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BOR - Papers in Press, published online ahead of print December 8, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.034298
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BIOLOGY OF REPRODUCTION 72, 862–871 (2005)
DOI: 10.1095/biolreprod.104.034298
© 2005 by the Society for the Study of Reproduction, Inc.

Epidermal Growth Factor-Mediated Inhibition of Follicle-Stimulating Hormone-Stimulated StAR Gene Expression in Porcine Granulosa Cells Is Associated with Reduced Histone H3 Acetylation1

Raluca Rusovici, Yvonne Y. Hui, and Holly A. LaVoie2,

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Steroidogenic acute regulatory protein (StAR) mediates cholesterol transport into the mitochondria and is essential for ovarian steroidogenesis. Epidermal growth factor (EGF) has been reported to inhibit FSH-stimulated differentiation in porcine granulosa cells. Previous studies have demonstrated FSH stimulates StAR mRNA accumulation and gene promoter activation in granulosa cells. Treatment of granulosa cells with FSH (5 ng/ml, 6 h) increased StAR mRNA, whereas coaddition of EGF (10 ng/ ml) significantly reduced (P < 0.05) FSH-stimulated mRNA accumulation by 62.7% ± 13.9%. Under these same conditions, FSH-stimulated cAMP accumulation in cultures was unaltered by coincubation with EGF. RNA stability studies showed that cotreatment with FSH and EGF did not alter the StAR mRNA half-life compared with FSH alone, t1/2 = 1.9–3.8 and 2.7–4.1 h, respectively. EGF significantly inhibited (P < 0.05) FSH-stimulated StAR heterogeneous nuclear RNA levels by 47.6% ± 6.8 %, implicating a repressive effect on transcription. Surprisingly, EGF (1–50 ng/ml) did not affect FSH stimulation of a 1423-base pair StAR gene promoter-luciferase construct in transient transfection assays in porcine granulosa cells. To evaluate FSH and EGF effects on the endogenous StAR gene, chromatin immunoprecipitation assays were performed in combination with real-time polymerase chain reaction. FSH increased histone H3 acetylation (lysines 9, 14) within the proximal region of the StAR gene promoter and coincubation with EGF blocked this effect. Dimethylation (lysine 9) of histone H3 was not influenced by treatments. In conclusion, EGF repression of FSH-stimulated StAR transcription in porcine granulosa cells is accompanied by reductions in histone H3 acetylation associated with the StAR gene promoter.

follicle-stimulating hormone, granulosa cells, growth factors, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the estrous and menstrual cycles, the ovarian follicle transforms from a structure designed primarily to synthesize and secrete estradiol into a gland dedicated to the manufacture of large quantities of progesterone. Prior to ovulation, the thecal cell compartment is endowed with the steroidogenic machinery to make progesterone, which in turn is converted to androgens and aromatized to estrogens by the granulosa cell layer [1]. However, before the gonadotropin surge, the granulosa cell compartment of the follicle lacks significant capacity to synthesize progesterone for several reasons. First, in some species, like pigs and humans, the preferred cholesterol substrate, low-density lipoprotein, has restricted access to the granulosa cells hindered by an intact basement membrane [2]. In addition, molecules critical for de novo synthesis of steroids, such a steroidogenic acute regulatory (StAR) protein, which transports cholesterol into the mitochondria, and cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), the first enzyme in the steroid biosynthetic pathway, are limited in granulosa cells [3, 4]. The midcycle surge in luteinizing hormone (LH) upregulates the expression of both mRNAs encoding StAR and P450scc [3, 5]. In the porcine ovary, the StAR mRNA exhibits the most dramatic increase during follicular luteinization [6].

In contrast with granulosa cells in developing follicles in vivo, granulosa cells isolated from antral follicles and placed in culture express detectable StAR mRNA and its protein [7, 8]. In cultured granulosa cells, StAR mRNA can be upregulated by activators of the cyclic AMP/protein kinase A (PKA) pathway and, in particular, follicle stimulating hormone (FSH) [7, 8]. Expression of StAR is regulated primarily at the transcriptional level [9]. Data from several species, including pig, have shown that the first 250 base pairs (bp) of the proximal promoter are critical for basal, FSH, and cAMP analogue-stimulated StAR gene transcription [9]. Additional studies have shown that transcription factors CEBP, GATA-4, and/or SF-1 participate in the FSH/ cAMP analogue-mediated stimulation of StAR promoter reporter gene constructs in cultured granulosa cells [10–13]. Data also support the association of these same transcription factors with the StAR gene promoter in vivo during hCG-induced differentiation of mouse granulosa cells [14]. In addition, recent studies have implicated chromatin remodeling in the regulation of StAR transcription as well. Christenson et al. first demonstrated that cAMP-stimulation of StAR gene transcription in MA-10 Leydig cells was associated with acetylation of histone H3 specifically within the proximal region of the StAR gene promoter [15]. These authors also demonstrated that in vivo luteinization of macaque granulosa cells was accompanied by an increase in acetylated histone H3 associated with the proximal StAR gene promoter [15]. In contrast, gonadotropin-induced activation of the StAR gene in mouse granulosa cells in vivo did not alter histone H3 acetylation but did reduce dimethylation of lysine 9 of histone H3, a modification linked with gene silencing [14].

In vivo, undifferentiated granulosa cells are exposed to plasma FSH and have FSH receptors [16], but do not express detectable StAR mRNA, although they do express the mRNA encoding P450scc (CYP11A mRNA) [17, 18]. Differences between granulosa StAR mRNA responses to FSH in vivo and in culture may be due to removal of granulosa cells from a restrictive follicular environment that may prevent premature luteinization in the presence of PKA activation. One hypothesis is that intraovarian factors present in vivo suppress FSH stimulation of StAR mRNA expression. Epidermal growth factor (EGF) has long been known to modify granulosa cell differentiation [19]. In the porcine ovary, EGF has been found in all cellular compartments of the follicle and its receptor is found primarily in granulosa and thecal cells with greatest binding in the granulosa cell layer [20, 21]. In species like the pig and human, where EGF stimulates granulosa cell proliferation in culture, it also inhibits several endpoints associated with cyclic AMP-mediated granulosa cell differentiation [22, 23]. EGF inhibits cAMP analogue-stimulated accumulation of StAR mRNA and progesterone in luteinized porcine granulosa cells, yet doesn't repress CYP11A message [24]. EGF also reduces FSH-stimulated P450 aromatase activity in human granulosa-lutein cells [25]. In addition, EGF suppresses FSH or cAMP analogue induction of LH receptors in rat and porcine granulosa-lutein cells [24, 26, 27]. Thus, EGF is a logical candidate in the pig for suppression of FSH activation of StAR gene expression.

