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Endocrinology, doi:10.1210/en.2003-0940
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Endocrinology Vol. 145, No. 2 799-808
Copyright © 2004 by The Endocrine Society

Valproate Potentiates Androgen Biosynthesis in Human Ovarian Theca Cells

Velen L. Nelson-DeGrave, Jessica K. Wickenheisser, Jennifer E. Cockrell, Jennifer R. Wood, Richard S. Legro, Jerome F. Strauss, III and Jan M. McAllister

Departments of Cellular and Molecular Physiology (V.L.N.-D., J.K.W., J.E.C., J.M.M.) and Obstetrics and Gynecology (R.S.L.), Pennsylvania State College of Medicine, Hershey, Pennsylvania 17033; and Center for Research on Reproduction and Women’s Health (J.R.W., J.F.S.), University of Pennsylvania, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Jan M. McAllister, Ph.D., Department of Cellular and Molecular Physiology, Pennsylvania State Hershey College of Medicine, 500 University Drive, C4723, Hershey, Pennsylvania 17033. E-mail: jmcallister{at}psu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In patients with epilepsy, treatment with valproate (VPA) has been reported to be associated with polycystic ovary syndrome-like symptoms including weight gain, hyperandrogenemia, and hyperinsulinemia. We examined the effect of VPA on androgen biosynthesis in ovarian theca cells isolated from follicles of normal cycling women to determine whether the hyperandrogenemia reported with VPA treatment could be a result of direct effects of VPA on the ovary. In long-term cultures of theca cells treated for 72 h with sodium valproate (30–3000 µM), we observed an increase in basal and forskolin-stimulated dehydroepiandrosterone (DHEA), androstenedione, and 17{alpha}-hydroxyprogesterone production compared with control values. In contrast, low doses of VPA treatment (i.e. 30–300 µM) had no effect on basal and forskolin-stimulated progesterone production, whereas higher doses of VPA (1000–3000 µM) inhibited progesterone production. The most pronounced effect of VPA on androgen biosynthesis was observed in the dose range of 300-3000 µM, which represent therapeutic levels in the treatment of epilepsy and bipolar disorder. Western analyses demonstrated that VPA treatment increased both basal and forskolin-stimulated P450c17 and P450scc protein levels, whereas the amount of steroidogenic acute regulatory protein was unaffected. In transient transfection studies, VPA was found to increase P450 17{alpha}-hydroxylase and P450 cholesterol side-chain cleavage promoter activity, whereas steroidogenic acute regulatory protein promoter activity was unaffected. Consistent with the ability of VPA to act as a histone deacetylase (HDAC) inhibitor in other cell systems, VPA (500 µM) treatment was observed to increase histone H3 acetylation and P450 17{alpha}-hydroxylase mRNA accumulation. The HDAC inhibitor butyric acid (500 µM) similarly increased histone H3 acetylation and DHEA biosynthesis, whereas the VPA derivative valpromide (500 µM), which lacks HDAC inhibitory activity, had no effect on histone acetylation or DHEA biosynthesis. These data suggest that VPA-induced ovarian androgen biosynthesis results from changes in chromatin modifications (histone acetylation) that augment transcription of steroidogenic genes. These studies provide the first biochemical evidence to support a role for VPA in the genesis of polycystic ovary syndrome-like symptoms, and establish a direct link between VPA treatment and increased ovarian androgen biosynthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VALPROIC ACID (VPA) is a short-chained fatty acid with antimanic properties that is widely used to treat epilepsy and bipolar disorders as well as migraines and generalized mood disorders (1, 2, 3). Long-term treatment of women with VPA has been reported to be associated with a polycystic ovary syndrome (PCOS)-like syndrome, which includes polycystic ovaries, hyperandrogenism, weight gain, insulin resistance, and increased levels of circulating insulin (3, 4, 5, 6, 7, 8). Although many investigators have recognized the association between VPA treatment and PCOS-like symptoms, both clinical and basic science studies examining the effects of VPA treatment on reproductive function are, for the most part, limited and conflicting. Although some investigators have proposed that seizure disorders predispose women to PCOS through neuroendocrine dysfunction (9, 10), other investigators have suggested that only women that have, or are predisposed to, PCOS respond inappropriately to VPA treatment (11, 12).

Whereas a number of investigators have used rodent, porcine, and monkey model systems to examine the effect of VPA treatment on ovarian androgen production and follicular development (13, 14, 15, 16), we found no reports in the literature that have focused on the direct effects of VPA on human ovarian or adrenal androgen biosynthesis. Although VPA is widely used in clinical practice, its mechanisms of action and the molecular basis of its teratogenicity are not well understood. Potential mechanisms for VPA-dependent changes in cellular function include alterations in protein kinase C activity and isozyme expression (17), inhibition of glycogen synthase kinase 3{alpha} and 3ß activity, increases in activator protein-1 binding activity and ß-catenin levels (18, 19), and induction of mitogen activated kinase and neurite cell growth (19). VPA has also been more recently reported to act as a histone deacetylase (HDAC) inhibitor (20, 21). However, the cellular mechanism by which VPA might induce a PCOS-like phenotype in women has not been examined.

