D had a slight reduction in binding for recombinant C/EBPß but did not affect GATA-4 binding, suggesting these nucleotides might influence C/EBPß binding to the neighboring site.

View larger version (52K): [in this window] [in a new window]   FIG. 4. In vitro-translated proteins for GATA-4/-6, C/EBPß, or SF-1 bind the –76- to –32-bp wild-type StAR oligonucleotide in EMSA (5% gels). A, The wild-type oligonucleotide (WT) and mutants B–D bind GATA-4 but not mutation A (GATA site); mutation of nucleotides B or C abolishes C/EBPß binding; and mutations D and E abolish SF-1 binding. B, Mutation A abolishes binding of both GATA-4 and GATA-6. Vector indicates expression vector without cDNA. The experiment was repeated at least twice for each recombinant factor.

View larger version (72K): [in this window] [in a new window]   FIG. 5. EMSA studies with treated (2–3 h) granulosa cell nuclear extracts and the –66- to –37-bp oligonucleotide (C/EBP-like site) or the –72- to –55-bp oligonucleotide (GATA site). A, Nuclear extracts from granulosa cells treated with vehicle (C), FSH (15 ng/ml, F), IGF-I (100 ng/ml, I), or their combination (F+I) were incubated with the radiolabeled –66- to –37-bp oligonucleotide and separated on a 5% native acrylamide gel. Treatments with FSH exhibit enhanced binding of nuclear extracts. B, Supershift and competition analyses using F+I-treated nuclear protein and radiolabeled –66- to –37-bp oligonucleotide and the indicated antibodies or cold competitor oligonucleotides (4.5% gels). C/EBPß and C/EBPß-2 represent two different antibodies. C, EMSA performed with F+I-treated nuclear extracts using the –72 to –55 bp showing FSH alone or in combination with IGF-I enhances DNA binding (5% gel). D, Supershift and competition analyses using F+I-treated nuclear protein and radiolabeled –72- to –55-bp oligonucleotide, and the indicated antibodies or cold competitor oligonucleotides reveal specific GATA binding with a prevalence of GATA-4 binding (5% gel). Each experiment was repeated at least three times.

View larger version (20K): [in this window] [in a new window]   FIG. 6. Promoter activation by overexpressed GATA-4 and C/EBPß is significantly reduced with the triple mutant. Granulosa cells were cotransfected with the wild-type p-1423StAR/luc construct or the plasmid containing MUT-ABC and expression vectors for GATA-4 and C/EBPß. After a preexpression period of 13 h in medium containing 3% serum, cells were incubated in serum-free medium containing vehicle or 1 mM 8-Br-cAMP for 4 h. Firefly luciferase activity was normalized for protein content of the lysate. Values are expressed relative to the maximum treatment (GATA-4 + C/EBPß + 1 mM 8-Br-cAMP). Data indicate the mean ± SEM for three to four individual experiments performed with duplicate wells. a, Treatment was significantly different, compared with vehicle-treated vector control (P < 0.001) and 8-Br-cAMP-treated vector control (P < 0.05); b, group is significantly greater than the same vector combination treated with vehicle (P < 0.01). All expression vector combinations and treatments with MUT-ABC were significantly inhibited, compared with the wild-type vector under the same conditions (P < 0.001).

View larger version (44K): [in this window] [in a new window]   FIG. 7. GATA-4 and C/EBPß phosphorylation status in treated granulosa cells. Immunoblots of granulosa cells treated for 2 h with vehicle (C) or hormones, IGF-I (100 ng/ml; I), FSH (15 ng/ml; F), or their combination (F+I). Granulosa nuclear protein (50 µg or 30 µg, respectively) was resolved by SDS-PAGE and probed with GATA-4 (A) or C/EBPß antibodies (B). B (middle panel), A lighter exposure of the blot in the top panel. For A and B, images labeled ponceau represent a region of the same PVDF membrane (43-kDa region) stained with ponceau S before immunoprobing and is included as a loading reference. Equal amounts of total whole-cell phosphoprotein (10 µg) from treated granulosa cells were analyzed by immunoblot (C and D). C, The membrane was first probed with anti-p-PKA-substrate, stripped and reprobed with anti-GATA-4, and stripped and finally probed with anti-Sp1 (105 kDa) for reference. A second blot run in parallel was probed with anti-pSER105GATA-4. D, A phosphoprotein immunoblot probed with anti-C/EBPß. Image of ponceau S-stained upper portion of the membrane is included as a loading reference. For A–D experiments were repeated three times. E, An immunoblot showing a GATA-4 immunoprecipitation of treated granulosa cell nuclear protein probed with anti-p-PKA-substrate (upper band indicated). The graph summarizes the results of immunoprecipitated GATA-4 blots probed with anti-p-PKA-substrate or anti-pSER105GATA-4 and then for total GATA-4. Relative expression indicates the phosphospecific band was first normalized for its corresponding total GATA-4 band, and then data within each experiment was normalized to vehicle control. Data represent mean ± SEM, n = 3. The asterisks indicate that the treatments were significantly greater than vehicle (C) but not each other. *, P < 0.05.