The present study confirmed that EGF is capable of suppressing StAR mRNA expression in FSH-responsive primary porcine granulosa cell cultures, thus, experiments were carried out to address the mechanism by which EGF repression occurs. Studies were undertaken to determine if EGF repression occurs at StAR gene transcriptional or posttranscriptional levels and whether histone H3 modifications within the chromatin are affected by FSH and/or EGF in cultured granulosa cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

Ovine FSH (NIDDK o-FSH-20) was obtained from the National Hormone and Pituitary Program (Bethesda, MD). Human recombinant EGF, 5-azacytidine, trichostatin A, sodium butyrate, protease inhibitors, and 5,6-dichloro-1ß-D-ribofuranosylbenzimidazole (DRB) were purchased from Sigma Aldrich (St. Louis, MO). LipofectAMINE, cell culture media, trizol, and all synthesized oligonucleotides were obtained from Gibco/Invitrogen (Carlsbad, CA). The ptk-RL/luc vector and all luciferase assay reagents were obtained from Promega Corp. (Madison, WI). General chemicals were purchased from Fisher Scientific (Fairlawn, NJ). Ovaries from prepubertal gilts were obtained from Greenwood Packing Plant, Inc. (Greenwood, SC).

Primary Granulosa Cell Culture

Granulosa cells were isolated from small and medium-sized (1- to 5-mm) follicles of immature porcine ovaries by needle aspiration as previously described [28]. Cells were plated in bicarbonate-buffered minimal essential medium (MEM) with antibiotics (penicillin, streptomycin, amphotericin, and nystatin) supplemented initially with 3% FCS for 39–43 h to permit cell anchorage, with a medium change after the first 24 h [29]. After the initial 39–43 h, medium was changed to serum-free MEM containing antibiotics for 5 h before treatment, with the exception of transfection studies described below. For RNA and chromatin immunoprecipitation assays (ChIP) studies, 8 x 106 viable granulosa cells were plated in 6-well culture plates (Falcon, Franklin Lakes, NJ). For transfection studies, 5 x 106 viable cells were plated in 12-well culture dishes. All cell culture experiments were performed with duplicate wells.

RNA Isolation and cDNA Synthesis

Total RNA was isolated from granulosa cells using Trizol reagent according to the manufacturer's suggestions. For heterogeneous nuclear RNA (hnRNA) studies, each RNA sample was subjected twice to DNase I treatment using the RNAeasy Micro kit (Qiagen, Inc., Valencia, CA) before reverse transcription to minimize amplification of genomic DNA. RNA samples (100 ng) were subjected to cDNA synthesis using Taqman Reverse Transcription Reagents (Applied Biosystems, Warrington, UK).

Real-Time Polymerase Chain Reaction

For real-time polymerase chain reaction (PCR), 2 µl of appropriate cDNA reaction and oligonucleotide primers corresponding to different regions of the porcine StAR gene were used in combination with SYBR Green Master Mix (Applied Biosystems). In all cases, optimal primer concentrations were determined empirically. For mature StAR mRNA studies, primers were designed to amplify a 183-bp amplicon encompassing exon 4 of the porcine StAR gene and each primer spanned an exon-exon boundary. Both the upstream primer (5'-GGAGAGCCGGCAGGAGAATG-3') and downstream primer (5'-CTTCTGCAGGATCTTGATCTTCTTG-3') were used at 300 nM. For hnRNA studies, primers were designed to amplify a 103-bp amplicon spanning the splice site between intron 4 and exon 5 of the porcine StAR gene ([30], GenBank accession no. U53020). Real-time PCR was performed with 300 nM each of upstream (5'-CCCACCGCATGGTCGAGTAGTG-3') and downstream (5'-CGCCTCTGCAGCCAACTCATG-3') primer. For amplification of the cDNA encoding P450scc (a product of the CYP11A gene), the primers were designed to amplify a 182-bp amplicon encompassing exon 2 and each primer spanned an exon-exon boundary (GenBank accession no. X13768). Real-time PCR was performed with 300 nM each of upstream (5'-GTCCCATTTACAGGGAGAAGCTCG-3') and downstream (5'-GGCTCCTGACTTCTTCAGCAGG-3') primer. Amplification was performed using the iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA). Amplification efficiencies were determined for all primer sets. Samples were amplified in duplicate or triplicate and the average threshold cycle (Ct) was used for quantification. For mRNA and hnRNA studies, the porcine S16 ribosomal protein mRNA was used as an internal amplification control. Primers to amplify S16 cDNA have been described previously [31]. Relative quantification of treatment samples was determined according to the mathematical formula

{bire-72-04-14-eq1}

In this equation, E represents the efficiencies of primers for both target (StAR gene) and S16 internal control (i) and Ct represent the threshold cycle for each sample [32]. For most experiments, samples were normalized to the FSH treatment rather than vehicle control. All PCR products (including those for ChIP) were also amplified without SYBR green and sequenced to confirm the identity of the amplicon (University of Maine DNA sequencing facility, Orono, ME).

Messenger RNA Half-Life Studies

The RNA polymerase II inhibitor, DRB, was used for mRNA half-life studies. After the initial attachment period and 5 h incubation in serum-free medium as described above, granulosa cells were treated with vehicle or FSH ± EGF in serum-free MEM plus antibiotics for 6 h. After this initial treatment period, either 100 µM DRB or an equivalent amount of dimethyl sulfoxide (solvent) was added to the culture media as previously described [33]. Cells were lysed at 0, 1, 2, 4, and 6 h and RNA isolated as described above and subjected to reverse transcription and real-time PCR with StAR and S16 mRNA primers.