As a consequence of examining theca cells propagated from normal cycling women and women with PCOS, we have previously established that increased androgen production is a stable phenotype of PCOS theca cells. This stable increase in androgen production results from selective alterations in the expression of P450 17{alpha}-hydroxylase (CYP17) and P450 cholesterol side-chain cleavage (CYP11A) while not affecting other components of the steroid biosynthetic pathway such as steroidogenic acute regulatory protein (StAR) gene expression (22, 23, 24). In these studies, we investigated the extent that VPA treatment affects human thecal CYP17, CYP11A, and STAR gene expression and overall steroid biosynthesis. We evaluated whether normal and PCOS theca cells differentially respond to VPA treatment. We also examined whether changes in histone acetylation are involved in VPA-augmented androgen synthesis. As a consequence of these studies, we hoped to gain important new information about the mechanisms underlying VPA-induced increases in ovarian androgen biosynthesis, which could be used to examine whether there is a disruption of these pathways in patients with PCOS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Theca cell isolation and propagation
Human theca interna tissue was obtained from follicles of women undergoing hysterectomy under a protocol approved by the Institutional Review Board of the Pennsylvania State University College of Medicine. Individual follicles were dissected away from ovarian stroma. The isolated follicles were size selected for diameters ranging from 3 to 5 mm so that theca cells derived from follicles of similar size from normal and PCOS subjects could be compared. The dissected follicles were placed into serum-containing medium and bisected. Under a dissecting microscope, the theca interna was stripped from the follicle wall, and the granulosa cells were removed with a platinum loop. The cleaned theca shells were dispersed with 0.05% collagenase I, 0.05% collagenase IA, and 0.01% deoxyribonuclease in medium containing 10% fetal bovine serum (FBS) (25). Dispersed cells were placed in culture dishes that had been precoated with fibronectin by incubation at 37 C with culture medium containing 5 µg/ml human fibronectin. The growth medium used was a 1:1 mixture of DMEM and Hams F-12 medium containing 10% FBS, 10% horse serum, 2% UltroSer G, 20 nM insulin, 20 nM selenium, 1 µM vitamin E, and antibiotics. From each follicle, 12 35-mm dishes of primary theca interna cells were grown until confluent, removed from the dish with neutral protease (pronase-E, protease type XXIV; Sigma, St. Louis, MO) in DMEM-F12 (1:1), frozen, and stored in liquid nitrogen (one 35-mm dish per vial) in culture medium that contained 20% FBS and 10% dimethylsulfoxide (DMSO). In all experiments, cells were thawed and propagated in the growth medium described above. To obtain successive passages of normal and PCOS theca cells, cells were thawed, propagated, and frozen at consecutive passages. The cells were grown in 5% O2, 90% N2, and 5% CO2. Reduced oxygen tension and supplemental antioxidants (vitamin E and selenium) were employed to prevent oxidative damage.

The PCOS and normal ovarian tissue came from age-matched women, 38–40 yr old. The diagnosis of PCOS was made according to established guidelines (26, 27), including hyperandrogenemia; oligoovulation; and the exclusion of 21-hydroxylase deficiency, Cushing’s syndrome, and hyperprolactinemia. All of the PCOS theca cell preparations studied came from ovaries of women with fewer than six menses per year and elevated serum total testosterone or bioavailable testosterone levels, as described previously (22, 27). Each of the PCOS ovaries contained multiple subcortical follicles of less than 10 mm in diameter. The control (normal) theca cell preparations came from ovaries of fertile women with normal menstrual histories, menstrual cycles of 21–35 d, and no clinical signs of hyperandrogenism. Neither PCOS nor normal subjects were receiving hormonal medications at the time of surgery. Indications for surgery were dysfunctional uterine bleeding, endometrial cancer, and pelvic pain. The passage conditions and split ratios for all normal and PCOS cells were identical. Experiments comparing PCOS and normal theca were performed using fourth-passage (31–38 population doublings) theca cells isolated from size-matched follicles obtained from age-matched subjects. Sera and growth factors were obtained from the following sources: FBS and DMEM/F12 were obtained from Irvine Scientific (Irvine, CA): horse serum was obtained from Hyclone (Logan, UT), UltroSer G was from Reactifs IBF (Villeneuve-la-Garenne, France), and other compounds were purchased from Sigma.

Steroid biosynthesis
For evaluation of steroid production, theca cells were grown until subconfluent and transferred into serum-free medium in the presence or absence of forskolin (20 µM) with and without varying concentrations of VPA. At 72 h the media were collected, and RIAs for dehydroepiandrosterone (DHEA), {Delta}4-androstenedione ({Delta}4A), 17{alpha}-hydroxyprogesterone (17OHP4), and progesterone (P4) were performed without organic solvent extraction using Diagnostic Products (Los Angeles, CA) and ICN Biochemicals (Irvine, CA) RIA kits as described previously (22).

