Alterations in Mitogen-Activated Protein Kinase Kinase and Extracellular Regulated Kinase Signaling in Theca Cells Contribute to Excessive Androgen Production in Polycystic Ovary Syndrome

Velen L. Nelson-Degrave, Jessica K. Wickenheisser, Karen L. Hendricks, Tomoichiro Asano, Midori Fujishiro, Richard S. Legro, Scot R. Kimball, Jerome F. Strauss, III and Jan M. McAllister

Departments of Cellular and Molecular Physiology (V.L.N.-D., J.K.W., K.L.H., S.R.K., J.M.M.) and Obstetrics and Gynecology (R.S.L., J.M.M.), Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; the Department of Diabetes and Metabolism (T.A., M.F.), Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan; and the Center for Research on Reproduction and Women’s Health (J.F.S.), University of Pennsylvania, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Jan M. McAllister, Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine 500 University Drive H166, Hershey, Pennsylvania 17033. E-mail: jmcallister{at}psu.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have investigated the involvement of the MAPK signaling pathway in increased androgen biosynthesis and CYP17 gene expression in women with polycystic ovary syndrome (PCOS). A comparison of MAPK kinase (MEK1/2) and ERK1/2 phosphorylation in propagated normal and PCOS theca cells, revealed that MEK1/2 phosphorylation was decreased more than 70%, and ERK1/2 phosphorylation was reduced 50% in PCOS cells as compared with normal cells. Infection with dominant-negative MEK1 increased CYP17 mRNA and dehydroepiandrosterone (DHEA) abundance, whereas constitutively active MEK1 reduced DHEA production and CYP17 mRNA abundance. Similarly, the MEK inhibitor, PD98059, increased CYP17 mRNA accumulation and CYP17 promoter activity to levels observed in PCOS cells. Remarkably, in theca cells maintained in the complete absence of insulin, ERK1/2 phosphorylation was decreased in PCOS theca cells as compared with normal theca cells, and CYP17 mRNA and DHEA synthesis were increased in PCOS theca cells. These studies demonstrate that in PCOS cells reduced levels of activated MEK1/2 and ERK1/2 are correlated with increased androgen production, irrespective of the insulin concentration. These findings implicate alterations in the MAPK pathway in the pathogenesis of excessive ovarian androgen production in PCOS.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
POLYCYSTIC OVARY SYNDROME (PCOS) is a disorder that affects approximately 5–10% of reproductive aged women and is characterized by excess androgen production and infertility (1, 2, 3). The presence of an elevated level of circulating free testosterone, primarily from increased production of androgens by the ovaries, is the classical endocrine phenotype of women with PCOS (2). There is general agreement that the ovarian theca cell is the primary source of excess androgen biosynthesis in women with PCOS (4, 5, 6). Theca cells express a variety of genes encoding components of the steroidogenic pathway that are involved in androgen biosynthesis, in response to the pituitary gonadotropin, LH (7). Of these, the expression of the cytochrome P450 17{alpha}-hydroxylase (CYP17) gene, which encodes a single cytochrome P450 (P450c17) with both 17{alpha}-hydroxylase and C17, 20 lyase activities, is essential for the production of androgens in theca cells (8).

We have previously reported that basal and forskolin-stimulated androgen production is elevated in theca cells isolated from the ovaries of women with PCOS and propagated for successive population doublings (9, 10). We established that increased CYP17 gene transcription, mRNA accumulation, and associated increases in P450 17{alpha}-hydroxylase/C17,20 lyase activity are stable characteristics of PCOS theca cells that persist in long-term culture, and thus do not appear to be a consequence of the hormonal milieu that the cells were exposed to in vivo (9, 10, 11, 12).

To examine the molecular mechanism(s) involved in increased androgen biosynthesis and CYP17 gene expression in women with PCOS, we have begun to investigate specific components of the MAPK signaling cascade in ovarian theca cells propagated from normal cycling women and women with PCOS. Although regulation of steroidogenic enzyme expression in theca cells has been largely attributed to LH-dependent increases in adenylate cyclase, more recent studies have provided evidence that alternative signaling pathways, including the MAPK and protein kinase B (AKT) pathways, are associated with LH-induced changes in steroid biosynthesis (13). Similar studies have also demonstrated a role for MAPK signaling in FSH-induced steroid biosynthesis and steroidogenic acute regulatory protein (STAR) gene expression in granulosa cells (14, 15, 16).

