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. 2008 Jul 24;149(11):5557–5567. doi: 10.1210/en.2008-0484

GATA4 Reduction Enhances 3′,5′-Cyclic Adenosine 5′-Monophosphate-Stimulated Steroidogenic Acute Regulatory Protein Messenger Ribonucleic Acid and Progesterone Production in Luteinized Porcine Granulosa Cells

Yvonne Y Hui 1, Holly A LaVoie 1
PMCID: PMC2584590  PMID: 18653717

Abstract

Previous studies with cultured granulosa cells implicated GATA4 in gonadotropin regulation of the steroidogenic acute regulatory protein (STAR) gene. Caveats to these prior studies exist. First, GATA4 levels are reduced in granulosa-luteal cells after the LH surge when GATA6 expression is relatively high. Second, STAR mRNA expression is negligible in granulosa cells until after the LH surge. Both exogenous GATA4 and GATA6 can transactivate STAR gene promoter constructs. We used an RNA interference (RNAi) approach to determine the contributions of GATA4 and GATA6 to cAMP analog regulation of the endogenous STAR gene in luteinizing granulosa cells. STAR mRNA was stimulated by cAMP under control RNAi conditions. Surprisingly, GATA4 reduction by its respective RNAi approximately doubled the cAMP induction of STAR mRNA. At 24 h cAMP treatment, this augmentation was abolished by co-down-regulation of GATA4+GATA6. GATA6 down-regulation by itself did not alter STAR mRNA levels. GATA4+GATA6 co-down-regulation elevated basal CYP11A mRNA at 24 h treatment but did not affect its induction by cAMP. Basal levels of HSD3B mRNA were reduced by GATA4 RNAi conditions leading to a greater fold induction of its mRNA by cAMP. Fold cAMP-stimulated progesterone production was enhanced by GATA4 down-regulation but not by GATA4+GATA6 co-down-regulation. These data implicate GATA6 as the facilitator in cAMP-stimulated STAR mRNA and downstream progesterone accumulation under reduced GATA4 conditions. Data also demonstrate that basal levels of GATA4/6 are not required for cAMP induction of the STAR gene. The altered ratio of GATA4 to GATA6 after ovulation may allow GATA6 to enhance STAR mRNA accumulation.

OVARIAN SOMATIC CELLS express two highly homologous GATA zinc-finger transcription factors, GATA4 and GATA6 (reviewed in Refs. 1 and 2). In the ovary, their function was first linked to the transcription of steroidogenic acute regulatory protein (STAR) gene (3), whose protein product mediates the rate-limiting cholesterol entry into the mitochondria initiating steroidogenesis (4). Gonadotropin-stimulated increases in granulosa cell STAR mRNA were associated with GATA4 binding to the proximal STAR promoter region that contains a consensus GATA site, TTATCT (3,5,6). GATA4 was also found to cooperate with CCAAT-enhancer binding protein-β (CEBPβ) in granulosa cells to drive FSH-stimulated STAR promoter transactivation (3,5). Elimination of the proximal GATA site reduced FSH-mediated activity by 30–40%, showing GATA was an enhancer rather than an essential element (3,5).

Several caveats exist to ovarian studies implicating GATA4 in STAR gene regulation. In vitro studies in granulosa cells show FSH can drive the STAR promoter, yet in most species examined, STAR mRNA levels in vivo are negligible until the LH surge when granulosa cells begin luteinization (7,8,9). During the postovulatory luteinization period, GATA4 levels have been shown to decline and remain low in corpora lutea (10,11,12,13,14). In contrast, GATA6 expression is fairly strong during the postovulatory period (10,11,13). Interestingly, we found both GATA4 and GATA6 overexpression could activate STAR gene promoter constructs in primary cultures of luteinizing granulosa cells (13). In addition, other genes encoding steroidogenic enzymes needed for increased postovulatory progesterone production such as CYP11A1 (CYP11A) and HSD3B2 [ovarian types 1 (pig) or 2 (human) referred to as HSD3B] were also transactivated by GATA4 and GATA6 in clonal cells (15,16,17). Most of the studies implicating GATA4 and GATA6 in transactivation of the genes for these steroidogenic proteins have been performed using reporter gene assays with overexpressed proteins and may not recapitulate regulation of the native gene. Therefore, the contribution of GATA4 vs. GATA6 in regulating the endogenous STAR gene and other genes mediating steroidogenesis remains unclear.

Genetic models to study the respective contributions of GATA4 and GATA6 to gene regulation have been limited because deletion of either gene in mice results in embryonic lethality (18). More recent studies in mice with GATA4/6 reductions or repression have shown that GATA4 and GATA6 do have both unique and redundant functions in the development of heart, liver, or pancreas (19,20,21). Genetic animal models to study GATA4- and GATA6-specific actions in the mature ovary are not yet available. We have taken an initial approach to study the contribution of GATA4 and GATA6 to STAR gene regulation through RNA interference (RNAi)-mediated reduction of GATA4, GATA6, or their combination in luteinizing porcine granulosa cell cultures. In addition, we explored the impact of GATA down-regulation on other aspects of steroidogenesis, namely CYP11A and HSD3B mRNA expression and progesterone production.

Materials and Methods

Materials

General chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) and Fisher Scientific (Fairlawn, NJ). Cell culture reagents, Lipofectamine 2000, Trizol, and all synthesized oligonucleotides were purchased from Invitrogen Corp. (Carlsbad, CA). HALT protease and phosphatase inhibitor cocktails were purchased from Pierce Biotechnology (Rockford, IL). Cell culture plates were obtained from Falcon (Franklin Lakes, NJ). Amersham enhanced chemiluminescence reagents and Hybond-P polyvinylidene difluoride membranes were obtained from GE Healthcare (Piscataway, NJ). Abattoir ovaries from prepubertal gilts were purchased from Greenwood Packing Plant, Inc. (Greenwood, SC).