Western analysis
Fourth-passage theca cells were grown until subconfluent and transferred into serum-free medium with and without forskolin (20 µM) and/or VPA (500 µM) for 48 h. After treatment, theca cells were harvested in ice-cold modified radioimmunoprecipitation assay buffer (30 mM Tris, 150 mM NaCl, 50 mM NaF, 0.5 mM EDTA, 0.5% deoxycholic acid, 1.0% Nonidet P-40, and 0.1% sodium dodecyl sulfate) containing 1 mM sodium orthovanadate, 0.5 mM phenylmethylsufonyl fluoride, 1 mM dithiothreitol, 1.0 mM benzamidine, 1 µM microcystin, 2 µg/ml leupeptin, and 2 µg/ml pepstatin A. Protein concentration was determined using a DC protein assay (Bio-Rad Laboratories, Hercules, CA). Whole-cell lysates were separated on a 10% SDS-PAGE and transferred to polyvinyl difluoride membrane, and Western analysis was performed. StAR, , P450 side-chain cleavage enzyme (P450scc), and P450c17 antisera were used as previously reported (28, 29). P450scc and P450c17 antisera were generously provided by Dr. Walter Miller (University of California, San Francisco, San Francisco, CA). All autoradiograms were scanned and densitometric analysis was performed using Image Quant version 1.2 (Molecular Dynamics, Sunnyvale, CA). Data were normalized using antisera specific to the 70-kDa subunit of DNA-dependent protein kinase (Ku70) from Santa Cruz Biotechnology (Santa Cruz, CA) (30). Acetylated (K9, K14) histone H3 (acetyl-H3) and histone H3 (H3) antisera were obtained from Upstate Biotechnology (Lake Placid, NY). The amount of H3 acetylation was normalized to total histone, as described previously (31, 32).

Transient transfection analysis
Subconfluent cultures of theca cells were transfected with reporter gene constructs as we have previously described (24) using the modified calcium-phosphate method of Graham and Eb (33). One hour before transfection, the cells were transferred into DMEM high-glucose medium containing 20 mM HEPES and 2% heat inactivated calf serum, and moved to a 3% CO2, 95% ambient air 37 C incubator. DNA/Ca2P04 solution containing 20 µg reporter plasmid and 1 µg pSV-bgal/100-mm dish in HEPES phosphate buffer was added to the media. After incubation for 6 h, cells were transferred into 2% calf serum in DMEM containing 20 mM HEPES and treated as described, and 72 h after forskolin treatment, the cells were harvested using trypsin/EDTA, pelleted, and resuspended in reporter lysis buffer for luciferase assays. Luciferase assays were performed using the luciferase assay system (Promega, Madison, WI). ß-Galactosidase activities were determined by Galacto-Light Plus chemiluminescent assay (Tropix, Bedford, MA) and used to normalize luciferase activities.

Quantitation of CYP17 and StAR mRNA
For quantitative real-time PCR, total mRNA was isolated (22) from fourth-passage theca cells that were grown to subconfluence, transferred into serum-free medium, and treated as indicated. RNA (1 µg) samples were then reverse transcribed using oligo (dT), and 200 U Stratascript reverse transcriptase (Stratagene, La Jolla, CA). CYP17 mRNA abundance was determined by quantitative real-time PCR using a CYP17-specific fluorescent probe (5' 6-FAM-TCGCGTCCAACAACCGTAAGGGTATC-3' BHQ-1) (Biosearch Technologies, Novato, CA). CYP11A mRNA abundance was determined using a CYP11A-specific fluorescent probe (5'-6-FAM-TCCACCTTCACCATGTCCAGAATTTCCA-3' BHQ-1). StAR mRNA abundance was determined using a StAR-specific fluorescent probe (5' 6-FAM-CGGAGCTCTCTACTCGGTTCTCGGC-3' BHQ-1). The abundance of TATA-box binding protein (TBP) mRNA was also determined for each cDNA sample using a TBP-specific fluorescent probe (5' JOE-TGTGCACAGGAGCCAAGAGTGAAGA-3' BHQ-1) (Biosearch Technologies). Briefly, 1 µl of each cDNA sample described above was combined with 300 nM CYP17-specific forward (5'-GGCCTCAAATGGCAACTCTAGA-3') and CYP17-specific reverse (5'-CTTCTGATCGCCATCCTTGAA-3') primers and 300 nM TBP-specific forward (5'-CACGGCACTGATTTTCAGTTC-3') and TBP-specific reverse (5'-TCTTGCTGCCAGTCTGGACT-3') primers and the reagents from the Brilliant QPCR kit (Stratagene) in a final volume of 50 µl. The same cDNA (1 µl) in a separate reaction was used to quantitate the StAR mRNA using 300 nM StAR-specific forward primer (5'-CCACCCCTAGCACGTGGAT-3') and a StAR-specific reverse primer (5'-TCCTGGTCAC TGTAGAGTCTCTTC-3').