Cyclic AMP Accumulation Measurement

At the termination of transfection experiments, 1 ml of media was collected from each well and immediately stored at –20°C until assayed. The concentration of cyclic AMP in the medium was determined in duplicate using the cAMP ELISA kit (Oxford Biomedical Research Inc., Oxford, MI). Total extracellular cAMP in the medium was normalized for the protein concentration of cell lysate supernatants.

Transfection of Porcine Granulosa Cells

For transfection studies, a previously described vector containing a 1553-bp fragment of the porcine StAR gene promoter (–1423 to +130 bp) linked to a firefly luciferase reporter gene (p-1423StAR/luc) was used [29]. Transient transfections of these reporter constructs were carried out as previously described [29]. Briefly, transfection medium (1 ml/well) was prepared in serum-free MEM without antibiotics using 2 µg total plasmid DNA and 12 µl LipofectAMINE. Cells were cotransfected with the p-1423StAR/luc construct (1.95 µg) and renilla luciferase control vector ptk-RL/luc (0.05 µg). After a 5-h incubation with transfection mixture, the medium was replaced with serum-free MEM containing antibiotics and the indicated hormone treatment or vehicle and incubated an additional 6 h. The 6-h time point was found in preliminary experiments to give the maximal response to FSH (5 ng/ml). Several preliminary experiments were performed to confirm that the ptk-RL/luc internal control was unaffected by hormone treatments. At the end of the treatment period, cells were rinsed with Dulbecco PBS (D-PBS), lysed, and stored at –70°C until assayed. Luciferase activity in cellular lysates was measured using Promega Dual Luciferase Reporter Assay System and a Turner TD-20e luminometer (Turner Designs, Sunnyvale, CA). Firefly luciferase values were normalized to their corresponding renilla luciferase values.

Chromatin Immunoprecipitation Assay (ChIP)

After the initial attachment period and a subsequent 5-h incubation in serum-free medium, granulosa cells were treated with vehicle or FSH ± EGF in serum-free MEM plus antibiotics for 4 h. Two preliminary time-course experiments were performed at 2, 4, and 6 h for each antibody. Four-hour treatment showed the most consistent results and was chosen for the remainder of experiments. ChIP assays were performed using the Chromatin Immunoprecipitation Kit (Upstate Biotechnology, Inc., Lake Placid, NY) according to the manufacturer's instructions. Briefly, at the end of the treatment period, formaldehyde (1%) was added to the culture dishes for 10 min to cross-link DNA and associated proteins. Cells were washed once with D-PBS containing protease inhibitors (1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml pepstatin A) and scraped in 1 ml/well D-PBS solution. Cells were transferred into a microfuge tube and pelleted at 4°C. Cell pellets were stored at –80°C until further use. Pellets were resuspended in 1200 µl of ChIP SDS lysis buffer with protease inhibitors. The lysates were sonicated at 35% relative output, five times for 30 sec with 10-sec intervals with a Fisher Sonic Dismembrator Model 300 (Fisher Scientific) to generate 200- to 1000-bp DNA fragments (confirmed by agarose gel electrophoresis). Aliquots (200 µl) of the supernatant were used with each antibody tested and diluted 1:10 in ChIP dilution buffer containing protease inhibitors. Twenty microliters of each supernatant were saved as input DNA, diluted 1:10 in ChIP dilution buffer, and stored at –20°C. The chromatin solution was precleared with salmon sperm DNA/protein A 50% agarose slurry (Upstate Biotechnology, Inc.) for 30 min before overnight incubation with specific antibodies. Antibodies included rabbit anti-acetyl-histone H3 (lysines 9, 14) (1:2000, #06-599; Upstate Biotechnology) or rabbit anti-dimethyl-histone H3 (lysine 9) (1:2000, #07-52; Upstate Biotechnology). Parallel samples were incubated with an equivalent concentration of normal rabbit IgG (sc-2027; Santa Cruz Biotech, Santa Cruz, CA) as a negative control. For immunoprecipitation, salmon sperm DNA/protein A-agarose slurry was added and incubated for 1–2 h. The chromatin-antibody/protein A-agarose complexes were washed and chromatin eluted according to the manufacturer's instructions. NaCl (0.2 M) was added to eluted samples and input chromatin to reverse cross-links at 65°C for 4 h. The samples were then digested with proteinase K (40 µg/ml), extracted twice with phenol/chloroform, and precipitated. DNA was resuspended in 13 µl of sterile water and 2-µl aliquots were used for real-time PCR. Primers for ChIP analysis of the porcine StAR promoter corresponded to nt 1464–1442 and nt 1126–1148 of GenBank sequence AF038553 and were designed to amplify a 338-bp region spanning the proximal promoter region of the porcine StAR gene. Upstream primer (5'-CAGGCTGCTTGAGAACCACTGTC-3') was used at 300 nM and downstream primer (5'-CCTTCCTGAGCCTGTCAGAGTTG-3') at 900 nM with 2 µl of immunoprecipitated or input chromatin in real-time PCR as described above. Primers for ChIP analysis of the porcine CYP11A gene promoter corresponded to nt 2292–2268 and nt 2158–2133 of GenBank sequence L34259 and were designed to amplify a 160-bp region spanning the proximal promoter region of the porcine CYP11A gene previously shown to include the cAMP-responsive region that binds Sp1 [34]. Upstream primer (5'-CTGATCTCGTAGAACTGGAATGGTGG-3') and downstream primer (5'-CCAGCTTATAACCACCAGCTCAAGG-3') were used at 300 nM. The average (of triplicate wells) Ct for each treatment was used for quantification using the equation described above. For ChIP studies, samples were normalized for their corresponding input DNA values (instead of S16 mRNA) as described by Hiroi et al. [14].