GATA RNAi and transfection of porcine granulosa cells

Cellular GATA4 and GATA6 reduction was performed using Stealth RNAi molecules. Stealth RNAi molecules were designed using the BLOCK-iT RNAi Designer software (Invitrogen) and GenBank coding sequences for porcine GATA4 (accession no. NM_214293), porcine GATA6 (accession no. NM_214328), and firefly luciferase (accession no. AB220162). Double-stranded Stealth RNAi molecules were synthesized by Invitrogen. We initially tested three different RNAi molecules, different transfection conditions, and different recovery times. The following GATA4 and GATA6 RNAi molecules were found to most efficiently reduce their respective mRNA in granulosa cells under the conditions described below. The RNAi sense strand sequences were GATA4, 5′-GAACCUUAACAAAUCGAAGACGUCA-3′; GATA6, 5′-GCAAUGCUUGUGGACUCUACAUGAA-3′; and firefly luciferase control, 5′-CCGGACUGUUUAUAGGUGUAGGUGU-3′.

Granulosa cells were isolated from 1- to 5-mm ovarian follicles of prepubertal gilts by needle aspiration as previously described (22). Cells were plated in MEM containing 3% fetal calf serum and antibiotics (2.5 μg/ml amphotericin B, 50 U/ml nystatin, 10 U/ml penicillin, 10 μg/ml streptomycin, 50 μg/ml gentamicin) at a density of 8 × 106 live cells per well in six-well culture plates. After 40–42 h culture, cells were rinsed with serum-free MEM without antibiotics and transfected with 200 nm RNAi using 5 μl Lipofectamine 2000 for 24 h. The control transfection consisted of 200 nm firefly luciferase RNAi; GATA4 transfection consisted of 100 nm GATA RNAi plus 100 nm firefly luciferase RNAi; GATA6 consisted of 100 nm GATA6 RNAi plus 100 nm firefly luciferase RNAi; and the combined GATA4+GATA6 consisted of 100 nm GATA4 plus 100 nm GATA6 RNAi. After transfection, medium was replaced with complete medium, and cells were incubated for 72 h before treatment. Cells were treated (time = 0 h) in serum-free medium containing antibiotics with vehicle (water) or cAMP analog, 8-Br-cAMP (1 mm; Sigma) for 6 and 24 h.

RNA isolation and real-time PCR

Total RNA was isolated from granulosa cells using Trizol reagent according to the manufacturer’s instructions. RNA was subjected to deoxyribonuclease I treatment using the RNeasy Micro kit (QIAGEN, Inc., Valencia, CA). RNA was quantified spectrophotometrically. RNA samples were reverse transcribed into cDNA using TaqMan reverse transcription reagents and protocol (Applied Biosystems, Foster City, CA).

Real-time PCR was carried out using 20 ng cDNA, specific primer sets, and SYBR Green Master Mix (Applied Biosystems) with 10 nm fluorescein using the iCycler iQ Real-Time PCR Detection System as recommended by the manufacturer (Bio-Rad Laboratories, Inc., Hercules, CA). Primers for porcine STAR, CYP11A, and S16 were described previously (23,24). PCR primers for porcine ovarian HSD3B yielding a 177-bp amplicon were derived from GenBank accession no. NM_001004049; the upstream primer was 5′-TTCTAAGCTCCAGAGCAAGATCAAG-3′, the downstream primer was 5′-TGGGTACCTTTCACATTGACCTTCATG-3′, and each was used at 300 nm. PCR primers for GATA4 (160-bp amplicon) were upstream 5′-TGAAGCTCCATGGTGTCCC-3′ and downstream 5′-CTGCTGGAGTTGCTGGAAG-3′ and for GATA6 (110 bp amplicon) were upstream 5′-AGAAACGCCGAGGGTGAAC-3′ and downstream 5′-CGTTTCCTGGTCTGAATTCCC-3′ and used at 900 nm each. Accession numbers for GATA4 and GATA6 messages are listed above. Annealing temperatures were 55 C for GATA4 and GATA6; 58 C for STAR, HSD3B, and S16; and 60 C for CYP11A primers. Amplification efficiencies were determined for all primer sets. Relative quantification using S16 mRNA as an internal control was performed according to methods of Pfaffl as previously described (23,25). All amplicons were subcloned and sequenced to confirm their identity.

Whole-cell extracts and immunoblots

Whole-cell extracts from granulosa cells were rinsed with D-PBS and scraped in a buffer containing 50 mm Tris (pH 7.4), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 1 mm EDTA, 1% HALT protease, and phosphatase inhibitor cocktails. Lysates were incubated on ice and subsequently spun at 13,600 × g in a microfuge. Protein was quantified using Bio-Rad protein dye reagent assay (Bio-Rad Laboratories, Hercules, CA).

In vitro-translated porcine GATA4 and GATA6 or COS-1 cells transiently expressing each porcine GATA protein were used as controls on some immunoblots. COS-1 cells were transfected with pcDNA3.1 expression vectors for porcine GATA4 or GATA6 by methods previously described (24). Posttransfection COS-1 cells were allowed to express GATA4 and GATA6 for 24 h before isolation of nuclear protein as described below. GATA proteins were in vitro translated from their respective expression vectors using the TNT T7 coupled wheat germ extract system (Promega Corp., Madison, WI).