To quantitate CYP11A mRNA, we used 300 nM CYP11A-specific forward primer (5'-GAGGGAGACGGGCACACA-3') and CYP11A-specific reverse primer (5'-TGACATAAACCGACTCCACGTT-3'). The gene-specific two-step PCR was performed in triplicate for each cDNA sample and serial diluted cDNA standards in an Mx4000 thermocycler (Stratagene), using the Mx4000 multiplex quantitative PCR system according to the manufacturer’s instructions. An arbitrary value of template was assigned to each serial dilution (i.e. 1000, 300, 100, 30, 10, 3, 1) and plotted against the cycle threshold value (y-axis = cycle threshold; x-axis = value, log scale) to generate a standard curve. Each unknown was assigned an arbitrary value based on the slope and y-intercept of the standard curve. The same process was performed for TBP, which was used to normalize each reaction. The mean target value for each unknown was divided by the mean TBP value for each unknown to generate a normalized value for the target for each sample. The average normalized value and SE for each target was determined using data from the normal samples with and without VPA for each time point used in the experiment.

Statistical analysis
Each experiment was performed using triplicate dishes. After combining the results from individual experiments, nonparametric Mann-Whitney U tests (i.e. control vs. VPA; forskolin vs. forskolin + VPA) were performed using PRISM 4.0 (SAS Institute, Inc., Cary, NC). P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effects of VPA on DHEA, {Delta}4A, 17OHP4, and P4 production
To examine the effects of VPA on steroid biosynthesis, fourth-passage theca cells were treated with increasing concentrations of VPA (1–3000 µM) in the presence or absence of a maximal dose of forskolin (20 µM) for 72 h. The effects of VPA treatment on basal and forskolin-stimulated DHEA, {Delta}4A, 17OHP4, and P4 production are shown in Fig. 1Go. The data presented are the cumulative results obtained from theca cells isolated from four different normal patients. Both basal and forskolin-stimulated DHEA and {Delta}4A production were increased in response to VPA treatment (30–3000 µM). Both basal and forskolin-stimulated 17OHP4 production were also increased after treatment with 30–3000 µM VPA, compared with control values in the absence of VPA. At a concentration of 300 µM VPA, forskolin-stimulated 17OHP4 accumulation was observed to be maximal, whereas treatment with VPA at concentrations above 1000–3000 µM resulted in the subsequent decline in 17OHP4 accumulation. In contrast, lower doses of VPA (30–100 µM) had no effect on basal or forskolin-stimulated P4 production, whereas concentrations of VPA in the 30–3000 µM range inhibited both basal and forskolin-stimulated P4 accumulation.



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  		FIG. 1. The effects of VPA on DHEA, {Delta}4A, 17OHP4, and P4 production. DHEA, {Delta}4A, 17OHP4, and P4 production was examined in fourth-passage theca cells that were grown until subconfluent and transferred into serum-free medium containing an increasing concentration of VPA (1.0–3000 µM) with and without 20 µM forskolin. After 72 h of treatment, the media were collected, steroid production was evaluated by RIA, and the data were normalized to cell number. Results are presented as the mean ± SEM of steroid levels from triplicate theca cell cultures from four patients.

 
VPA augments P450c17 and P450scc but not StAR protein
To examine the direct effects of VPA treatment on P450c17, P450scc, and StAR protein levels fourth-passage theca cells were grown to subconfluent and treated with and without a therapeutically relevant dose of 500 µM sodium VPA (10, 34) in the presence and absence of a maximal dose of forskolin (20 µM) for 48 h. Western analysis was performed as described in Materials and Methods. In Fig. 2AGo, we present representative data showing the effects of VPA treatment on basal and forskolin-stimulated P450c17, P450scc, and StAR abundance in theca cells isolated from a single patient. In Fig. 2BGo, cumulative data from four different patients was normalized by Ku70 and is presented as percentage increase over control values. Treatment of fourth-passage theca cells with 20 µM forskolin for 48 h augmented immunoreactive P450c17, P450scc, and StAR about 2-fold, about 5–10-fold, and about 2-fold, respectively, above control values (Fig. 2Go). VPA (500 µM) treatment augmented both basal and forskolin-stimulated P450c17 and P450scc abundance about 2-fold but had no effect on either basal or forskolin-stimulated StAR.



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  		FIG. 2. The effects of VPA on basal and forskolin-stimulated P450c17, P450scc, and StAR protein abundance. Immunoblot analysis of P450c17, P450scc, and StAR protein in whole-cell lysates (35 µg/lane) harvested from fourth-passage theca cells that were grown until subconfluent and treated for 48 h with vehicle (C) or 20 µM forskolin (F) with and without 500 µM VPA. Antibodies specific for human P450c17, P450scc, and StAR were used. Data were normalized to Ku70. The data shown in A are representative of those obtained from theca cells isolated from a single patient. In B, cumulative data from four different patients are presented as percentage increase over control values. P450c17 abundance was increased in response to VPA treatment under basal (a; P < 0.05) and forskolin-stimulated (b; P < 0.05) conditions.