SDS/PAGE and Immunoblot Analysis

Granulosa cells were treated as described above for ChIP assays. At the end of the 4-h treatment period, cells were rinsed with D-PBS, harvested in SDS lysis buffer (final concentration 150 mM Tris-HCl pH 8.75, 3% SDS), and boiled for 3 min. An aliquot of the protein extract was saved for quantification and the remaining lysate was adjusted to a final concentration of 30% glycerol, 15% ß-mercaptoethanol, and 0.04% bromophenol blue and stored at –70°C [35]. Protein concentrations were determined with the BCA protein assay kit (Pierce, Rockford, IL). Equivalent amounts (16 or 20 µg) of granulosa cell protein extracts were analyzed by 15% Tris-glycine or 16.5% Tris-tricine SDS-PAGE gels and electrotransferred onto polyvinylidene fluoride Hybond membranes (Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were blocked in 0.6 mg/ml gelatin in Tris-buffered saline containing 0.05% Tween-20. Primary antibodies were incubated in gelatin blocking solution overnight at 4°C. The antibodies used for ChIP assay were also used for immunoblotting and included rabbit anti-acetyl-histone H3 (lysines 9, 14) (1:3000) and rabbit anti-dimethyl-histone H3 (lysine 9) (1:2000). After washing, membranes were incubated with HRP-coupled goat anti-rabbit secondary antibody (1:3000; Zymed Lab, So. San Francisco, CA) in gelatin solution for 1 h. Immunoreactive bands were detected by enhanced chemiluminescence (Amersham). Membranes were stripped before reblocking and probed with rabbit anti-histone H3 (1:200, sc-10809; Santa Cruz Biotech) to evaluate total histone H3 per treatment. For immunoblot data, densitometric analyses of immunoreactive bands were performed using UnScan-It Gel software version 5.1 (Silk Scientific Corporation, Orem, UT). The optical density units for the modified histone immunoreactive bands were normalized for total histone H3 and expressed relative to the control treatment on the same blot.

Statistical Methods

Data are presented as the mean and SEM of three or more independent experiments. Normalized data were subjected to ANOVA. When significant, means were compared using the post hoc Tukey multiple comparison test. P < 0.05 was considered significant. To calculate messenger RNA half-life, all values were expressed relative to the time-zero control values. Half-life ranges were calculated by the equation t1/2 = 0.693/(Kd ± SEM) [33]. Statistical comparisons and slopes for the regression lines of mRNA half-life data were determined using GraphPad Prism 3.0 software (GraphPad Software, San Diego, CA). ELISA raw data were analyzed using the TableCurve 2D software (Systat Software, Point Richmond, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
EGF has been previously shown to suppress StAR mRNA but not CYP11A mRNA in luteinized porcine granulosa cells treated with cAMP analogue [24]. We wanted to confirm whether EGF was capable of inhibiting FSH-stimulated StAR mRNA in our less differentiated cell model. Preliminary studies were performed to determine the FSH and EGF concentrations and the time needed to observe an effect on StAR mRNA accumulation. Follicular fluid concentrations of EGF in different species have been reported to range from the low picograms per milliliter up to approximately 30 ng/ml [36, 37]. Our preliminary studies revealed 10 ng/ml EGF was the lowest dose exhibiting statistically significant inhibition of StAR mRNA in combination with FSH and it was also a maximal dose. Subsequent experiments used a submaximal dose of FSH (5 ng/ml) with maximal dose(s) of EGF (10 and/or 50 ng/ml). Total RNA was extracted from granulosa cells treated with vehicle, FSH, EGF (10, 50 ng/ml), or their combinations and subjected to reverse transcription and real-time PCR analysis. Relative StAR mRNA levels (normalized for corresponding S16 mRNA values) are shown in Figure 1A. Consistent with previous studies, FSH significantly (P < 0.05) stimulated StAR mRNA compared with vehicle control [7, 8]. StAR mRNA accumulation was significantly inhibited (62.8% and 52.2%, P < 0.05) by cotreatment with EGF (10 and 50 ng/ml, respectively) and FSH. EGF by itself did not affect StAR mRNA expression. Unlike the StAR mRNA, analysis of the mRNA for the CYP11A gene showed significant stimulation in the presence of FSH, EGF (10 ng/ml), and their combination (P < 0.05) when compared with vehicle control (Fig. 1B). The coaddition of EGF with FSH resulted in similar CYP11A mRNA expression compared with FSH alone and was less than additive.



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  		FIG. 1. FSH-stimulated StAR messenger RNA (mRNA) accumulation is inhibited by EGF. A) Total RNA from granulosa cells treated for 6 h with vehicle or FSH (5 ng/ml) alone or in the presence of EGF (10, 50 ng/ml) was reverse transcribed and subjected to real-time PCR analysis with oligonucleotide primers encompassing exon 4 of StAR cDNA as described in Materials and Methods. All values were corrected for S16 mRNA values and normalized to the FSH treatment. Data represent the mean + 1 SEM of four individual experiments. Letters indicate treatment is significantly different than the same treatment without FSH. Bars with different letters differ significantly, P < 0.05. B) Amplification of exon 2 of CYP11A cDNA was performed as described in (A). Data represent the mean + 1 SEM of three individual experiments. The letter c indicates treatments were significantly different from control but not each other, P < 0.05

In ovarian granulosa cells, addition of cAMP analogues stimulates StAR mRNA production [7, 8, 38, 39]. Moreover, FSH-stimulated StAR gene activity is associated with cAMP accumulation in the medium [29]. To determine whether the mechanism behind EGF inhibition of FSH-stimulated StAR mRNA accumulation was due to an upstream effect on FSH-stimulated cAMP levels, cAMP accumulation in the medium was measured by ELISA. Figure 2 shows levels of extracellular cAMP in the media of granulosa cells treated for 6 h with vehicle, FSH, EGF, or their combination. Basal extracellular cAMP accumulation was low and was significantly (P < 0.05) increased by FSH treatment. EGF by itself had no effect on cAMP accumulation. Coincubation of EGF with FSH did not reduce cAMP accumulation when compared with FSH alone.