The MAPKs are mediators of signal transduction from the cytosol to the nucleus (17). MAPKs are all proline-directed, serine-threonine kinases that are phosphorylated (i.e. activated) on threonine and tyrosine in response to a wide variety of stimuli, including cytokines, growth factors, hormones, cellular stress, and cell adherence (18, 19, 20). The signals that link G protein-coupled receptors to the MAPK pathway are complex and may involve the Ras pathway as well as other convergent protein kinase cascades (21). The Ras/MAPK kinase (MEK)/ERK pathway is an important signaling cascade involved in the control of cell proliferation and differentiation. Activation of the ERK1/2 or p42/44 MAPK signaling cascade results from the phosphorylation and activation of MEK1/2 (21). Activation of the ERK pathway stimulates the expression and activity of a number of transcription factors, including members of the Jun, and Fos families, in a cell type-specific manner (17, 20, 22, 23). Pharmacological inhibitors of MEK1 [i.e. PD98059 (PD) and U0126], which prevent the activation of ERK1/2 by MEK1/2, have been widely used throughout the literature to examine the role of the ERK1/2 pathway in cellular signaling and differentiation (21, 24, 25).

Although some investigators have suggested that cAMP-stimulated activation of the MEK/ERK signaling cascade augments ovarian steroid biosynthesis (13, 15), others have demonstrated that inhibition of the MEK/ERK signaling cascade is associated with increased steroid biosynthesis (16, 26). In human adrenocortical H295 cells, a reduction in the activation state of the ERK1/2 has also been associated with increased CYP17 gene expression (27, 28). In the present study, we have begun to evaluate the extent that components of the MEK/ERK signaling cascade may be involved in increased androgen biosynthesis and CYP17 gene expression in normal and PCOS theca cells. We compared the phosphorylation states of MEK1/2 and ERK1/2 in theca cells propagated from multiple normal and PCOS patients. We used an adenovirus-mediated transfection/infection procedure to over express both the dominant-negative and constitutively active forms of these MEK mutants in theca cells to evaluate the effects of the resultant state of MAPK activation on androgen biosynthesis and CYP17 mRNA abundance. We also examined the effects of the pharmacological MEK1/2 inhibitor, PD, on CYP17 gene transcription. Because insulin resistance and hyperinsulinemia in PCOS have been hypothesized to contribute to increased androgen production by the ovary, we also examined the extent to which insulin contributes to ERK1/2 phosphorylation in normal and PCOS theca cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Phosphorylation of MEK1/2 and ERK1/2 Is Reduced in PCOS Theca Cells
To investigate whether there are differences in MEK/ERK signaling in normal and PCOS theca cells maintained in long-term culture, we compared the phosphorylation states of MEK1/2 and ERK1/2 in fourth-passage theca cells. We examined both the phosphorylated and total forms of MEK1/2 (Fig. 1Go) and ERK1/2 (Fig. 2Go) by Western blot analysis in cells that were treated with and without forskolin (20 µM) for 24 h. Our analysis of whole cell lysates harvested from theca cells isolated from five independent normal and PCOS patients, showed that the phosphorylation state of MEK1/ 2 was decreased more than 70% in PCOS theca cells as compared with normal theca cells (Fig. 1Go). We observed a 50% reduction in the phosphorylation state of ERK1/2 in PCOS cells as compared with normal cells (Fig. 2Go). ERK1/2 phosphorylation was observed to be decreased in response to forskolin treatment in both normal and PCOS theca cells.



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Fig. 1. The Activation State of MEK1/2 Is Decreased in PCOS Theca Cells as Compared with Normal Theca Cells

Fourth-passage theca cells propagated from normal and PCOS patients were grown to confluence and transferred into serum-free medium with vehicle (C) or 20 µM forskolin (F). 24 h thereafter the cells were harvested, and immunnoblot analysis was performed using 35 µg of whole cell extract and antibodies specific for phosphorylated MEK1/2 (Phospho-MEK 1/2) and total MEK. Upper panel, Representative immunoblot data of whole cell extracts prepared from theca cells isolated from normal and PCOS patients. Bottom panel, Quantitation of the activation state of MEK1/2 in theca cells isolated from five independent normal and PCOS patients is presented. Both basal (a, P < 0.05) and forskolin (b, P < 0.05)-stimulated MEK1/2 phosphorylation was decreased in PCOS theca cells as compared with normal cells (*, P < 0.05).