Proteins (20 μg) were resolved by 10% SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes. Membranes were blocked in 0.6 mg/ml gelatin in Tris-buffered saline containing 0.05% Tween 20 (TTBS). Primary antibodies were incubated overnight at 4 C in gelatin-TTBS blocking solution. Because of the high homology in some portions of GATA4 and GATA6 (short-form) proteins, their similarity in molecular mass (52–55 kDa), and the unknown sequence specificity of commercially available antibodies, several different products were tested in immunoblotting. The antibodies tested were GATA4 sc-9053 and GATA4 sc-25310 (Santa Cruz Biotechnology, Santa Cruz, CA) and GATA4 ab25992 (Abcam Inc., Cambridge, MA). We were not able to use goat GATA4 or GATA6 antibodies for immunoblots due to high background experienced with porcine protein. We found cross-reactivity of GATA4 sc-9053 with porcine GATA6 expressed in COS cells, and GATA4 sc-25310 and GATA4 ab25992 antibodies were unable to detect low levels of GATA4 by on immunoblots. We therefore used the GATA4 sc-9053 antibody preabsorbed with COS cell nuclear extracts expressing porcine GATA6 to minimize detection of GATA6 on membranes. For GATA6 immunodetection, we tested the antibodies GATA6 sc-9055 (Santa Cruz) and GATA6 H-00002627-A01 (Novus Biologicals, Littleton, CO). Only GATA6 sc-9055 was able to efficiently detect recombinant GATA6 on immunoblots, and it also cross-reacted with denatured porcine GATA4. Therefore, the GATA6 sc-9055 antibody was preabsorbed with denatured recombinant porcine GATA4-GST protein before use to minimize cross-reactivity. Primary antibodies for GATA4 and GATA6 were used at a concentration of 2–4 μg/ml. The secondary antibody used for rabbit primary antibodies was goat antirabbit (1:2000 to 1:4000 Zymed Laboratories, South San Francisco, CA). Immunoreactive bands were detected by enhanced chemiluminescence.

Immunoblots were used to detect the active 17-kDa fragment of caspase-3, an indicator of caspase-3-dependent apoptosis, and performed as previously described using 20 μg cytoplasmic protein (26). The mouse IgG1 anti-caspase-3 (Active-Motif, Carlsbad, CA) used also detects the inactive 35-kDa caspase-3 fragment.

Nuclear protein isolation and EMSA

Granulosa cell nuclear protein isolation was performed at the end of the treatment period using a hypotonic lysis method as previously described (5) with the modification that HALT protease and phosphatase inhibitor cocktails were used at a final concentration of 1% (vol/vol) each. Cytoplasmic fractions were saved also for caspase-3 immunodetection. Protein was quantified as described above. The final concentration of DNA binding reaction used for EMSA was 5% glycerol, 0.5 mm dithiothreitol, 100 mm KCl, 10 mm Tris (pH 7.5), 12.5 mm HEPES, 2 μg poly deoxyinosine-deoxycytosine (Sigma), 0.25 mm EDTA, 0.025% HALT protease inhibitor, and 0.025% phosphatase inhibitor. Double-stranded oligonucleotides were end labeled using T4 polynucleotide kinase (Promega) and [γ-32P]ATP (PerkinElmer, Waltham, MA). For supershift and competition assays, nuclear extracts (3–6 μg) were preincubated with antibodies (1.5–6 μg) or 50-fold excess cold competitor oligonucleotides for 30 min on ice before addition of 100,000 cpm high-specific-activity oligonucleotide. Reactions were further incubated on ice for 30 min. The reactions were electrophoresed on 4.5% native polyacrylamide gels for 2 h 15 min at 150 V, dried, and visualized by autoradiography using Biomax MR film (Kodak, Rochester, NY). Antibodies used for gel shift assays were GATA4 sc-1237X, GATA4 sc-25310X, GATA6 sc-7245X, GATA6 sc-9055X, CEBPβ sc-150X, CEBPβ sc-746X (cross-reactive with CEBPα, -δ, and -ε), and normal rabbit, mouse, and goat IgGs (Santa Cruz). Also GATA4 ab25992 (Abcam) and GATA6 H-00002627-A01 (Novus Biologicals) were used. The GATA-2X, AP-1, and NFκB consensus oligonucleotides were obtained from Santa Cruz. The CEBP-2X containing two copies of the CEBP consensus sequence was described in our previous study (5). An oligonucleotide corresponding to the −75- to −49-bp region of the porcine STAR promoter containing the known GATA and CEBP binding elements was generated by annealing sense and antisense strands for the following sequence: 5′-GTTTTTTTATCTCCAGATGATGAAACA-3′.

Progesterone and DNA measurements

Progesterone concentrations in the cell culture medium were measured by RIA using the ImmuChem Progesterone 125I kit (MP Biomedicals, Costa Mesa, CA) and standards diluted in culture medium. The RIA has a sensitivity of 0.15 ng/ml and less than 1% cross-reactivity with other relevant steroid hormones (27). Progesterone was normalized to DNA content of wells.

Total DNA content per well was measured by Hoechst dye method using the DNA quantification kit fluorescence assay (Sigma) (28). Cells were scraped in 1× assay buffer and frozen at −80 C. Cells were sonicated briefly on ice before assay.

Data analyses

Densitometric analyses of immunoreactive bands and EMSA bands were performed using UN-SCAN-IT gel version 5.1 software (Silk Scientific, Orem, UT). Progesterone, DNA, mRNA, and protein data were analyzed by one-way repeated-measures ANOVA followed by Newman-Keuls multiple comparison test. Fold changes under basal conditions and cAMP-stimulated conditions were also analyzed in separate ANOVAs. Comparison of vehicle and 8-Br-cAMP raw progesterone data (nanograms per milliliter and nanograms per microgram DNA) for each RNAi was performed by paired t test. Statistical comparisons were performed using GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego, CA).