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  		FIG. 2. EGF did not affect FSH-induced cAMP accumulation in porcine granulosa cells. Granulosa cell cultures treated for 6 h with vehicle (C), FSH (5 ng/ml, F), EGF (10 ng/ml, E), or their combination (F+E). Total extracellular cAMP in the medium was measured by ELISA and normalized for protein concentration as described in Materials and Methods. Data represent the mean + 1 SEM of three individual experiments normalized to FSH treatment. Asterisks indicate the treatments are significantly higher than treatments without FSH but not each other, P < 0.05

As our data demonstrated that EGF inhibits FSH-stimulated StAR mRNA accumulation, we investigated whether EGF increases the turnover of StAR mRNA when combined with FSH. Granulosa cells were treated for 6 h with vehicle, FSH, EGF, or their combination. DRB was added after the initial 6-h treatment (time 0) to suppress new mRNA synthesis for a period of at least 6 h, as determined previously [33]. Total RNA was collected at 0, 1, 2, 4, and 6 h after the initial treatment period, reverse transcribed, and subjected to real-time PCR analysis. As shown in Figure 3, the slopes of the decay curves for StAR mRNA in the presence of FSH alone and for FSH combined with EGF were similar. This data shows that EGF does not modify the half-life of FSH-stimulated StAR mRNA. Table 1 summarizes the half-life ranges determined for all treatments (in the presence of DRB).



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  		FIG. 3. EGF coincubation with FSH does not alter StAR mRNA stability. Granulosa cells were treated for 6 h with vehicle, FSH (5 ng/ml, F), EGF (10 ng/ml, E), or their combination (F+E). Only FSH and FSH+EGF data are shown. DRB was added after the initial 6-h treatment (time 0) to suppress new mRNA synthesis. Total RNA was collected at 0, 1, 2, 4, and 6 h. RNA was reverse transcribed and subjected to real-time PCR analysis with oligonucleotide primers encompassing exon 4 of StAR cDNA as described in Materials and Methods. All values were normalized for S16 mRNA values and normalized to the vehicle control (time 0). Lines represent linear regressions of data points derived from three individual experiments. The slopes of the linear regression lines do not significantly differ


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  		TABLE 1. StAR mRNA half-life

The abundance of hnRNA is indirectly representative of the rate of gene transcription [40]. To determine whether EGF suppression of the FSH-stimulated StAR mRNA accumulation might occur at the level of StAR transcription, StAR hnRNA was measured in primary porcine granulosa cell cultures treated for 6 h with vehicle, FSH, EGF, or their combination. Double-DNase treated total RNA was reverse transcribed and subjected to real-time PCR analysis. Primers for PCR analysis were designed to amplify both intron and exon portions (intron 4 and exon 5) of the StAR gene, which detects hnRNA abundance but not mRNA. Figure 4 shows StAR hnRNA levels in granulosa cell treated for 6 h. FSH alone significantly (P < 0.05) increased hnRNA levels. A significant decrease (P < 0.05) in FSH-stimulated StAR hnRNA was observed when EGF was coincubated with FSH.



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  		FIG. 4. EGF inhibits FSH-stimulation of StAR heterogenous nuclear (hn) RNA production. Total RNA from granulosa cells treated for 6 h with vehicle (C), FSH (5 ng/ml, F), EGF (10 ng/ml, E), or their combination (F+E). DNase-treated total RNA was reverse transcribed and subjected to real-time PCR analysis with primers amplifying a region spanning intron 4 and exon 5 of the StAR gene as described in Materials and Methods. All values were corrected for S16 mRNA values and normalized to the FSH treatment. Data represent the mean + 1 SEM of four individual experiments. The asterisk indicates that the treatment was significantly different from all other treatments, P < 0.05

Previous studies by our laboratory have demonstrated FSH stimulates reporter gene constructs containing the porcine StAR gene 5'-flanking region [29]. FSH activation of such constructs has been localized to adjacent GATA and C/EBP sites in the proximal promoter [12]. To evaluate whether EGF could suppress FSH-stimulated activation of the StAR promoter reporter gene vector, porcine granulosa cells were transiently cotransfected for 5 h with the p-1423StAR/luc and ptk-RL/luc vectors. After transfection, cells were treated for 6 h with vehicle, FSH, EGF, or their combination. Figure 5 shows FSH significantly (P < 0.05) stimulated the porcine StAR promoter construct, as expected. However, EGF (10 ng/ml) did not significantly affect FSH-stimulated promoter activity. In addition, EGF at 1 and 50 ng/ml did not significantly reduce FSH-stimulated promoter activity (n = 3, data not shown). We also preincubated cells with EGF (10 ng/ml) for 1 h before addition of FSH, but EGF still failed to inhibit FSH-driven promoter activity (n = 2, data not shown).



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  		FIG. 5. EGF does not significantly affect FSH-stimulation of the 1553-bp porcine StAR promoter construct in transient transfection assays. Porcine granulosa cells were cotransfected for 5 h with the p-1423StAR/luc and ptk-RL/luc constructs as described in Materials and Methods. After transfection, cells were treated for 6 h with vehicle (C), FSH (5 ng/ml, F), EGF (10 ng/ml, E), or their combination (F+E). Data represent the mean + 1 SEM of five individual transfection experiments. Asterisks indicate the treatments are significantly higher than treatments without FSH but not each other, P < 0.05

Transiently transfected promoter constructs do not necessarily replicate the native chromatin structure [41]. Because porcine granulosa cells cannot be stably transfected without changing their phenotype and responsiveness to FSH, it was not possible to explore the promoter construct under stably transfected conditions. To determine the role that chromatin remodeling, histones, and their posttranslational modifications play in the EGF response, ChIP assays were conducted. Preliminary time-course experiments at 2, 4, and 6 h were performed to determine the optimal treatment time of granulosa cell cultures with vehicle, FSH, EGF, or their combination. Four hours was found to yield the most consistent and reproducible results. Immunoprecipitated DNA was subjected to real-time PCR with primers amplifying an amplicon within the proximal region of the 5'-flanking region of the porcine StAR gene. Because recent studies have shown that, in some cellular settings, increased acetylated histone H3, a marker of transcriptional activity, can be associated with the proximal StAR promoter, and dimethylation of histone H3 at lysine 9, associated with StAR gene silencing, is decreased in other cellular settings, these modifications were evaluated [14]. Figure 6A shows that a 4-h treatment with FSH significantly (P < 0.05) stimulated acetylation of histone H3 on lysines 9 and 14 within the proximal StAR promoter and this effect was significantly inhibited by coincubation with EGF (P < 0.05). EGF alone did not significantly affect histone H3 acetylation. Figure 6A also shows that histone H3 associated with the known regulatory region of the CYP11A gene promoter [34] was similarly acetylated with all treatments. Because the primer efficiencies for the StAR and CYP11A promoter fragments were identical (efficiency = 2) and the CYP11A antiacetylated histone H3 precipitated DNA had lower Ct values than StAR during amplification relative to input we can conclude that the amount of basal acetylation associated with the CYP11A gene was higher than that for the StAR gene. Figure 6B shows that dimethylation of histone H3 on lysine 9 was not significantly affected by hormone treatments and that levels are similar to control. Use of normal rabbit IgG in place of the primary antibody showed no significant differences between treatments (n = 3, data not shown).