Because the phospho-PKA-substrate antibody is not specific for GATA-4, we wanted to confirm that the PKA phosphorylated band(s) was GATA-4. We immunoprecipitated GATA-4 from treated granulosa cell nuclear extracts and immunoblotted with anti-phospho-PKA-substrate or pSER¹⁰⁵GATA-4 followed by anti-GATA-4 (Fig. 7E). Two anti-phospho-PKA-substrate reactive bands comigrated with GATA-4. One of these bands was unique to cells treated with FSH alone or FSH + IGF-I. A single immunoreactive GATA band was observed with anti-pSER¹⁰⁵GATA-4. Densitometric analysis of these blots (Fig. 7E) revealed no change in the relative abundance of Ser105 phosphorylated GATA-4 but did reveal significant increases in one phospho-PKA reactive site for cells treated with FSH or FSH + IGF-I (P < 0.05). We were unable to detect quantitative differences in phosphorylation between FSH and FSH + IGF-I treatments.

To determine whether luteal cells extracts exhibit the same complexes as those observed with treated granulosa cells, EMSA was performed with nuclear extracts isolated from early to midluteal phase corpora lutea. Both GATA and C/EBP are part of the high-molecular-weight complex (Fig. 8, A and B). A less prevalent lower-molecular-weight complex could be competed off with both C/EBP or GATA consensus sequences as well and was clearly shifted by the GATA-4 antibody. As mentioned above, this band may represent GATA binding with a lower-molecular-weight isoform(s) of C/EBPß. Only minimal shifts were observed with the GATA-6 antibody.

View larger version (73K): [in this window] [in a new window]   FIG. 8. EMSA showing nuclear extracts from corpora lutea exhibit GATA-4 and C/EBPß binding to the –76- to –32-bp StAR oligonucleotide. Nuclear protein from healthy corpora lutea (15 µg) was used (A and B). A, Competition assay in which 50-fold molar excess of the indicated cold oligonucleotide was preincubated with nuclear extract before addition of labeled DNA. Both GATA and C/EBP are part of the major complex (thick arrow) and part of a minor lower-molecular-weight band (thin arrow). B, Supershift assay shows GATA-4 and C/EBPß shift or reduce the intensity of the major complex (thick arrow). GATA-4 also clearly shifted from the lower-molecular-weight band (thin arrow). The experiment was repeated at least three times (with corpora lutea from different animals).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

SF-1 alone or in combination with cAMP analogs can transactivate the StAR promoter (18, 19, 25). Up to six potential SF-1 sites have been identified in the StAR promoters of different species (18, 42, 43, 44, 45). Although recombinant porcine SF-1 was able to bind the proximal SF-1 site at –48 to –40 bp, we failed to provide sufficient data to implicate this proximal SF-1 sequence or the site at –112 to –103 bp in basal or hormone-regulated activity. We were unable to supershift SF-1 from the proximal site in treated granulosa cell extracts using two SF-1 antibodies that we verified (not shown) could recognize recombinant porcine SF-1. We have not evaluated all of the putative SF-1 sites in our isolated region of the porcine StAR promoter; however, what is evident from the present data is that intact SF-1 sites in the presence of the GATA and C/EBP mutations (sites ABC) are insufficient for hormone-stimulated transactivation. The analogous proximal SF-1 site in the human promoter was found to be important for both basal and cAMP stimulation in granulosa-luteal cells (18). Our data showing that a second mutation (E) in the same SF-1 site abolished recombinant SF-1 binding but had no effect on promoter activity suggest this proximal site is not critical for function in porcine granulosa cells.