Results

Our goal was to determine whether GATA4, GATA6, or their combination contributes to the regulation of basal or cAMP-stimulated STAR gene expression in luteinizing granulosa cells. To address this question, we knocked down porcine GATA4, GATA6, and their combination using homologous RNAi molecules. We then treated the granulosa cells with 8-Br-cAMP to mimic gonadotropin effects on STAR gene expression. The cAMP analog was used to bypass any effects of GATA down-regulation on gonadotropin receptors. Cell cultures were terminated after 6 and 24 h treatment and analyzed for GATA4 and GATA6 mRNA gene expression, protein expression, and the ability of nuclear extracts to bind to a GATA oligonucleotide to verify GATA down-regulation. The results of these experiments are shown in Fig. 1. In vehicle-treated cells, GATA4 RNAi alone or in combination with GATA6 RNAi effectively reduced GATA4 mRNA by 77 ± 4 and 67 ± 3%, respectively, at 6 h and 76 ± 2 and 68 ± 3%, respectively, at 24 h, compared with the control RNAi (P < 0.001; Fig. 1A). In 8-Br-cAMP-treated cells, GATA4 RNAi alone or in combination with GATA6 RNAi reduced GATA4 mRNA by 93 ± 11 and 95 ± 11%, respectively, at 6 h and 119 ± 16 and 115 ± 17%, respectively, at 24 h compared with the control RNAi (P < 0.001). In vehicle-treated cells, GATA6 RNAi alone or in combination with GATA4 RNAi effectively reduced GATA6 mRNA by 69 ± 4 and 68 ± 4%, respectively, at 6 h and 71 ± 5 and 66 ± 5%, respectively, at 24 h compared with the control RNAi (P < 0.001; Fig. 1B). In 8-Br-cAMP-treated cells, GATA6 RNAi alone or in combination with GATA4 RNAi reduced GATA6 mRNA by 106 ± 6 and 119 ± 11%, respectively, at 6 h and 91 ± 15 and 95 ± 14%, respectively, at 24 h compared with the control RNAi (P < 0.001). Additionally, in the presence of the control or GATA6 RNAi, cAMP analog significantly increased GATA4 mRNA with 24 h treatment (P < 0.05). GATA6 mRNA was increased by 8-Br-cAMP treatment in the control (6 and 24 h) and GATA4 (6 h) RNAi cultures (P < 0.05).

Figure 1.

Figure 1

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GATA4 and GATA6 RNAi effectively reduce their respective messages and proteins and decrease total GATA DNA binding in luteinizing granulosa cells. After an initial attachment period, porcine primary granulosa cell cultures were transfected with control (C), GATA4 (G4), GATA6 (G6), or combined GATA4 and GATA6 (G4+G6) RNAi molecules for 24 h and then allowed to recover for 72 h before treatment with 8-Br-cAMP (1 mm) or vehicle for 6 and 24 h. All bars represent mean and sem. A and B, RNA was collected from cells after treatment with vehicle or 8-Br-cAMP, and GATA mRNAs were measured. GATA mRNA levels were normalized for S16 mRNA and are expressed relative to vehicle-treated control RNAi levels (n = 6 experiments). C and D, Summary of immunoblots for GATA4 and GATA6 proteins (n = 3–4 experiments). GATA4 and GATA6 proteins were normalized to the vehicle-treated control RNAi condition on each blot. E, EMSA using nuclear protein from RNAi-conditioned granulosa cells and a GATA-2X consensus oligonucleotide. Reductions in total GATA DNA binding occurred with individual GATA down-regulation and binding was dramatically reduced by GATA4+GATA6 coreduction. Results were similar in three different experiments. *, Percentage reduction in the GATA mRNA (P < 0.001) or protein (P < 0.05) was significantly lower than the control RNAi with the same treatment; a, treatment with 8-Br-cAMP is significantly higher than the same RNAi receiving vehicle (P < 0.05).

The intensities of immunoreactive GATA bands on immunoblots were normalized for their respective vehicle-treated control on each blot and are summarized in Fig. 1, C and D, respectively. GATA4 and GATA6 protein were significantly reduced by their respective RNAi molecules (P < 0.05). GATA4 protein reduction ranged from 53–82% in cells receiving the GATA4 RNAi alone and with GATA6 co-down-regulation. GATA6 protein reduction ranged from 54–77% in cells receiving the GATA6 RNAi alone and with GATA4 coreduction. The small increases in mRNA observed with cAMP treatment for specific RNAi conditions were not observed for protein. Examples of immunoblots are shown in supplemental Fig. 1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

GATA down-regulation was also verified by assessing the ability of nuclear extracts from each condition to bind to an oligonucleotide containing two consensus sites for GATA (GATA-2X). EMSA revealed reductions in total GATA-DNA binding with each GATA RNAi condition (Fig. 1E). In the GATA4+GATA6 reduced samples, greater than 70% (as assessed by densitometry) of total GATA-DNA binding was eliminated under all treatments and at both time points.