 


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Fig. 2. The Activation State of ERK1/2 Is Decreased in PCOS Theca Cells as Compared with Normal Theca Cells

Fourth-passage theca cells propagated from normal and PCOS patients were grown to confluent and transferred into serum-free medium with vehicle (C) or 20 µM forskolin (F). Twenty-four hours thereafter, the cells were harvested, and immunoblot analysis was performed using 35 µg of whole cell extract and antibodies specific for phosphorylated ERK1/2 (phospho ERK1/2) and total ERK1/2 (ERK1/2). Upper panel, Representative immunoblot data of whole cell extracts prepared from theca cells isolated from normal and PCOS patients. Bottom panel, Quantitation of the activation state of ERK1/2 in theca cells isolated from five independent normal and PCOS patients is presented. Forskolin treatment resulted in the inhibition of ERK1/2 phosphorylation in both normal and PCOS theca cells. Both basal (a, P < 0.05) and forskolin (b, P < 0.05)-stimulated ERK1/2 phosphorylation was decreased in PCOS theca cells as compared with normal cells.

 
MEK and ERK Regulate Thecal Androgen Synthesis
To determine whether the MEK and ERK signaling pathways influence thecal androgen biosynthesis, an adenoviral transfection/infection system was used to examine the effects of a dominant-negative MEK1 molecule (DN-MEK1) (29) on dehydroepiandrosterone (DHEA) production and CYP17 mRNA accumulation in normal theca cells. In these experiments, a half-maximal dose of 7.5 µM forskolin was used to examine possible stimulatory effectors (9). As shown in Fig. 3AGo, there was a dose-dependent increase in DHEA biosynthesis in response to infection with DN-MEK1 under both basal and forskolin-stimulated conditions, as compared with a control adenovirus expressing ß-galactosidase (ß-Gal). At a dose of 10–30 plaque forming-units (pfu) of DN-MEK1, which was found to maximally stimulate DHEA biosynthesis 5-fold over forskolin-stimulated conditions, we observed a 50% reduction in ERK1/2 phosphorylation under both basal and forskolin-stimulated conditions (Fig. 3BGo). We also observed an approximately 2-fold increase in total ERK1/2 after infection with DN-MEK1/2, which effectively reduced the phosphorylation state of ERK1/2. In parallel studies, infection with 10–30 pfu of DN-MEK1 adenovirus significantly increased both basal and forskolin-stimulated CYP17 mRNA abundance (Fig. 3CGo) and 17{alpha}-hydroxylase enzyme activity approximately 2-fold (Fig. 3DGo).



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Fig. 3. Inhibition of MEK/ERK Pathway Using a Dominant-Negative MEK1 Adenovirus Augments Thecal Androgen Biosynthesis, 17{alpha}-Hydroxylase Enzyme Activity, and CYP17 mRNA Expression

A, To examine the effect of increasing amounts of an adenovirus encoding DN-MEK on thecal androgen biosynthesis, fourth-passage theca cells were grown until subconfluent and were infected with increasing concentrations (3–300 pfu) of either DN-MEK adenovirus or a control ß-Gal. Seventy-two hours after treatment with and without forskolin (7.5 µM), the media were collected, and DHEA production was evaluated by RIA. Data were normalized to cell number and results are presented as the mean ± SEM of steroid levels from triplicate theca cell cultures. B, To examine ERK1/2 phosphorylation, fourth-passage theca cells were infected with 10 pfu of either a recombinant adenovirus expressing as a control (ß-Gal) or DN-MEK1 and treated with and without 7.5 µM forskolin for 24 h. Thirty-five micrograms of protein isolated from whole cell lysates were subjected to SDS-PAGE and then immunoblotted with antiphospho-ERK (Thr202/Tyr204) antibody. Representative immunoblots from four independent experiments are shown. C, In parallel studies to those performed above, at 24 h CYP17 mRNA abundance was evaluated using quantitative real-time PCR analysis. mRNA accumulation was normalized by TBP mRNA abundance and is depicted graphically as the mean ± SEM. Infection with a DN-MEK1 adenovirus increased both basal (a, P < 0.05) and forskolin- (b, P < 0.05) stimulated CYP17 mRNA abundance. D, 17{alpha}-Hydroxylase enzyme activity was evaluated after infection with DN-MEK or ß-Gal adenovirus followed by treatment with and without 7.5 µM forskolin for 72 h, as described in Materials and Methods. Infection with a DN-MEK1 adenovirus increased both basal (a, P < 0.05) and forskolin- (b, P < 0.05) stimulated 17{alpha}-hydroxylase enzyme activity.