 
VPA enhances CYP17 and CYP11A but not STAR gene expression
To evaluate the effects of VPA treatment on CYP17, CYP11A, and StAR mRNA accumulation, real-time quantitative PCR analysis was performed. Total RNA (1 µg) was isolated from fourth-passage theca cells that were grown to subconfluence and transferred into serum-free medium in the presence or absence of 20 µM forskolin, with and without 500 µM VPA for 24 h. In agreement with our previous findings, forskolin treatment coordinately increases CYP17, CYP11A, and StAR mRNA (Fig. 3Go). VPA treatment was observed to increase both basal and forskolin-stimulated CYP17 and CYP11A mRNA accumulation 3-fold and 5-fold, respectively. In contrast, VPA treatment had no significant effect on basal or forskolin-stimulated StAR mRNA accumulation.



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  		FIG. 3. The effects of VPA on basal and forskolin-stimulated CYP17, CYP11A, and StAR mRNA accumulation. CYP17, CYP11A, and StAR mRNA abundance was evaluated in fourth-passage theca cells that were grown until subconfluent and transferred into serum-free medium with vehicle (C) or 20 µM forskolin (F) with and without 500 µM VPA for 24 h. All data were obtained using quantitative real-time PCR analysis. mRNA accumulation was normalized by TBP mRNA abundance for each sample and is depicted graphically as the mean ± SEM of these values. CYP17 and CYP11A mRNA abundance were increased in response to VPA treatment under basal (a; P < 0.05) and forskolin (b; P < 0.05) stimulated conditions.

 
To investigate whether VPA treatment affected CYP17, CYP11A, and StAR promoter activities, fourth-passage theca cells were transiently transfected with reporter constructs containing either -235/+44 bp of the human CYP17 promoter, -2327/+49 bp of the CYP11A promoter, or -885/+39 bp of the human StAR promoter. The effects of VPA (500 µM) on basal and forskolin-stimulated CYP17, CYP11A, and StAR promoter function were examined in theca cells isolated from three different patients. CYP17, CYP11A, and StAR promoter function were increased about 2-fold in response to forskolin treatment (Fig. 4Go). VPA treatment enhanced both basal and forskolin-stimulated CYP17 and CYP11A promoter activity about 2-fold (Fig. 4Go), whereas StAR promoter function was unaffected (Fig. 4Go). Combined, these data demonstrate that VPA treatment selectively increases CYP17 and CYP11A gene expression.



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  		FIG. 4. Effect of VPA on basal and forskolin-stimulated CYP17, CYP11A, and StAR promoter activity. To examine the effects of VPA on basal and forskolin-stimulated promoter regulation, fourth-passage theca cells were transiently transfected with 20 µg/dish of a pGL3 reporter plasmid containing -235 to +44 bp of the CYP17 promoter (-235 CYP17), -2327 to +49 bp of the CYP11A promoter (-2327 CYP11A), or the pGL2 reporter plasmid containing -885 to +39 bp of the StAR promoter (-885 StAR). After transfection, the cells were treated with vehicle (C) or 20 µM forskolin (F) with and without 500 µM of VPA. Then 72 h later the cells were harvested and luciferase (LUC) activity assayed. Data are presented as relative LUC activity that has been corrected for ß-galactosidase activity. Data represent the mean ± SEM of experiments performed with triplicate cultures of theca cells isolated from four normal patients. CYP17 and CYP11A promoter function was increased in response to VPA treatment under basal (a; P < 0.05) and forskolin (b; P < 0.05) stimulated conditions.

 
The effects of VPA treatment on steroid biosynthesis in normal and PCOS theca cells
To evaluate whether normal and PCOS theca cells differentially respond to VPA treatment, VPA-stimulated DHEA, {Delta}4A, 17OHP4, and P4 biosynthesis was examined under basal and forskolin-stimulated conditions (Fig. 5Go). In agreement with our previously published data, both basal and forskolin-stimulated DHEA (35), 17OHP4, and P4 (22) production was increased about 10-fold in PCOS theca cells, compared with normal theca cells. In contrast, in PCOS theca cells, {Delta}4A synthesis was increased about 25-fold under basal conditions and about 100-fold after forskolin treatment, compared with normal theca cells. Both basal and forskolin-stimulated DHEA and {Delta}4A production were significantly increased in response to VPA treatment in both normal and PCOS theca cells. The magnitude of VPA-stimulated DHEA and {Delta}4A production were also markedly increased in PCOS theca, compared with normal theca, cells. We also observed that VPA treatment enhanced both basal and forskolin-stimulated 17OHP4 production in normal theca cells. However, in PCOS theca cells, we observed that VPA treatment increased basal 17OHP4 production and had no effect on forskolin stimulated 17OHP4 production. In addition, VPA treatment had no effect on basal or forskolin-stimulated P4 production in normal theca cells, whereas in PCOS theca cells, VPA treatment inhibited forskolin-stimulated P4 biosynthesis. The observed decrease in P4 production in PCOS theca cells more than likely results from an increase in CYP17 gene expression and the flux of steroids down the {Delta}5 steroid pathway. Based on the observed increase in VPA-stimulated {Delta}4A production in both normal (Figs. 1Go and 5Go) and PCOS (Fig. 5Go) theca cells, it does not appear that VPA inhibits 3ß-hydroxysteroid dehydrogenase enzyme activity/expression. These data suggest that VPA treatment increases androgen biosynthesis in women with and without PCOS. Furthermore, the increased magnitude in VPA-stimulated DHEA and {Delta}4A production in PCOS theca cells, compared with normal cells, suggests that VPA treatment may have more adverse effects in women with PCOS.