Although this is the second study to show that a C/EBP site adjacent to the GATA binding site is involved in FSH action, we identified a new downstream nucleotide sequence responsible for C/EBPß binding. In our initial EMSA competition studies, we used an oligonucleotide containing an optimal C/EBP sequence employed by other laboratories for studies of C/EBP (21, 36). This consensus oligonucleotide was able to compete the specific high-molecular-weight band off of the porcine StAR promoter –76- to –32-bp region as efficiently as the cold oligonucleotide corresponding to this region. Closer examination of the optimal C/EBP oligonucleotide sequence revealed a perfect GATA consensus sequence and subsequent EMSA studies with GATA-4 antibodies (not shown) confirmed its binding; thus, we designated the oligonucleotide GATA-C/EBP. Additionally, consensus oligonucleotides containing either two GATA or C/EBP optimal sequences were also able to compete for the high-molecular-weight complex binding to the –72- to –32-bp region, suggesting the complex is formed by participation of both molecules. We confirmed that the GATA-4 site was localized to –72- to –55-bp region containing the GATA DNA element previously described (20, 22). The C/EBPß site was localized within the –66- to –37-bp region of the promoter that contains a perfectly conserved TGATGA element that overlaps with nucleotides conforming to a C/EBP sequence. Mutation of this element by itself caused a significant reduction in hormone-stimulated activity, and almost complete reduction (FSH 5 ng/ml > 25 ng/ml) occurred when combined with the GATA mutation. A TGATGA element upstream of the GATA site in the murine promoter was important for protein-DNA complex formation to –87- to –64-bp region and basal activity of reporter gene constructs (20) and participates in C/EBPß binding in rat granulosa cells (22). However, the murine sequence is not conserved across species, whereas the proximal TGATGA sequence is perfectly conserved among known mammalian sequences. One possible interpretation of this finding is that an element similar to this needs to exist adjacent to the GATA site for gonadotropin function and the murine promoter exhibits redundancy in this sequence.

Several studies have now shown that C/EBPß can bind the StAR promoters of different species; however, each study has revealed distinct C/EBP sites (17, 21, 22). Studies of the human StAR promoter showed recombinant C/EBPß and C/EBP could bind the two C/EBP-like elements at –119 to –110 (C1) and –50 to –41 bp (C4). Moreover, both sites were important for basal regulation of the human promoter in granulosa-luteal cells but retained similar fold-activation by 8-Br-cAMP when mutated (21). Furthermore, DNA binding studies showed C/EBPß formed a complex with the former but not the latter site in granulosa-luteal cell extracts. Studies of the murine StAR promoter showed an analogous sequence to the human C1 site to be a high-affinity C/EBPß site and initially proposed an additional sequence at –87 (C2) as a low-affinity site with both being equally important for basal activity in MA-10 cells (17). Subsequent studies in MA-10 cells showed the C2-containing region (–96 to –67) did not bind C/EBPß in cAMP-treated nuclear extracts but rather preferentially bound CREM (20, 24). Another study of the murine promoter showed that an additional C/EBPß site at –81 to –72 bp (C3) acted with the GATA site to promote FSH-stimulated transactivation of the murine promoter in rat granulosa cells (22). We now present data that indicate a proximal C/EBPß element overlapping the C4 site is important for hormone-stimulated activity of the porcine promoter in homologous primary gonadal cells. Our study does not indicate that the porcine C1 site (–125 to –115 bp) by itself significantly participates in basal or hormonally driven activation of the promoter and is most likely due to the fact that the region between –76 to –32 contains all the required elements for activity. The involvement of these apparently redundant C/EBPß sites may depend on the particular cellular and hormonal environment. In addition, the sequence similarity of C/EBP sites to ATF and AP-1 sequences provide the possibility that under certain cellular settings, that these sites could be occupied by other factors (21, 24).

Although potential C/EBPß binding sites vary among species, the GATA site is perfectly conserved in sequence and location (17). To date only the murine sequence has been investigated for its ability to bind GATA-4 in gonadal cells (20, 22, 46). Our studies show that GATA-4 binding to its respective DNA site is regulated by treatments with FSH in granulosa cell nuclear extracts and is enhanced in a time-dependent manner in the presence of IGF-I. In addition, GATA-4 binding to the StAR sequence continues in luteal nuclear extracts isolated from porcine corpora lutea in the early to midluteal phase. Silverman et al. (22) demonstrated GATA-4 binding in whole ovarian extracts from rats primed with PMSG/hCG but did not determine whether hormone priming increased GATA binding. Moreover, it is unknown whether GATA-4 binding exists in rat luteal extracts because GATA-4 expression has not been reported for the rat ovary. However, GATA-4 expression is down-regulated in the mouse corpus luteum, whereas GATA-6 is strongly expressed (47). Our previous studies with porcine luteal nuclear extracts showed GATA-4 DNA binding activity to a consensus oligonucleotide predominated over GATA-6 binding (32). Although both GATA-4 and GATA-6 can activate our porcine StAR reporter construct when overexpressed in granulosa cells (32), our data implicate GATA-4 as the major GATA protein regulating the StAR promoter in these cells.