 
We used an adenovirus expressing constitutively active MEK1 (CA-MEK1) to examine whether an increase in ERK1/2 activity could reduce DHEA synthesis and CYP17 mRNA accumulation. These studies required using a different adenoviral vector system than that used for DN-MEK1 (Fig. 3Go) (30) and therefore required using a LacZ control adenovirus. These experiments were performed with a maximal stimulatory dose of 20 µM forskolin so that we could more fully examine the possible inhibitory effects of the MEK1 signaling pathway. As shown in Fig. 4Go (upper panel), infection of theca cells with CA-MEK1 resulted in an increase in ERK1/2 phosphorylation and a 50% reduction in total ERK1/2. Both basal and forskolin-stimulated DHEA production and CYP17 mRNA abundance were significantly decreased more than 95% and more than 70%, respectively, after infection with CA-MEK1 as compared with control LacZ adenovirus (Fig. 4Go, lower and middle panels). These data demonstrate that activation of ERK1/2 directly inhibits thecal CYP17 mRNA accumulation and androgen biosynthesis. Similar results were obtained after infection of PCOS theca cells with CA-MEK1 (data not shown).



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Fig. 4. Constitutively Active MEK Stimulates ERK Phosphorylation and Inhibits DHEA and CYP17 mRNA Abundance

Fourth-passage theca cells were infected with a recombinant adenovirus containing LacZ (control) or CA-MEK1 and treated with and without 20 µM forskolin. Upper panel, Twenty-four hours after treatment, the cells were harvested and 35 µg of protein isolated from whole cell lysates were subjected to SDS-PAGE and then immunoblotted with anti-phospho-ERK (Thr202/Tyr204) antibody. Representative immunoblots from four independent experiments are shown. Middle panel, Seventy-two hours after treatment, the media were collected and DHEA 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 at least four experiments. Infection with CA-MEK1 inhibited both basal (a, P < 0.05) and forskolin-stimulated (b, P < 0.05) DHEA production. Bottom panel, Fourth-passage theca cells were infected with a recombinant adenovirus containing LacZ or CA-MEK1 and treated with and without 20 µM forskolin. At 24 h, the cells were harvested and CYP17 abundance was evaluated using quantitative real-time PCR analysis. mRNA accumulation was normalized by TBP mRNA abundance and is depicted graphically as the mean ± SEM. Infection with a CA-MEK1 adenovirus inhibited both basal (a, P < 0.05) and forskolin (b, P < 0.05) stimulated CYP17 abundance.

 
To investigate whether inactivation of ERK1/2 signaling directly affects CYP17 gene expression, we examined the effects of the MEK/ERK pathway-specific pharmacological inhibitor, PD (24) on CYP17 mRNA abundance and promoter function. Evaluation of CYP17 mRNA abundance after PD treatment demonstrated a time-dependent increase in CYP17 mRNA abundance, which was consistent with the results observed after infection with DN-MEK1 (Fig. 3Go). CYP17 mRNA abundance increased approximately 2-fold in response to 24 h of PD treatment (Fig. 5Go, upper panel). Experiments were performed to assess the effects of PD treatment on DHEA production, however in agreement with the studies of Munir et al. (31), we found that PD treatment of theca cells either had no effect or mildly inhibited 17{alpha}-hydroxylase enzyme activity as well as DHEA or 17{alpha}-hydroxyprogesterone production. From our analysis, it appears that unlike DN-MEK1, PD treatment may nonspecifically inhibit some component of the cytochrome P450 complex.