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  		FIG. 5. The effects of VPA on steroid biosynthesis in normal and PCOS theca cells. To compare the effect of VPA treatment on overall steroid biosynthesis normal and PCOS theca cells, VPA-stimulated DHEA, {Delta}4A, 17OHP4, and P4 biosynthesis was examined. Fourth-passage theca cells isolated from normal and PCOS women were treated in the presence or absence (C) of 20 µM forskolin (F) with and without VPA (500 µM). After 72 h of treatment, the media were collected, and DHEA, {Delta}4A, 17OHP4, and P4 production was evaluated by RIA, and the data were normalized to cell number. Results are presented as the mean ± SEM of steroid levels from triplicate theca cell cultures from five independent normal and four independent PCOS patients. DHEA, {Delta}4A, and 17OHP4 production were increased in response to VPA treatment under basal (a; P < 0.05) and forskolin (b; P < 0.05) stimulated conditions.

 
VPA increases histone H3 acetylation
There are data in the literature to suggest that VPA-induced changes in cellular function result from the inhibition of histone deacetylase(s) (20, 21). To investigate whether VPA treatment affects HDAC activity in ovarian theca cells, time-course experiments were performed to examine H3 acetylation. Fourth-passage theca cells were grown to subconfluence and transferred into serum-free medium in the presence or absence of VPA (500 µM) for 3, 6, 12, 24, and 48 h, and Western analysis was performed using-whole cell lysates. Acetyl-H3 was normalized by H3. Valproate-stimulated H3 acetylation was observed to increase above control values after 1 h of treatment, and maximal stimulation was observed after 6–12 h (Fig. 6AGo). In conjunction with these studies, replicate cultures of treated cells were also used to examine the time course of VPA-induced CYP17 mRNA accumulation using quantitative real-time PCR analysis. VPA-stimulated CYP17 mRNA accumulation was first observed to increase between 12 and 24 h, and maximal stimulation was achieved between 24 and 48 h (Fig 6BGo). CYP11A mRNA accumulation was similarly increased in response to VPA treatment, whereas StAR mRNA accumulation was not affected (data not shown).



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  		FIG. 6. The time course of VPA-induced histone H3 acetylation and CYP17 mRNA accumulation. A, Immunoblot analysis of histone H3 acetylation in whole-cell extracts (35 µg/lane) harvested from fourth-passage theca cells treated with 500 µM VPA ({blacktriangledown}) or vehicle ({square}) for 1, 3, 6, 12, and 24 h. The data presented were normalized to histone H3 and are presented as relative abundance. B, CYP17 mRNA abundance in normal theca cells cultured for 3, 9, 12, 24, and 48 h in the absence ({square}) or presence ({blacktriangledown}) of 500 µM VPA as determined by quantitative real-time PCR. CYP17 mRNA levels were normalized by TBP mRNA levels for each sample and are depicted graphically as the mean ± SEM of normalized values.

 
Western analysis was performed to compare basal and forskolin-stimulated acetyl-H3 after treatment with VPA; butyric acid, a known HDAC inhibitor; and valpromide, a VPA derivative that lacks HDAC inhibitory activity. As shown in Fig. 7Go, 24 h treatment of theca cells with either VPA (500 µM) or butyric acid (500 µM) resulted in a more than 10-fold increase in acetyl-H3. In contrast, acetyl-H3 was unaffected after treatment with forskolin or valpromide. Similar changes in histone H4 acetylation were also observed (data not shown).



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  		FIG. 7. The effects of VPA, butyric acid, and valpromide on histone H3 acetylation. Immunoblot analysis of histone H3 acetylation in whole-cell extracts (35 µg/lane) harvested from fourth-passage theca cells that were grown to subconfluence and treated with vehicle, 500 µM VPA, 500 µM valpromide, or 500 µM butyric acid in the presence (+) and absence (-) of 20 µM forskolin for 24 h.