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  		FIG. 6. Evaluation of histone H3 modifications associated with the proximal region of the porcine StAR gene promoter. ChIP assays were performed using lysates isolated from primary cultures of granulosa cells treated for 4 h with vehicle (C), FSH (5 ng/ml, F), EGF (10 ng/ml, E), or their combination (F+E) as described in Materials and Methods. Real-time data for each treatment was normalized for its respective input DNA and all treatments were normalized to the FSH treatment. A) FSH stimulated acetylation of histone H3 on lysines 9 and 14 within the proximal StAR promoter, which was inhibited by coincubation with EGF (left). Results are presented as mean + 1 SEM, n = 4 experiments. The asterisk indicates treatment was significantly different from all other treatments, P < 0.05. The known regulatory region of the CYP11A gene was constitutively acetylated on H3 and unregulated by treatments (right). Results are presented as mean + 1 SEM, n = 3 experiments. B) Dimethylation of histone H3 on lysine 9 within the proximal StAR promoter was not significantly affected by hormone treatments. Results are presented as mean + 1 SEM, n = 6 experiments. C) Diagram showing the location of primers used for ChIP relative to the known FSH/cyclic AMP-regulated regions of the porcine StAR and CYP11A gene promoters. Numbers indicate position relative to the transcriptional start sites (+1)

We also tested histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and sodium butyrate (NaB) and DNA methyltransferase inhibitor 5-azacytidine (5-aza-C) [42] on their ability to modify basal and FSH/EGF effects on 6-h StAR mRNA accumulation. Preincubation for 1 h with NaB (0.1 mM) significantly (P < 0.001) increased the levels of FSH-stimulated StAR mRNA accumulation but not FSH plus EGF mRNA levels (Fig. 7). Higher NaB concentrations were actually inhibitory to treatments that elevated StAR mRNA (P < 0.05). In addition, pretreatment of granulosa cells with TSA (50 ng/ml) inhibited FSH-stimulated StAR mRNA accumulation (P < 0.05, n = 3, data not shown). No significant differences in StAR mRNA accumulation were found between treatments with or without 5-aza-C (0.1–10 µM; n = 3; data not shown).



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  		FIG. 7. Low concentrations of sodium butyrate (NaB, 0.1 mM) augment FSH-stimulated StAR mRNA accumulation whereas higher concentrations block hormone-stimulated activity. Total RNA from granulosa cells was preincubated with NaB for 1 h followed by a 6-h treatment with vehicle, FSH (5 ng/ml), EGF (10 ng/ml), or their combination, was reverse-transcribed, and subjected to real-time PCR analysis with oligonucleotide primers encompassing exon 4 of StAR cDNA as described in Materials and Methods. All values were corrected for S16 mRNA values and normalized to the FSH treatment (without NaB). Data represent the mean + 1 SEM for three individual experiments. The letter a indicates treatment was significantly higher than the same treatment without NaB (P < 0.001). The letter b indicates the treatment was significantly lower than the same treatment without NaB (P < 0.05)

To determine whether inhibition of FSH-stimulated histone H3 acetylation is accompanied by lower levels of acetylated histone H3 protein content in cells treated with the combination of FSH and EGF, we analyzed treated granulosa extracts by immunoblotting. Figure 8A shows the abundance of acetylated or dimethylated histone H3 and total histone H3 in treated granulosa cells. Figure 8B shows the results of densitometric analyses of immunoblot data. The optical densities for the modified histone H3 band were normalized to densitometric values obtained for total histone H3 antibody. No significant differences were observed between treatments in total cellular acetylated or dimethylated histone H3.



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  		FIG. 8. Total cellular histone H3 modifications in granulosa cells. Immunoblots of granulosa cells were treated for 4 h with vehicle (C), FSH (5 ng/ml, F), EGF (10 ng/ml, E), or their combination (F+E) as described in Materials and Methods. A) Granulosa whole-cell extracts (16–20 µg) were resolved by 16.5% Tris-tricine gels and probed with histone H3 acetylated at lysines 9 and 14 or dimethylated histone H3 at lysine 9 antibodies. The same membranes were stripped and reprobed with total histone H3 antibody. B) All immunoreactive bands (17 kDa) were analyzed by densitometry and values were corrected for total histone H3 antibody and normalized to vehicle control on the same blot. Acetylated and dimethylated histone H3 protein levels did not significantly differ among treatments. Results are presented as mean + 1 SEM, n = 4 experiments


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
EGF is well known to suppress FSH-stimulated granulosa cell differentiation in cultured porcine granulosa cells. One endpoint associated with luteinization is increased progesterone production, which is preceded by increases in StAR mRNA. This study has revealed a new mechanism by which EGF can control FSH-mediated porcine granulosa cell differentiation by suppressing StAR gene transcription at the level of chromatin.

Our initial data indicated that EGF attenuated FSH-stimulated StAR mRNA accumulation after 6 h of combined treatment in our porcine granulosa cell model. This effect was specific for StAR as the FSH-stimulated CYP11A message was not attenuated by coincubation with EGF. These results are consistent with data in luteinized porcine granulosa cells in which EGF (10 nM) eliminated the StAR response to cAMP analogue, dibutyryl cAMP after 6 h, whereas the CYP11A mRNA was not affected [24]. In addition, EGF alone did not affect StAR mRNA, whereas it stimulated CYP11A mRNA, also similar to previous reports in differentiating and luteinized porcine granulosa cells [24, 43]. By contrast, in mouse Leydig tumor cells, where EGF stimulates steroid synthesis, EGF is capable of stimulating StAR mRNA accumulation and acts additively with submaximal cAMP stimuli [44]. The divergent effects of EGF on StAR message in these steroidogenic cells are most likely due to their specific intracellular signaling pathways.