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Fig. 5. Inhibition of the ERK1/2 Pathway with the MEK1 Inhibitor PD Augments CYP17 mRNA Accumulation and Promoter Function

Upper panel: Fourth-passage theca cells were treated with and without the MEK1/2 inhibitor PD (25 µM). At 4, 8, 16, 24, and 48 h, the cells were harvested and CYP17 abundance was evaluated using quantitative real-time PCR analysis. mRNA accumulation was normalized by TBP mRNA abundance and is depicted graphically as the mean ± SEM. Bottom panel: Fourth-passage theca cells were transiently transfected with a pGL3 LUC construct containing –750 to +44 bp of the CYP17 promoter (–750 CYP17/LUC). After transfection, the cells were treated with an increasing concentration of PD (0.03–30 µM) with and without a submaximal dose of forskolin (7.5 µM). Seventy-two hours thereafter, the cells were harvested and LUC activity assayed. Data are presented as relative LUC activity that has been corrected for ß-Gal activity. Data represent the mean ± SEM of experiments performed with triplicate cultures of theca cells isolated from four normal patients. Inhibition of ERK1/2 signaling using the MEK1 inhibitor PD augmented basal mRNA accumulation and both basal and forskolin-stimulated –750 CYP17/LUC promoter activity in a time and dose-dependent manner (P < 0.05).

 
To examine the effects of PD on the transcriptional regulation of the CYP17 gene, normal theca cells were transiently transfected with a luciferase (LUC) reporter plasmid containing –750/+44 bp of the 5' promoter of the human CYP17 gene. After transfection, the cells were treated with increasing concentrations of PD (0.03–30.0 µM) in the presence and absence of a half-maximal dose of forskolin (7.5 µM). As shown in Fig. 5Go (lower panel), PD treatment stimulated both basal and forskolin-stimulated CYP17 promoter activity in a dose-dependent manner with an ED50 of approximately 0.5 µM. These experiments were performed in triplicate cultures of theca cells isolated from four independent normal patients. Similar results were observed with the MEK1/2 inhibitor U0126 (data not shown) (25).

Inhibition of the ERK1/2 MAPK Pathway with the MEK1 Inhibitor, PD, Differentially Regulates CYP17 Promoter Activity in Normal and PCOS Theca Cells
In view of our data demonstrating that the phosphorylation states of MEK1/2 and ERK1/2 are decreased in PCOS theca cells compared with normal theca cells, we investigated whether pharmacological inhibition of ERK1/2 using PD had similar or differential effects on CYP17 promoter regulation in normal and PCOS theca cells. In these experiments, normal and PCOS theca cells propagated from five independent normal and PCOS patients were transiently transfected with a pGL3 LUC construct containing –750 to +44 bp of the CYP17 promoter (–750 CYP17/LUC). After transfection, the cells were treated in the presence or absence of a half-maximal dose of forskolin (7.5 µM), with and without PD (25 µM). In agreement with our preliminary data presented in Fig. 6Go (upper panel), both basal and forskolin-stimulated CYP17 reporter activity were increased in PCOS theca cells as compared with normal theca cells (11). Treatment of normal cells with PD resulted in an approximately 3-fold increase in basal, and a more than 2-fold increase in forskolin-stimulated reporter activity. In contrast, in PCOS theca cells PD treatment resulted in a less than 1.5-fold increase in both basal and forskolin-stimulated reporter activity. These data demonstrate that CYP17 gene expression in normal theca cells involves direct regulation by the ERK pathway. In effect, PD treatment of normal theca cells increased CYP17 promoter activity to the levels observed in PCOS theca cells. In agreement with these findings, in parallel studies PD treatment was observed to inhibit ERK1/2 phosphorylation in normal theca cells to a greater extent than in PCOS theca cells (Fig. 6Go, lower panel).