We and others (13,29) have previously shown that overexpression of GATA4 and GATA6 individually in transient transfection assays can stimulate reporter gene constructs harboring the 5′-flanking regions of the STAR gene from different species. Because reporter gene assays do not always reflect regulation of the endogenous gene, we next sought to determine the effect of GATA4, GATA6, or their combined reduction on STAR mRNA expression in the luteinizing granulosa cells (Fig. 2). No effect of GATA reduction on STAR mRNA was measurable under basal (vehicle-treated) conditions. Under control RNAi conditions, 8-Br-cAMP significantly stimulated STAR gene expression at both 6 and 24 h by 7.3 ± 0.8-fold and 6.5 ± 1.5-fold, respectively (P < 0.05). Unexpectedly, GATA4 RNAi conditions enhanced the ability of the cAMP analog to stimulate STAR mRNA expression with an increase of 13.1 ± 1.9-fold and 14.6 ± 5.0-fold at 6 and 24 h, respectively (P < 0.05). This represented approximately double the cAMP responsiveness under GATA4 RNAi conditions compared with control. GATA6 reduction alone did not significantly alter the ability of the cAMP analog to stimulate STAR mRNA accumulation. Combining GATA6 RNAi with GATA4 RNAi produced lower mean cAMP-stimulated STAR mRNA levels compared with GATA4 RNAi-only conditions, which was significant at 24 h (P < 0.05). At 24 h, the 8-Br-cAMP response in the GATA4+GATA6 reduced condition was similar to control and GATA6 RNAi conditions. In other words, GATA6 RNAi eliminated the ability of GATA4 RNAi to enhance cAMP-stimulated STAR mRNA expression at 24 h. We have previously shown that GATA4, GATA6, and CEBPβ can bind to an oligonucleotide spanning the −76- to −32-bp region of the porcine STAR gene promoter and that their binding sites reside within the −75- to −49-bp region based on mutagenesis studies (5). Mutations in this region of the porcine promoter showed this sequence was responsible for most of the cAMP-stimulated activity of reporter gene constructs in granulosa cells. We examined the effect of GATA reduction on the ability of granulosa cell nuclear proteins to bind to a −75- to −49-bp STAR promoter oligonucleotide (Fig. 3). We identified three regions containing one or more specific complexes that were designated regions A, B, and C. We did not see differences between vehicle and 8-Br-cAMP-treated nuclear extract binding to these regions at our selected time points. We did observe differences in the intensity or migration of regions A, B, and C depending on RNAi conditioning. In control and GATA4 RNAi conditions, the region A complex was larger (migrated differently) than under GATA6 or GATA4+GATA6 conditions. This mobility change was more pronounced at 6 h than 24 h. The intensity in region B also differed with respect to RNAi conditions. Region B had a double band of which the top band was more intense in control and GATA4 than in the GATA6 and GATA4+GATA6 RNAi cultures. The intensity of this upper band was greater in GATA4 RNAi cultures than control cultures at 6 h. Region C also showed two complexes. At 6 h, the bands in region C were lowest in control and GATA4 RNAi cultures and intense in both the GATA6 and GATA4+GATA6 RNAi cultures. At 24 h, the complexes in region C revealed that same pattern over multiple experiments with GATA6 and GATA4+GATA6 RNAi cultures being similar in intensity, control RNAi cultures being lower in intensity, and GATA4 RNAi cultures being the lowest. In summary, the enhancement of STAR mRNA by GATA4 RNAi was associated with increased intensity of complexes in region B at 6 h and low intensity of complexes in region C at both time points.

Figure 2.

Figure 2

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Effect of GATA reduction on STAR mRNA expression. Cells were subjected to control, GATA4, and/or GATA6 RNAi and treated as described in Fig. 1. RNA was collected from cells at 6 and 24 h after treatment, and STAR mRNA was measured. All bars represent mean and sem for n = 6 experiments. STAR mRNA levels were normalized for S16 mRNA and expressed relative to vehicle-treated control RNAi levels. Numbers above bars represent the mean fold induction of mRNA in response to 8-Br-cAMP. Different letters over bars indicate the fold induction with 8-Br-cAMP differs between groups (P < 0.05). No significant differences in basal levels were detected between groups.

Figure 3.

Figure 3

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GATA reduction alters the protein-DNA binding pattern to the −75- to −49-bp STAR promoter oligonucleotide. The oligonucleotide contains known GATA and CEBP binding elements. Nuclear extracts were isolated from granulosa cells transfected and treated as described in Fig. 1. EMSA were performed with nuclear extracts. Regions A, B, and C contain complexes that varied with RNAi and time. The top panel shows all bands with all treatments. The lower panel is a darker exposure of region A of the same gels. The DNA-protein complex in region A was larger (migrated differently) in all samples with control (C) or GATA4 (G4) RNAi conditions and most pronounced at 6 h. The complexes in region B were greatest in control and GATA4 RNAi samples. Two complexes in region C were most intense in GATA6 (G6) and GATA6+GATA4 (G4+G6) RNAi conditions at 6 and 24 h and were lowest in GATA4 RNAi conditions at 24 h. ns, Nonspecific band as indicated by competition studies. Results were similar in three different experiments.

We examined the composition of these complexes through competition assays with 50-fold excess cold oligonucleotides (Fig. 4A). Competition with cold −75- to −49-bp oligonucleotide revealed region A, B, and C complexes to be specific, with a nonspecific band between regions A and B. Competition with GATA-2X or CEBP-2X but not the unrelated NFκB oligo reduced the intensity of complexes in region A in control and GATA4 RNAi cells. GATA-2X reduced residual region A binding in GATA6 and GATA4+GATA6 RNAi cells, whereas CEBP-2X had minimal effect (Fig. 4A and not shown). Cold GATA-2X and CEBP-2X had minimal effect on complexes in regions B and C. These data suggest that in the absence of GATA6 RNAi, CEBP and a GATA molecule are bound in the complexes within region A. This idea is supported by antibody supershift studies. CEBPβ antibody was able to supershift part of region A in control and GATA4 RNAi cultures but not in GATA6 and GATA4+GATA6 RNAi cultures (Fig. 4B and not shown).

Figure 4.

Figure 4

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Competition and supershift EMSA studies with the −75- to −49-bp STAR promoter oligonucleotide. Granulosa cell nuclear extracts and labels are described in Fig. 3. A, Competition with 50-fold excess of cold oligonucleotide reveals complexes in A, B, and C are specific. The complex in region A can be reduced by competition with cold GATA-2X or CEBP-2X oligonucleotides in control and GATA4 RNAi nuclear extracts and is unaffected by an unrelated oligonucleotide, NFKB. Under GATA6 RNAi conditions, region A is partially competed by the GATA-2X oligo and CEBP-2X has minimal effect. Left and right panels represent two different gels. The left panel was 24 h vehicle treatment, and the right panel has 6 h 8-Br-cAMP treatment. B, Antibodies to CEBPβ supershifted part of complex A in GATA4 RNAi conditions (and control RNAi, not shown) but not with GATA6 RNAi conditions. Antibodies were CEBPβ* (sc-150) and CEBPβ** (sc-746X). Treatments with 24 h vehicle are shown. C, Under GATA4 RNAi conditions (and control RNAi, not shown), GATA antibodies implicate GATA6 as the major GATA factor binding to the −75- to −49-bp oligonucleotide in region A. Twenty-four-hour 8-Br-cAMP treatment is shown. Antibodies were GATA4* (sc-25310X), GATA4** (ab-25992), GATA6* (sc-7245X), and GATA6** (sc-9055X). No np indicates no nuclear protein was in the reaction and reveals a nonspecific band shift with the GATA4** antibody. Each antibody or competition was repeated at least three times with different experiments with similar results.