Tremblay et al. (40) found that transfected GATA-4 or GATA-6 alone but not CEBPß alone stimulated the murine StAR promoter in heterologous CV-1 cells and that the combination of GATA-4 and C/EBPß showed cooperative transactivation only in the presence of PKA expression vector. These studies implicated a PKA phosphorylation event in transactivation mediated by these factors. In agreement with previous observations in CV-1, our studies with exogenous GATA-4 and C/EBPß in primary steroidogenic cells exhibited the greatest transactivation in cells overexpressing both factors when coincubated with cAMP analog. Studies with MA-10 Leydig cells demonstrated cAMP-stimulated phosphorylation of endogenous GATA-4 on a PKA consensus sequence (40), and subsequent in vitro phosphorylation experiments showed that Serine 261 of GATA-4 could be phosphorylated by PKA (37). In addition, murine GATA-4 harboring a mutation in the Serine 261 site had reduced capacity to activate a murine StAR promoter construct (37). Studies have not been performed to verify that this site is phosphorylated in vivo. A second weaker in vitro PKA site phosphorylation was observed in the carboxyl terminus of murine GATA-4 (37). Our immunoblot data indicate that at least two sites can be phosphorylated within endogenous GATA-4 in granulosa cells including basal phosphorylation of Serine 105 and FSH-regulated phosphorylation at a PKA site. GATA-4 phosphorylation can increase DNA binding activity (46, 48). Our present data show an increase in GATA-4 PKA-site phosphorylation temporally coincided with increased GATA binding to the –73- to –55-bp and –76- to –32-bp oligonucleotides. These data infer that PKA-mediated phosphorylation of GATA-4 may be responsible for increased DNA binding. Additional phosphorylations of GATA-4 may also be present (38). Analysis of the primary amino acid sequence of porcine GATA-4 reveals four potential PKA phosphorylation consensus sequences as well as the conserved Serine 105 (MAPK site). Also, our immunoprecipitation studies suggest that basal phosphorylation on an additional PKA recognition site is present in granulosa cells.

Phosphorylation of GATA-4 at Serine 105 is regulated by MAPKs in cardiomyocytes and is involved in hypertrophic responses (38, 39). In our studies, use of phosphospecific antisera for phosphorylated Serine 105 of GATA-4 revealed constitutive phosphorylation with all treatments. FSH and LH can activate the ERK cascade in granulosa cells via mechanisms that do not necessarily involve MAPK kinase stimulation (49, 50). In some granulosa cell lines, ERK inhibition actually enhances gonadotropin-stimulated StAR protein abundance (51). Although it is not known whether StAR transactivation is a downstream target of ERKs in granulosa cells, this pathway regulates StAR expression in Y1 adrenal cells by phosphorylation and enhanced binding of SF-1 to sequences of the murine StAR promoter (44). Regulation of GATA-4 on Serine 105 site may occur in granulosa cells under other conditions, and additional studies are needed to clarify the role of specific GATA-4 phosphorylation sites in the transactivation of StAR in primary cells.

C/EBPß activity can be regulated by transcription, alternative translation initiation, dimerization partners, and posttranslational modifications (41). Differential phosphorylation and dephosphorylation can enhance or reduce C/EBPß DNA binding and/or transactivation (52). Other studies have shown that C/EBPß exists as a repressed molecule and phosphorylation of the repression domain increases its transactivation potential (53, 54). We observed minimal changes (only with FSH + IGF-I, 38-kDa isoform) in total phosphorylation at 2 h treatment, a time point in which GATA-4 showed a net increase in phosphorylation in treatments with FSH and FSH + IGF-I. In our studies, a net increase in total C/EBP phosphorylation would not be observed whether different amino acids were simultaneously phosphorylated and dephosphorylated. Another possibility is that analysis of total phosphoprotein may actually underestimate phosphorylation of these molecules because net phosphorylation is increased with some treatments. We hypothesize that differential phosphorylation of C/EBPß may regulate its binding activity and cooperation with GATA, but additional studies to identify regulated phosphorylation sites are needed to support this idea.