Under identical culture conditions, we previously demonstrated that FSH (5 ng/ml, NIDDK o-20) moderately increased cAMP accumulation in the medium with a 4-h treatment [29]. Consistent with this previous report, extracellular cAMP accumulation was significantly increased at the 6-h FSH treatment, and EGF did not modify cAMP levels. In the granulosa cells of most species, EGF itself does not increase cAMP levels [45], with the exception of rabbit granulosa cells, in which EGF elicited release of cAMP [46]. Cotreatment of EGF with FSH did not reduce FSH-stimulated cAMP accumulation. One study using granulosa cells from estradiol-treated rats showed EGF repression of progesterone synthesis was accompanied by reductions in cAMP formation; however, the same study found no differences in cAMP accumulation between FSH and FSH plus EGF between 1–12 h, consistent with our observations [47]. These data suggest that, with short-term (6-h) treatment, EGF repression of FSH-stimulated endpoints is not due to suppression of cAMP levels.

Although a report in rat granulosa cells showed EGF may suppress FSH receptor mRNA in the presence of FSH [48], studies in porcine granulosa cells have demonstrated increased FSH receptor binding with EGF treatment [49]. Thus, it is unlikely that EGF reduces FSH-stimulated StAR expression by decreasing FSH binding, and this is supported by data showing similar cAMP levels between FSH and FSH plus EGF and that FSH-stimulated CYP11A mRNA was not decreased by coincubation with EGF.

Changes in StAR mRNA accumulation can be the result of altered transcription, message stability, or both. In the present study, no difference in StAR mRNA half-life could be detected between FSH treatment alone or FSH plus EGF treatment. Our estimation of StAR half-life with FSH treatment (2.7–4.1 h) using DRB as an mRNA synthesis inhibitor was similar to the 3–3.5 h found for StAR mRNA half-life in basal and cAMP analogue-treated MA-10 cells in the presence of actinomycin D [50]. These studies support the concept that StAR mRNA turnover is not a major site for hormonal regulation.

Nascent or heterogenous nuclear RNA levels are an indirect measure of transcriptional activity [40]. In the present study, we showed that FSH stimulates StAR hnRNA abundance measured at 6-h treatment. Another study using cultured porcine thecal cells also showed LH stimulated StAR hnRNA abundance at the same time point [30]. Hiroi et al. also found that nascent StAR RNA was increased rapidly in hCG-primed mouse granulosa cells in vivo, a setting in which StAR mRNA increases rapidly as well [14]. In our cells, EGF by itself did not modify StAR hnRNA basal levels consistent with our observations for mRNA. Treatment of porcine granulosa cells with the combination of EGF and FSH significantly decreased the levels of FSH-stimulated StAR hnRNA, with a significant reduction similar to that found for mRNA (48% and 62%, respectively).

StAR hnRNA results supported the hypothesis that the inhibitory effect of EGF was transcriptional. The –1423-bp StAR gene promoter reporter construct has been previously shown to be activated by FSH and cis elements have been localized to the proximal region of the promoter [12, 29]. However, we were unable to measure an inhibitory effect of EGF on FSH-stimulation of the –1423-bp porcine StAR gene promoter construct in transiently transfected granulosa cells even with EGF preincubation. A possible explanation for these results was that EGF affects a region of the StAR promoter outside of the first 1423 bp of the 5'-flanking region of the StAR gene. This seemed unlikely, as most of the StAR promoter responsiveness to cAMP stimuli resides within the first 250 bp of the promoter for all species examined [12, 51]. Another explanation for the findings was that the transiently transfected promoter construct did not possess the in vivo chromatin structure, as previous studies have shown for other transiently transfected constructs [41]. In other words, histones proteins may not be appropriately associated with the promoter construct in our cell model.

Association of modified histones (through acetylation, methylation, or other posttranslational events) with chromatin can determine whether a gene is transcriptionally active or silent [52]. Quantitative ChIP assays are an important tool for analyzing interactions between histones and native chromatin [53]. Two studies have looked at the association of different proteins (including modified histones) with the StAR gene promoter or coding sequence in MA-10 Leydig or hCG-primed mouse granulosa cells [14, 15]. Our study is the first to demonstrate that FSH stimulation of StAR mRNA accumulation is associated with increased acetylation of histone H3 within the proximal StAR gene promoter. EGF reduced or blocked the ability of FSH to increase histone H3 acetylation in the proximal region of the endogenous StAR promoter. Acetylation of histone H3 at lysines 9 and 14 is linked to transcriptional activity [52]. FSH stimulation of lysines 9 and 14 histone acetylation associated with the proximal StAR promoter after 4 h of treatment is in agreement with data for the murine gene in MA-10 cells [14]. Hiroi et al. also saw that this modification occurs in MA-10 cells as early as 15 min after 8-Br-cAMP treatment and is maintained as well at later time points (2–4 h) [14]. This rapid modification in MA-10 cells is accompanied by significant increases in StAR mRNA seen within 30 min of treatment. In contrast, in preliminary studies, we did not detect significant histone H3 modification before 4 h of treatment and this may be due to the slower increase in StAR mRNA in our granulosa cells that is minimal at 2 h of FSH treatment (data not shown). Our data also contrast with in vivo data in granulosa cells from eCG-treated mice stimulated with hCG, that showed no changes in histone H3 acetylation compared with non-hCG treated cells. Several potential explanations for this difference exist, including that the mouse granulosa cells were exposed to hormone in an in vivo setting, unlike our cultured cells. Also, prior exposure of the mouse granulosa cells to eCG, which acts like an FSH stimulus to murine granulosa cells, may have already altered the chromatin structure. Additionally, the type of hormonal stimulus (FSH versus hCG) may differentially regulate histone H3. Most likely there are also species differences because treatment of macaques with an ovulatory dose of hCG to induce granulosa cell luteinization resulted in a dramatic increase in histone H3 acetylation compared with nonluteinized granulosa cells [15].