Samples of the EMSA results with the most specific GATA antibodies are shown in Fig. 4C. Complex A was reduced approximately 50% (by densitometry) by preincubation of GATA4 RNAi nuclear extracts with GATA6 antibodies and was minimally affected by GATA4-specific antibodies. This was similar in control RNAi extracts (not shown). There was minimal reduction (<10%) in region A intensity under GATA6 RNAi-only and GATA6+GATA4 RNAi conditions with any GATA4/6 antibody used in Fig. 4 (not shown). Although our interpretation is limited by the antibodies employed, our results suggest that our luteinized cells have predominantly GATA6 in complex A.

Because previous transfection studies in clonal cells have implicated GATA4/6 in regulating additional genes involved in progesterone biosynthesis (15,16,17), we examined CYP11A and HSD3B mRNA expression under our RNAi conditions. We also wanted to determine whether the effect seen on STAR mRNA extended to other cAMP-regulated steroidogenic genes that have additional regulatory mechanisms in granulosa cells. Figure 5 shows the effect of control and GATA RNAi conditions on CYP11A and HSD3B mRNA expression in granulosa cells in response to vehicle or 8-Br-cAMP treatment. cAMP analog stimulated the expression of both mRNAs under all RNAi conditions at both time points (P < 0.05). The cAMP-stimulated fold increase in CYP11A mRNA expression was not significantly affected by any RNAi condition at either time point (Fig. 5A). Basal levels of CYP11A were higher in the GATA4+GATA6 coreduced group at 24 h compared with all other RNAi groups with vehicle treatment (P < 0.05). At 6 h, there was no significant alteration in HSD3B mRNA fold induction by 8-Br-cAMP; however, there was a significant reduction in basal HSD3B message under GATA4 RNAi-only conditions (Fig. 5B, P < 0.05). At 24 h, basal levels of HSD3B mRNA were also reduced in GATA4 RNAi cultures compared with GATA6 and GATA6+GATA4 RNAi cultures (P < 0.05). This reduction in basal levels led to an 18.0 ± 2.8-fold increase in cAMP-stimulated HSD3B mRNA in the GATA4 RNAi condition and was significantly higher than cAMP-stimulated increases measured under control (11.3 ± 2.2), GATA6 (7.8 ± 1.7), and GATA4+GATA6 (7.4 ± 1.5) RNAi conditions (P < 0.05).

Figure 5.

Figure 5

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Effect of GATA reduction on CYP11A and HSD3B mRNA expression. Cells were subjected to control or GATA4 and/or GATA6 RNAi and treated as described in Fig. 1. RNA was collected from cells at 6 and 24 h after treatment, and mRNA was measured. A and B, all bars represent mean and sem for n = 6 (6 h) or n = 5 (24 h) experiments. In all graphs, 8-Br-cAMP-treated means were significantly higher than their respective vehicle treatment for the same RNAi (P < 0.05). A, CYP11A mRNA levels normalized for S16 mRNA and expressed relative to vehicle-treated control RNAi levels. a, mRNA was significantly different from all other RNAi cultures treated with vehicle (P < 0.05); *, fold response to cAMP analog was significantly higher than all other groups at the same time point. B, HSD3B mRNA levels normalized for S16 mRNA and expressed relative to vehicle-treated control RNAi levels. a and *, Same as described in A; b, mRNA was significantly different from vehicle-treated GATA6 or GATA4+GATA6 RNAi cultures (P < 0.05).

We measured progesterone levels in the media to determine whether alterations in STAR or other steroidogenic gene transcripts had downstream physiological consequences on cAMP-stimulated steroidogenesis. Figure 6, A and B, shows the effect of GATA reduction on progesterone accumulation in granulosa cells. No significant effects of 8-Br-cAMP or GATA RNAi on progesterone were observed after 6 h treatment. At 24 h, cAMP analog significantly stimulated progesterone accumulation (compared with vehicle, P < 0.05) under control and GATA4 RNAi conditions and was borderline significant under combined GATA4+GATA6 RNAi conditions (P = 0.076 for nanograms per milliliter and P = 0.063 for nanograms per microgram DNA). Compared with vehicle, 8-Br-cAMP treatment did not significantly increase progesterone accumulation in GATA6 RNAi conditions, but the fold response to the cAMP analog did not differ from the control. Under GATA4 RNAi-only conditions, 24 h 8-Br-cAMP resulted in a significant (fold) augmentation (P < 0.05) in progesterone accumulation compared with all other RNAi conditions. We visually noted that granulosa cultures receiving GATA4 RNAi appeared denser at 24 h, and thus we measured DNA content of wells to normalize progesterone values (Fig. 6C). GATA4 and GATA4+GATA6 RNAi cultures had the highest amount of DNA content per well, and GATA6 RNAi had the lowest (P < 0.05). Even after normalizing for cellular DNA, cAMP-stimulated progesterone levels (fold) were significantly augmented with GATA4 RNAi conditions, and the increase was diminished by co-down-regulation of GATA6.

Figure 6.

Figure 6

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Effect of GATA reduction on progesterone accumulation and DNA content per well in cultured granulosa cells. Cells were subjected to control or GATA4 and/or GATA6 RNAi and treated as described in Fig. 1. Progesterone and DNA were collected at 6 and 24 h after treatment and measured. A–C, All bars represent mean and sem for n = 3 experiments. The 6- and 24-h cultures originated from the same experiments. A, Progesterone concentrations in media collected from cultured granulosa cells. Numbers above bars indicate the mean fold increase in progesterone in response to 8-Br-cAMP. *, Fold response to cAMP analog was significantly higher than other groups; a, 8-Br-cAMP treatment was significantly higher than vehicle for the same RNAi; b, 8-Br-cAMP treatment compared with vehicle for this RNAi bordered significance (0.05 < P < 0.10). B, Progesterone normalized for cellular DNA content per well. Fold increases and asterisks are presented as in A. C, DNA content per well. Letters indicate treatment significantly differs (P < 0.05) from vehicle-treated control RNAi (c), from vehicle-treated GATA6 RNAi (d), or 8-Br-cAMP-treated GATA6 RNAi (e).