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Fig. 6. Inhibition of the ERK1/2 MAPK Pathway with the MEK1 Inhibitor PD Differentially Regulates CYP17 Promoter Activity in Normal and PCOS Theca Cells

Upper panel: Fourth-passage theca cells isolated from normal or PCOS patients were transiently transfected with a pGL3 LUC construct containing –750 to +44 bp of the CYP17 promoter. After transfection, the cells were treated with vehicle (C) or 7.5 µM forskolin (F) with and without the 25 µM PD. Seventy-two hours thereafter, the cells were harvested and LUC activity assayed. Data are presented as relative LUC activity that has been corrected for ß-Gal activity. Data represent the mean ± SEM of experiments performed with triplicate cultures of theca cells isolated from five independent normal and PCOS patients. In normal theca cells, inhibition of ERK1/2 signaling using PD augmented both basal (a, P < 0.05) and forskolin (b, P < 0.05) stimulated –750 CYP17/LUC promoter activity. In contrast, –750 CYP17/LUC promoter activity was not significantly increased in response to PD treatment in PCOS theca cells. Bottom panel: To compare the effects of PD on ERK1/2 phosphorylation in normal and PCOS theca cells, fourth-passage theca cells propagated from normal and PCOS patients were grown to confluent and transferred into serum-free medium with vehicle (C) or 7.5 µM forskolin (F) in the presence or absence of 25 µM PD. Twenty-four hours thereafter, the cells were harvested, and immunnoblot analysis was performed using 35 µg of whole cell extract and antibodies specific for phosphorylated ERK1/2 (Phospho-ERK 1/2) and total ERK1/2.

 
The Activation State of ERK1/2 Is Decreased, and CYP17 mRNA Abundance and DHEA Biosynthesis Are Increased in PCOS Theca Cells as Compared with Normal Theca Cells Irrespective of the Insulin Concentration
Given the controversial role of insulin in regulating androgen biosynthesis in normal and PCOS theca cells, experiments were performed to assess the effects of insulin on ERK phosphorylation and overall androgen biosynthesis. In these experiments, theca cells propagated from multiple normal and PCOS patients were grown to subconfluent, and transferred into serum-free medium without insulin for 24 h, then treated for an additional 24 h with serum-free medium, with and without 2 or 20 nM insulin, in the presence and absence of 7.5 µM forskolin. An insulin concentration of 2 nM (i.e. 10 ng/ml) is equivalent to the normal plasma concentration of insulin. As shown in Fig. 7Go, insulin treatment did not significantly affect the phosphorylation state of ERK1/2 in normal or PCOS theca cells. ERK1/2 phosphorylation was decreased in PCOS theca cells as compared with normal theca cells, at both concentrations of insulin tested, and in the absence of insulin. In agreement with the data presented in Fig. 2Go, treatment with 7.5 µM forskolin reduced ERK phosphorylation approximately 50% in both normal and PCOS theca cells in the absence and presence of insulin. Of significant interest is the observation that in the absence of insulin treatment, CYP17 mRNA abundance (Fig. 8Go, middle panel), DHEA accumulation (Fig. 8Go, lower panel), and 17{alpha}-hydroxylase enzyme activity (data not shown) were increased in PCOS theca cells as compared with normal theca cells. These data suggest that alterations in MEK/ERK signaling, CYP17 mRNA accumulation, and androgen biosynthesis may not be directly associated with insulin action.



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Fig. 7. The Activation State of ERK1/2 Is Decreased in PCOS Theca Cells as Compared with Normal Theca Cells Irrespective of the Insulin Concentration

Fourth-passage theca cells propagated from normal and PCOS patients were grown to confluent, transferred into serum-free medium without insulin for 24 h, and then treated with serum-free medium with vehicle (C) or 7.5 µM forskolin (F), without or with 2 or 20 nM insulin. Twenty-four hours thereafter, the cells were harvested, and immunoblot analysis was performed using 35 µg of whole cell extract and antibodies specific for phosphorylated ERK1/2 (Phospho-ERK1/2) and total ERK1/2. Representative immunoblot data of whole cell extracts prepared from theca cells isolated from normal and PCOS patients. Insulin treatment did not significantly affect the phosphorylation state of ERK1/2 in normal or PCOS theca cells.