 
To determine whether increases in H3 acetylation are associated with increased androgen biosynthesis, we compared the effect of 500 µM VPA, butyric acid, and valpromide on DHEA, {Delta}4A, and P4 production. In agreement with our H3 data, treatment with either VPA or butyric acid increased both basal and forskolin-stimulated DHEA production, whereas valpromide or DMSO treatment had no effect (Fig. 8Go, A and B). DMSO was used as the vehicle for valpromide treatment and had no effect on DHEA, {Delta}4A, or P4 production. Both basal and forskolin-stimulated {Delta}4A production were increased in response to VPA treatment; however, butyric acid had no statistical effect on {Delta}4A production (Fig. 8CGo). However, the lack of an effect of butyrate on {Delta}4A production may have been a consequence of insufficient substrate (i.e. DHEA). In agreement with the data presented in Fig. 1Go, P4 biosynthesis was not significantly increased in response to VPA, butyric acid, or valpromide treatment (Fig. 8CGo). Oleic acid and isobutyric acid had no effect on either basal or forskolin-stimulated 17OHP4, DHEA, {Delta}4A, or P4 accumulation at similar concentrations (data not shown).



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  		FIG. 8. Comparison of the effects of VPA, butyric acid, and valpromide on steroid biosynthesis. DHEA, {Delta}4A, and P4 production was examined in fourth-passage theca cells that were grown until subconfluent and transferred into serum-free medium containing 500 µM VPA, 500 µM valpromide, and 500 µM butyric acid in the presence (F) and absence (C) of 20 µM forskolin. DMSO was used as the vehicle for valpromide. Then 72 h after treatment, the media were collected and steroid production was evaluated by RIA. Data were normalized to cell number and are presented as the mean ± SEM from triplicate theca cell cultures that are representative of multiple patients. A, DHEA production in the presence and absence of forskolin. B, Basal DHEA production presented on an expanded axis. C, {Delta}4A production. D, P4 production. Both basal (a; P < 0.05) and forskolin-stimulated (b; P < 0.05) DHEA production was increased in response to VPA and butyric acid treatment. Forskolin-stimulated {Delta}4A synthesis was increased in response to VPA treatment (b; P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Very few studies have focused on the effect of VPA treatment on normal-cycling women and the potential contraindications of VPA treatment in reproductive age patients beyond the known teratogenic actions. Studies examining the effects of long-term VPA treatment in a number of animal species have yielded variable results. In the rat reproductive tract, prolonged VPA treatment has been reported to result in the formation of ovarian follicular cysts and a reduced number of corpora lutea (13). However, in the rat, VPA treatment resulted in a decline in overall androgen and P4 biosynthesis. In isolated pig follicles, in vitro treatment with VPA has been reported to result in an increased androgen to estrogen ratio (15). However, the results of these and subsequent studies using intact porcine follicles and isolated cells, showed that overall androgen production was in fact decreased in response to VPA treatment, and the observed increase in the T/E ratio resulted from a decrease in granulosa cell aromatase activity (15, 16). In monkeys treated with VPA for an extended period, investigators observed no changes in follicular development and/or androgen biosynthesis (14). It is interesting to note that these studies were performed using a dose of VPA 10-fold higher than that used to treat humans because of the observation that monkeys have a 10-fold higher metabolic clearance rate for VPA, as compared to humans (14).

The data presented in this article establish that human theca cells have the capacity to respond to a therapeutically relevant concentration of VPA with an increase in androgen biosynthesis and CYP17 and CYP11A gene expression (10, 34). VPA is one of the few compounds we have identified that can markedly potentiate steroidogenic enzyme expression as well as DHEA and {Delta}4A production to levels significantly higher than those observed after maximal treatment with forskolin. The findings that VPA augments steroidogenic enzyme expression under basal nonstimulated conditions as well as in the presence of a maximal saturating concentration of the adenylate cyclase activator, forskolin, are consistent with the notion that VPA treatment may convert normal theca cells to a PCOS phenotype. The observation that VPA treatment has no effect on StAR gene expression is also consistent with our previous data demonstrating that there are no differences in StAR expression in normal and PCOS theca cells maintained in long-term culture (22, 24). Although most of the responses we observed after VPA treatment of normal theca cells were consistent with those characteristic of PCOS theca cells in long-term culture (23, 24), one inconsistency we observed was that high doses of VPA treatment did not enhance P4 biosynthesis and in fact inhibited P4 production (Figs. 1Go and 5Go). The decrease in P4 production in theca cells at higher doses of VPA (Fig. 1Go) most likely results from an increase in CYP17 gene expression, flux of steroids down the {Delta}5 steroid pathway (i.e. conversion of pregnenolone to 17{alpha}-hydroxypregnenolone and DHEA), and subsequent conversion of DHEA to {Delta}4A via 3ß-hydroxysteroid dehydrogenase (23, 24).