Immunoblots for caspase-3 were performed to determine whether apoptosis could be detected at the end of the treatment period (see supplemental Fig. 2). No active caspase-3 fragment was detected in experiments with any treatment condition (n = 3 experiments each at 6 and 24 h) despite the fact that the inactive form of caspase-3 was readily detected and that the active fragment was readily detected in positive control samples.

Discussion

We previously showed that both GATA4 and GATA6 could transactivate porcine STAR promoter reporter plasmids in cultured granulosa cells and that GATA6 induction of STAR was slightly greater than GATA4 (13). Using a less luteinized granulosa cell model than the present studies we previously identified GATA4 by EMSA as the predominant GATA factor binding to a STAR promoter oligonucleotide containing all the major gonadotropin-/cAMP-regulated elements, although some GATA6 binding was also observed (5). Similar findings with GATA4 and the rat STAR promoter were also described in rat granulosa cells (3). In addition, chromatin immunoprecipitation assays demonstrated GATA4 binding to the proximal murine STAR promoter in granulosa cells in vivo immediately after an ovulatory human chorionic gonadotropin stimulus (6). Our current studies implicate GATA6 over GATA4 as the major STAR oligonucleotide-binding molecule under control and GATA4 reduced conditions. The difference in the current result vs. our previous study (5) may be due to previously flawed EMSA interpretation due to the cross-reactivity of GATA4/6 antibodies. Differences may also result from the use of FSH in previous studies compared with cAMP analog in the current study. It is likely that the more luteinized conditions of our cells in the current study may account for differences in GATA4 vs. GATA6 binding activity. In this study, granulosa cells were returned to serum-containing medium after RNAi transfection, and serum is known to promote luteinization (30). Serum was necessary to maintain high cAMP responsiveness of the STAR gene under subsequent serum-free conditions.

In our current study, we used GATA RNAi to determine the contribution of GATA4 and GATA6 to regulation of the porcine STAR gene. RNAi was found to effectively reduce both GATA4 and GATA6 expression and DNA binding capacity. Our data show that induction of STAR by cAMP does not require much if any GATA4 or GATA6 because STAR mRNA expression in the GATA4+GATA6-reduced condition was similar to the control RNAi. Unexpectedly, GATA4 reduction by itself actually augmented cAMP-stimulated STAR mRNA expression. Reduction of GATA6 in combination with GATA4 abolished this enhancement at the 24-h time point, implicating GATA6 as the augmenting factor or alternatively implicating a GATA6-regulated factor as the effector. One interpretation of these data is that GATA4 may somehow interfere with the ability of GATA6 to enhance STAR transactivation and that reducing GATA4 allows GATA6 to act more efficiently. We could not determine whether the proportion of GATA4 and GATA6 binding to the −75- to −49-bp STAR promoter oligonucleotide was altered between control and GATA4 RNAi cultures, but GATA6 appeared to be the predominant GATA molecule in region A under these conditions. The binding of CEBPβ in region A was eliminated by GATA6 and GATA4+GATA6 RNAi conditions. Other differences in EMSA were also evident. GATA6 and GATA4+GATA6 down-regulation reduced the intensity complexes in region A and region B and coordinately increased complexes in region C. Enhancement of cAMP-stimulated STAR mRNA by GATA4 was accompanied by a unique pattern in complexes in regions B and C, namely a more intense band shift in region B at 6 h and extremely low levels of complexes in region C at both 6 and 24 h. The identity of proteins in regions B and C were not identified, but competition assays show they are not GATA or CEBP family members (or AP-1, not shown). More extensive studies will be needed to identify these proteins and determine their regulation. No differences in the intensity of GATA-CEBP complexes were observed with cAMP treatment under control RNAi conditions. This finding is not inconsistent with our previous study that found only small increases in GATA-CEBP DNA binding complexes with gonadotropin treatment and only at discrete time points 2–4 h with constitutive binding at later time points studied (5). The cAMP responsiveness of the endogenous STAR promoter is likely due not only to GATA and CEBPβ binding but also to their posttranslational modifications and their ability to recruit other factors such as steroidogenic factor-1 (SF-1) or liver receptor homolog-1 (LRH-1) and coactivator cAMP response element-binding protein-binding protein (CBP) that contribute to ovarian-specific STAR gene regulation (5,6,31). cAMP responsiveness of the ovarian STAR gene may also be influenced by the dissociation of corepressors such as dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene-1 (DAX-1) or friend of GATA-2 (FOG-2) (14,31,32).