 


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Fig. 8. CYP17 mRNA Abundance and DHEA Biosynthesis Are Increased in PCOS Theca Cells as Compared with Normal Theca Cells in the Absence of Insulin

Fourth-passage theca cells propagated from normal and PCOS patients were grown to subconfluent, transferred into serum-free medium in the absence of insulin for 24 h, and then treated with serum-free medium with vehicle (C) or 20 forskolin (F) in the absence of insulin. Upper panel, For analysis of ERK1/2 phosphorylation, 24 h thereafter the cells were harvested, and immunoblot analysis was performed using 35 µg of whole cell extract and antibodies specific for phosphorylated ERK1/2 (Phospho-ERK1/2) and total ERK1/2. Representative immunoblot data of whole cell extracts prepared from theca cells isolated from four independent normal and PCOS patients. Middle panel, At 24 h the cells were harvested and CYP17 mRNA abundance was evaluated using quantitative real-time PCR analysis. mRNA accumulation was normalized by TBP mRNA abundance and is depicted graphically as the mean ± SEM. In the absence of insulin treatment, both basal (a, P < 0.05) and forskolin (b, P < 0.05)-stimulated CYP17 mRNA accumulation were significantly increased in PCOS theca cells as compared with normal theca cells. Bottom panel, Seventy-two hours after treatment, the media were collected and DHEA production was evaluated by RIA. Data were normalized to cell number and are presented as the mean ± SEM from triplicate theca cell cultures from four independent normal and PCOS patients. In the absence of insulin treatment, both basal (a, P < 0.05) and forskolin (b, P < 0.05)-stimulated CYP17 mRNA accumulation were significantly increased in PCOS theca cells as compared with normal theca cells.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The study of human theca cells maintained in long-term culture provides an opportunity to compare the steroidogenic abnormalities and their underlying causes in PCOS theca cells (9, 10). In addition to augmented transcription of the CYP17 gene (11, 12), recent microarray analysis of differential gene expression in normal and PCOS theca cells demonstrated that dysregulation of androgen biosynthesis is associated with selective differences in several gene networks that are involved in steroid hormone biosynthesis as well as insulin and glucose homeostasis (32, 33, 34)

In an attempt to examine the molecular mechanisms underlying dysregulated gene expression in the PCOS ovary, we have extended our studies on MAPK signaling in normal and PCOS theca cells. In this report, we present data to support the concept that increased CYP17 gene expression and overall androgen biosynthesis are associated with diminished MEK1/2 and ERK1/2 signaling in the PCOS ovary. We compared the phosphorylation states of MEK1/2 and ERK1/2 in the same normal and PCOS theca cell preparations that we previously examined in our CYP17 transcriptional studies (11, 12) and microarray analysis (32). The comparison of the phosphorylation states of MEK1/2 and ERK1/2 in normal and PCOS theca cells propagated in long-term culture revealed a gross reduction in the tone of MEK/ERK signaling in PCOS cells.

Infection of normal theca cells with a dominant-negative MEK1 adenovirus resulted in increased DHEA production, CYP17 mRNA accumulation, and 17{alpha}-hydroxylase enzyme activity, and treatment with the pharmacological MEK1 inhibitor, PD, augmented CYP17 gene transcription. In contrast, infection with a constitutively active MEK1 inhibited both DHEA synthesis and CYP17 mRNA accumulation. In our comparison of CYP17 promoter function in normal and PCOS theca cells, the observed lack of a response of PCOS theca cells to PD further supports the idea that the suppression of the MEK/ERK signaling pathway plays a pivotal role in regulating androgen synthesis in the PCOS ovary.

Although several investigators have examined the mechanisms by which insulin and IGF acutely stimulate the ERK1/2 signaling pathway in porcine and human theca cells (31, 35, 36), there is no information available with respect to the intracellular signaling pathways mediating androgen biosynthesis in normal and PCOS theca cells under basal and cAMP-stimulated conditions. In addition, most studies have focused on the acute regulation of MEK or ERK phosphorylation in response to a variety of stimuli, rather than examining MAPK signaling under steady-state conditions in differentiated cells. In contrast, we compared MAPK signaling using conditions in which normal and PCOS theca cells are fully differentiated. Data from array analysis suggests that cross talk between several signaling pathways, including the MAPK pathway, may be dysregulated in the PCOS ovary. For instance tribble 3, which inhibits Akt/protein kinase B (PKB) phosphorylation, exhibits decreased gene expression in PCOS theca cells, and cAMP-GEFII, which augments Akt/PKB phosphorylation, exhibits increased gene expression in PCOS theca cells (32).