Although many investigators are examining the genetic basis for PCOS, an equal number of investigators have recognized that PCOS-like symptoms may be manifested in response to environmental cues, such as prenatal exposure to androgens and weight gain (36, 37). Investigators have reported that there is an increased frequency of reproductive disorders in patients with epilepsy (1, 11). Thus, it is possible that this population of patients is more likely to be treated with VPA, and as a consequence investigators observed a higher incidence of PCOS-like symptoms. However, the finding that VPA increases steroid biosynthesis in theca cells isolated from both normal-cycling and PCOS patients suggests that VPA treatment could independently induce PCOS-like symptoms in the absence of a genetic predisposition for PCOS. The observation that PCOS theca cells respond more robustly to VPA treatment with an increase in DHEA and {Delta}4A production (Fig. 5Go) suggests that VPA treatment can further augment androgen biosynthesis in patients with PCOS. In view of our data as well as data demonstrating that frequency and/or amplitude of GnRH-stimulated gonadotrophin secretion is unaffected in response to VPA treatment (38), it appears that VPA increases ovarian androgen biosynthesis directly, rather than affecting hypothalamic pituitary function. It is also interesting to note the VPA treatment results in weight gain and that in a population of women with no clinical signs of PCOS, long-term VPA treatment was observed to be associated with annovulation and hyperandrogenemia, both of which were more prevalent in obese women (34, 39, 40).

The intracellular mechanisms by which VPA induces a PCOS-like phenotype in epileptic and bipolar patients have not been examined. In attempt to develop new modes of treatment for these diseases, a number of investigators have examined VPA signaling in mouse hippocampal cells and human neuroblastoma cells (17, 18). Although these studies suggest that a variety of signaling cascades may be involved in VPA action, no one has examined the mechanisms underlying VPA-induced changes in steroid biosynthesis. Because VPA affects human reproductive cells differently than those of other animal species, one may speculate that the mechanisms underlying VPA-stimulated androgen biosynthesis may be species specific. The finding that human theca cells robustly respond to VPA treatment has provided a novel human cell system to begin to examine the signaling components involved in VPA-stimulated androgen biosynthesis and the mechanisms by which VPA differentially regulates CYP17 and CYP11A gene expression at the transcriptional and posttranscriptional level.

In agreement with the studies of Phiel et al. (20) and Gottlicher et al. (21), both VPA and the HDAC inhibitor, butyric acid, increased histone H3 acetylation and histone H4 acetylation (data not shown), suggesting that VPA-dependent changes in steroid biosynthesis may result from changes in HDAC activity and modification of chromatin structure, which enhances transcription of genes encoding steroidogenic enzymes. Consistent with these findings is the observation that VPA and butyric acid enhance DHEA production, whereas the VPA analog, valpromide, which lacks HDAC inhibitory activity, has no effect. Although we present data demonstrating that VPA increases histone acetylation after 1 h of treatment, CYP17 mRNA accumulation did not increase until 12 h of treatment, suggesting that an additional intermediary factor(s) may be needed for induction of transcription of the CYP17 gene. In addition, the finding that forskolin treatment did not affect histone H3 acetylation, suggests that the mechanism(s) underlying VPA- and forskolin-stimulated androgen biosynthesis are distinct and may also involve changes in both transcriptional and posttranscriptional regulation.

The data presented here demonstrate that prolonged VPA treatment could directly affect ovarian steroid biosynthesis in women with and without PCOS. At present, the mechanisms underlying increased weight gain and insulin resistance in patients undergoing long-term VPA treatment are unknown. Furthermore, the effects of VPA treatment on other PCOS-like characteristics including endometrial hyperplasia and hyperlipidemia require examination. We anticipate that future examination of the changes in overall gene expression that underlie VPA-induced increases in ovarian androgen biosynthesis will provide valuable information about the network of genes or signaling components that are affected so that new modes of treatment that do not affect these pathways can be developed. We also anticipate identifying common pathways that underlie PCOS-like symptoms that will provide new approaches to PCOS therapy.


   Acknowledgments
 
We thank Dr. Sergei Grigoryev (Department of Biochemistry and Molecular Biology, Pennsylvania State College of Medicine, Hershey, PA) for expertise in histone H3 acetylation. We also thank Dr. Kathyrn LaNoue (Department of Cellular and Molecular Physiology, Pennsylvania State College of Medicine) for helpful input regarding branched chain amino acids and acknowledge Karen Hendricks and Allison Wolfe for technical expertise.


   Footnotes
 
This work was supported by NIH Grants HD34449 (to J.F.S., J.M.M., and R.S.L.), HD33852 (to J.M.M.), T32HD07305 (to J.R.W.), and T32GM08619 (to J.E.C.).

Abbreviations: {Delta}4A, {Delta}4-Androstenedione; acetyl-H3, acetylated histone H3; CYP11A, P450 cholesterol side-chain cleavage; CYP17, P450 17{alpha}-hydroxylase; DHEA, dehydroepiandrosterone; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; H3, histone H3; HDAC, histone deacetylase; Ku70, 70-kDa subunit of DNA-dependent protein kinase; 17OHP4, 17{alpha}-hydroxyprogesterone; P4, progesterone; PCOS, polycystic ovary syndrome; P450scc, P450 side-chain cleavage enzyme; StAR, steroidogenic acute regulatory protein; TBP, TATA-box binding protein; VPA, valproate.

Received July 24, 2003.

Accepted for publication October 16, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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