The messages of CYP11A and HSD3B that code for enzymes mediating cholesterol side-chain cleavage and the formation of progesterone from pregnenolone, respectively, were also examined for GATA RNAi effects. Both of their mRNAs are induced by gonadotropins and cAMP analog in cultured granulosa cells (33,34). Their gene promoters have been shown to be stimulated by GATA4 and/or GATA6 in transfection assays in clonal cells (15,16,17). CYP11A promoter activity has been examined in primary granulosa cells, and GATA4 rather than GATA6 appeared to be the regulating factor (35). Specificity protein-1 (Sp1), SF-1, LRH-1, cAMP response element-binding protein (CREB), and fos-related antigen 2 (Fra-2) also participate in ovarian CYP11A gene regulation (35,36,37,38). GATA regulation of the HSD3B promoter has not been examined in ovarian cells, but LRH-1, SF-1, and nerve growth factor 1B (NGFI-B) can mediate ovarian expression (39,40), and GATA4/6 can cooperate with SF-1 and LRH-1 to drive HSD3B promoter activity in Leydig and adrenal cell lines (17). Our data showed that the fold induction of CYP11A mRNA to cAMP analog was not affected by GATA reduction. At 24 h, basal CYP11A mRNA levels were higher only in the GATA4/6 RNAi condition, indicating these combined factors have a slight but significant inhibitory effect on basal but not cAMP-stimulated CYP11A mRNA. Basal levels of HSD3B with GATA4 RNAi were significantly lower than most or all other RNAi conditions. At 24 h, the reduction in basal levels with GATA4 RNAi conditions increased the HSD3B mRNA fold response to 8-Br-cAMP, but it did not increase the absolute levels nerve growth factor 1B of HSD3B mRNA. Basal levels of HSD3B mRNA with GATA4/6 co-down-regulation were not altered, suggesting GATA6 plays a role under GATA4 RNAi conditions in lowering basal HSD3B levels. Our conclusions from these data are that STAR, CYP11A, and HSD3B are not coordinately regulated by GATA4/6. In our model, the highest GATA4 levels impede maximal STAR mRNA induction by cAMP and affect mostly basal HSD3B mRNA, whereas combined high GATA4 and -6 levels suppress basal CYP11A mRNA at 24 h.

Progesterone was measured as a downstream indicator of GATA impact. The pattern of progesterone for the most part followed the pattern of STAR mRNA at 24 h, with the exception that cAMP-stimulated progesterone in the GATA6 RNAi cultures was not significant compared with its vehicle (even though the fold induction was not different from control). Progesterone production depends on many factors including the levels and activities of STAR protein, the components of the P450 cholesterol side-chain cleavage complex, and 3β-hydroxysteroid dehydrogenase. It was not our intent to examine the proteins in the steroidogenic pathway but rather to look at the major transcripts involved that had previous ties to GATA. Given the profile of progesterone production, it is assumed that the protein expression was altered to some extent in concert with the transcripts examined.

One unexpected finding was that DNA content per well in GATA4 and GATA4+GATA6 RNAi conditions was elevated at 24 h. This pattern was evident at 6 h but was not significant. DNA content per well was not affected by cAMP treatment and was affected only by the presence of GATA4 RNAi (alone or with GATA6 RNAi). We could not distinguish between cell retention to culture dishes and increased cell proliferation or cellular DNA content by our assay, but there were no significant reductions in DNA content at 24 h compared with 6 h, suggesting cells are not lost during this interval. There are mixed data in the literature as to the role of GATA factors in cell proliferation. Deletion of GATA4 in developing heart decreased cardiomyocyte proliferation (41). In cultured vascular smooth muscle and glomerular mesangial cells, GATA6 is down-regulated during cell cycle reentry, and overexpression of exogenous GATA6 induces cell cycle arrest (42,43). In granulosa tumors, higher GATA4 immunoreactivity correlated with tumor aggressiveness (44). In contrast, loss of GATA6 is prevalent in epithelial-derived ovarian tumors, and loss of GATA4 occurs in established proliferating ovarian cancer cell lines (45,46). When these studies are taken together, it appears as if GATA6 promotes cell quiescence but that the role of GATA4 in cell proliferation is cell type dependent. The mechanism of GATA4 RNAi enhancement of DNA in our cell model is unknown and will require further investigation.

Because GATA4 had been indirectly linked to apoptosis of granulosa cells in the ovary, we also examined our cultures for active caspase-3 as a marker of apoptosis (26). There were no detectable active caspase-3 fragments under any RNAi or treatment conditions in our studies. Previous studies by us and others have shown that GATA4 RNA or immunoreactivity was lost in granulosa cells of early atretic follicles but that of GATA6 was retained (10,13). GATA4 is believed to prevent apoptosis via regulation of Bcl-2 and Bcl-X because overexpression of GATA4 can activate their gene promoters in some cell types (47,48). The lack of active caspase-3 (apoptosis) could be due to one or more reasons. Perhaps GATA4 loss is a result of apoptosis rather than a cause of apoptosis. A likely possibility is that our cells, which have undergone luteinization in culture, may have become resistant to apoptosis upon GATA4 reduction. This latter concept is supported by data that show endogenous reductions in GATA4 levels during the post-LH surge period and lower GATA4 levels in luteal cells compared with granulosa cells (10,11,12,13,14). Granulosa cells of preovulatory follicles exposed to the LH surge undergo terminal differentiation and become resistant to apoptosis (49). So GATA4 may not be important for cell survival in terminally differentiated granulosa cells.

In summary, our data show RNAi as an effective approach to study the two resident GATA factors in the ovary. Our data support GATA6 as an enhancer of cAMP-stimulated STAR mRNA induction under conditions of reduced GATA4. Increased cAMP responsiveness of the STAR gene may result in the downstream enhancement of progesterone production (fold response) under GATA4 RNAi conditions. The accumulation of STAR mRNA in response to cAMP was either similar to or higher than the control RNAi, indicating that little or no GATA is required for cAMP induction of the STAR gene. Our data provide the first in situ evidence that GATA factors are not essential for cAMP-stimulated STAR mRNA accumulation and that GATA6 functions as a STAR gene enhancer when GATA4 is low. These results shed light on the induction of STAR in luteinizing granulosa cells and will be enhanced further when GATA RNAi can be applied to highly purified luteal cell cultures.

Supplementary Material

[Supplemental Data]
en.2008-0484_index.html (2.5KB, html)

Footnotes

This project was supported by National Research Initiative Competitive Grant 2007-35203-18060 from the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service and by Grant P20-MD00233 from the National Institutes of Health.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 24, 2008

Abbreviations: CEBPβ, CCAAT-enhancer binding protein-β; LRH-1, liver receptor homolog-1; RNAi, RNA interference; SF-1, steroidogenic factor-1; STAR, steroidogenic acute regulatory protein.

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