In contrast to studies in short-term cultured rat granulosa cells (22), we did not find increases in C/EBPß protein abundance with the short-term hormone treatment that coincided with increased C/EBPß DNA binding. The preculture period of porcine granulosa cells required for StAR promoter expression most likely provides sufficient time and conditions for the expression of C/EBPß; thus, an alteration in C/EBPß activity rather than abundance is most likely responsible for transactivation observed in the present study. However, this does not exclude the possibility that further luteinization of granulosa cells may up-regulate C/EBPß abundance or that changes in the ratio of active (34 and 38 kDa) to inactive (20 kDa) isoforms occurs with further differentiation (41). Our previous studies and current data show FSH provides a short transient increase (4–6 h) in StAR promoter activity, whereas LH activation of the promoter in luteinized cells reaches maximal activation at 24 h and exhibits higher basal activity indicative of differential regulation of transactivation based on differentiation status (14, 15). C/EBPß mRNA up-regulation is associated with in situ luteinization of porcine follicles (Gillio-Meina, C., and H. A. LaVoie, unpublished data). Other studies have demonstrated increased C/EBPß mRNA and protein in rodent granulosa cells in response to an ovulatory dose of gonadotropins (55, 56, 57). Our laboratory has previously shown GATA-4 and GATA-6 mRNAs are not coordinately increased with StAR in developing corpora lutea but rather show a peak in periovulatory granulosa cells (32). We plan to investigate these trans-acting factors in our biochemically luteinized LH-responsive cell model to address differences in regulation associated with terminal differentiation of these cells.

In the pig ovary, IGF-I levels parallel progesterone levels in follicular fluid and early corpora lutea (58, 59). It has been proposed that intraovarian IGF-I acts to promote gonadotropin-induced steroidogenesis and may provide a selective advantage to periovulatory follicles (16). In cultured porcine granulosa cells, IGF-I facilitates several gonadotropin-stimulated end points including StAR promoter construct activity, mRNA accumulation, and mature protein abundance (12, 14, 15). We also previously demonstrated that under our transfection assay conditions that IGF-I can enhance FSH-stimulated cAMP accumulation, and augment forskolin, 8-Br-cAMP, and PKA subunit-driven StAR promoter activity (14). Our current data further support a dependence of the IGF-I effect on an FSH-driven signal because all IGF-I enhancing activity was localized to FSH-dependent regions of the promoter. Mutation of the GATA site demonstrated significant reductions in IGF-I augmentation at both FSH concentrations, whereas the decrement in activity with submaximal concentrations of FSH alone did not reach significance. One possible interpretation of these data is that IGF-I augmentation activity is more highly dependent on GATA than the adjacent C/EBP-like sequence, which consistently demonstrated highly significant reductions in transactivation for both FSH alone and in combination with IGF-I. Comparison of DNA binding of treated granulosa cell extracts to the –73- to –55-bp region (GATA site) and the –66- to –37-bp (C/EBP-like site) revealed IGF-I could enhance FSH-stimulated GATA binding in a time-dependent manner but FSH ± IGF-I-stimulated binding to the C/EBP site was fairly similar. IGF-I therefore may shift the time course of PKA-stimulated events leading to earlier phosphorylation and activation of GATA-4. Although FSH-induced phosphorylation (± IGF-I) of GATA-4 on a PKA recognition site, significant differences in GATA-4 phosphorylation between FSH alone and FSH with IGF-I were not observed suggesting the likelihood that IGF-I facilitates other events.

In summary, we have provided data that FSH ± IGF-I act through adjacent GATA and C/EBP sites on the porcine StAR promoter. The proximity of these two elements and the phosphorylation status of their binding proteins may contribute to their ability to recruit cofactors such as p300/CBP (37, 60, 61) or dissociate corepressors, such as Friend of GATA-2 (62). Current studies are underway to determine how multihormonal regulation may influence the interaction of GATA-4 and C/EBP with cofactors in primary ovarian cells.

	Acknowledgments

 
We thank Ms. Sara Hanscom for technical assistance with the isolation of porcine SF-1, Dr. Steven L. McKnight for the C/EBP cDNA vector, and Dr. A. Parlow (NIDDK, Torrance, CA) for the gift of ovine FSH-20. In addition, we thank Caughman Meat Packing for the gift of adult porcine ovaries.

	Footnotes

 
This work was supported by National Institutes of Health Grant HD-038945 (to H.A.L.) and a BOYSCAST fellowship from the Department of Science and Technology, Government of India (to D.S.).

Abbreviations: AP-1, Activator protein-1; ATF, activating transcription factor; 8-Br-cAMP, 8-bromoadenosine-cAMP; C/EBP, CCAAT/enhancer-binding protein; CREM, cAMP response element modulator; hCG, human chorionic gonadotropin; PKA, protein kinase A; PMSG, pregnant mare serum gonadotropin; pPKA, phosphorylated PKA; SF-1, steroidogenic factor 1; SREBP-1, sterol-regulatory element binding protein 1; StAR, steroidogenic acute regulatory protein.

Received December 18, 2003.

Accepted for publication March 24, 2004.

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