Unlike the StAR gene promoter, the CYP11A-regulated promoter region did not show any differences in the association of acetylated histone H3 in the region previously shown to be activated by forskolin [34]. Other studies have shown CYP11A mRNA is more abundant in cultured granulosa cells than StAR mRNA under basal conditions (vehicle treatment) [7, 54]. Although cAMP activators can stimulate the accumulation of both StAR and CYP11A transcripts in cultured granulosa cells, a fundamental difference in their expression occurs in the granulosa cells of developing follicles. CYP11A mRNA is detected by Northern blot in granulosa cells isolated from medium and preovulatory porcine follicles, yet StAR mRNA is not detectable by this method [7, 17, 18], suggesting differences in histone H3 acetylation could account in part for differential expression of these messages in vivo.

Dimethylation at lysine 9 of histone H3 is associated with gene silencing by binding of repressor proteins that lead to the formation of transcriptionally inactive heterochromatin [52]. EGF alone or in combination with FSH did not significantly influence dimethylation at lysine 9 of histones associated with the porcine StAR gene promoter. Our vehicle-treated control dimethylated histone H3 levels tended to be higher than other treatments, but not significantly. Lack of alterations in dimethylated histone H3 at lysine 9 associated with the proximal StAR gene promoter was also observed in MA-10 Leydig cells under the same conditions in which histone H3 acetylation was increased [14]. Collectively, the majority of data show cAMP-mediated activation of the StAR gene is associated with increased histone H3 acetylation, but can in certain cellular contexts be transcriptionally derepressed by decreases in histone H3 lysine 9 dimethylation.

HDAC inhibitors, such as sodium butyrate, would be expected to potentiate mRNA accumulation by facilitating transcription through stabilizing acetylated histones [55]. NaB (0.1 mM) significantly increased the levels of FSH-stimulated StAR mRNA accumulation, supporting our ChIP data that FSH alters histone H3 acetylation within the StAR gene. However, higher NaB concentrations or TSA were actually inhibitory to treatments that elevated StAR mRNA. A similar effect of HDAC inhibitors was observed in cardiomyocyte responses to hypertrophic stimuli [56]. Because these inhibitors block several HDAC classes, they may affect other genes that need to be silenced during the granulosa cell differentiation process requisite for StAR gene expression. Aza-C did not modify StAR mRNA levels, consistent with our ChIP data and immunoblot data, suggesting that dimethylation is not a factor in StAR promoter inactivation in our model. However, these inhibitors are not selective for a particular gene and data must be interpreted with caution. In addition, these inhibitors may be unreliable for implicating histone acetylation or methylation in the regulation of StAR mRNA.

Our immunoblot data demonstrated that FSH, EGF, or their combination did not promote a detectable change in bulk levels of acetylated or dimethylated histone H3 compared with control. A similar pattern was reported for histone H3 acetylation after FSH treatment in rat granulosa cells, even when regional acetylation of histone H3 within several gene promoters could be measured by ChIP assays [57]. These studies support the idea that total cellular levels of histone H3 acetylation are fairly constant and increases in histone H3 acetylation are restricted to target genes.

EGF stimulates proliferation and inhibits differentiation in porcine granulosa cells [22]. The ability of EGF to inhibit steroidogenesis and differentiation is dependent on the presence or absence of other factors in the granulosa cell microenvironment, such as FSH, LH, or other growth factors [58–60]. Our present study shows EGF probably inhibits FSH-stimulated steroidogenesis through repression of StAR, the rate-limiting step in de novo steroid synthesis in granulosa cells. EGF repression of StAR mRNA accumulation was demonstrated to be at the level of chromatin, specifically a reduction in FSH-meditated histone H3 acetylation within the proximal region of the StAR gene promoter. EGF did not affect the transiently transfected promoter construct that harbors the GATA-4 and C/EBPß binding sites shown in part to mediate FSH activation of this construct, indicating EGF probably doesn't affect the ability of these trans-acting factors to associate with naked or relaxed DNA [12]. However, in the endogenous promoter, histone deacetylation by EGF may affect the accessibility of these factors to StAR gene DNA. Whether EGF inhibits FSH-stimulated histone acetylases or EGF stimulates histone deacetylases will require further study. In addition, further study will be needed to determine if signaling cross-talk between EGF and FSH signaling pathways occurs downstream of cAMP, such as via the mitogen-activated protein kinase pathway [61]. In conclusion, although our in vitro studies suggest EGF is an excellent candidate for suppressing StAR mRNA expression in the porcine follicle (in an FSH environment) before the LH surge, in vivo experiments are needed to confirm these studies.


   ACKNOWLEDGMENTS
 
We thank Carolina Gillio-Meina and Allison Benoit for help with granulosa primary cultures.


   FOOTNOTES
 
1 Supported in part by NIH grant HD-38945 and the University of South Carolina School of Medicine Research Development fund. Back

2 Correspondence: Department of Cell and Developmental Biology and Anatomy, School of Medicine, Building 1, University of South Carolina, Columbia, SC 29209. FAX: 803 733 3212; hlavoie{at}med.sc.edu Back

Received: 9 July 2004.

First decision: 27 July 2004.

Accepted: 16 November 2004.


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 DISCUSSION
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K.N. Fru, M. Cherian-Shaw, M. Puttabyatappa, C.A. VandeVoort, and C.L. Chaffin
Regulation of granulosa cell proliferation and EGF-like ligands during the periovulatory interval in monkeys
Hum. Reprod., May 1, 2007; 22(5): 1247 - 1252.
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R. G Lea, P. Wooding, I. Stewart, L. T Hannah, S. Morton, K. Wallace, R. P Aitken, J. S Milne, T. R Regnault, R. V Anthony, et al.
The expression of ovine placental lactogen, StAR and progesterone-associated steroidogenic enzymes in placentae of overnourished growing adolescent ewes
Reproduction, April 1, 2007; 133(4): 785 - 796.
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 J Mol EndocrinolHome page
H. Duan and C. R Jefcoate
The predominant cAMP-stimulated 3.5 kb StAR mRNA contains specific sequence elements in the extended 3'UTR that confer high basal instability
J. Mol. Endocrinol., January 1, 2007; 38(1): 159 - 179.
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