PCOS is also associated with insulin resistance, obesity, and type II diabetes (37). Insulin resistance and hyperinsulinemia in PCOS have been hypothesized to contribute to increased androgen production by the ovary. Although several investigators had initially proposed that hyperinsulinemia, resulting from the insulin-resistant state, produces ovarian hyperandrogenism by spillover occupancy and activation of the IGF-I receptors (38), the studies of Nestler et al. (6) have conclusively demonstrated that PCOS theca cells are not insulin resistant and are responsive to insulin. However, there are no apparent differences in insulin sensitivity between normal and PCOS theca cells, and insulin appears to act via its own receptor rather than through spillover occupancy of the IGF-I receptor (6). No differences were found in the ED50 of insulin-stimulated steroid biosynthesis in freshly isolated granulosa cells (39, 40) obtained from normal and PCOS ovaries. These data are in agreement with our data demonstrating that insulin treatment does not differentially affect ERK1/2 phosphorylation in normal or PCOS theca cells. Furthermore, in the absence of insulin, CYP17 mRNA abundance and DHEA accumulation was increased in PCOS theca cells as compared with normal theca cells, and the phosphorylation state of ERK1/2 was decreased in PCOS theca cells, as compared with normal theca cells.

In human adrenal cells, Sewer et al. (41) has established that there is cross talk between the MAPK and ACTH/cAMP pathways in the regulation of CYP17 mRNA synthesis. These authors demonstrated that a reduction in the activation state of the ERK1/2 is associated with increased CYP17 gene expression in adrenal cells (41). These authors also showed that protein kinase A phosphorylates and activates a dual-specificity phosphatase, MAPK phosphatase-1 (MKP-1), which mediates CYP17 gene transcription in response to ACTH/cAMP (28, 41). However, these investigators did not report the role of ERK1/2 or MKP-1 in overall adrenal androgen biosynthesis. Therefore, the possible involvement of MKP-1 in augmented CYP17 gene expression and increased androgen biosynthesis in normal and PCOS theca cells is unclear and will require future examination.

We are in the process of examining the relationship between the cohort of multiple signaling pathways and transcription factors that confer increased CYP17 gene expression in PCOS theca cells. The reduction in MEK1/2 and ERK1/2 phosphorylation in PCOS cells is also in agreement with our previously published data demonstrating that a relief of transcriptional repression may play a role in augmented transcriptional regulation of the CYP17 gene in the PCOS ovary (11). Analysis of the CYP17 promoter function in normal and PCOS theca cells demonstrated that a 16-bp sequence, spanning –174 to –158 bp of the CYP17 promoter confers increased expression of the CYP17 promoter in PCOS theca cells (11). We reported that transcription factor, NF-1 C, bound to this element and was able to repress CYP17 promoter function (11). Identification of the cohort of transcription factors that regulate this element will further our knowledge of the underlying cause(s) of increased CYP17 gene expression in PCOS theca cells and the extent to which defects in MAPK signaling and other signaling pathways are involved in the pathogenesis of PCOS.

In patients with insulin resistance and type II diabetes, there are reports of alterations in multiple components of the phosphoinositol 3-kinase/PKB and Ras/MAPK signaling pathways (42, 43, 44, 45, 46, 47, 48, 49, 50, 51). Yet, there are very few reports of the specific signaling defects observed in PCOS patients. In patients with PCOS and insulin resistance, there are reports to suggest that D-chiro-inositol signaling is altered (6, 52, 53, 54, 55). Dunaif et al. and Li et al. (56, 57) have reported that insulin receptor autophosphorylation is decreased in fibroblasts in approximately 50% of patients with PCOS. Whereas in PCOS adipocytes, Ciaraldi et al. (58, 59) reported that insulin resistance is accompanied by normal function of the insulin receptor, but involves a novel postreceptor defect in the insulin signal transduction between the receptor kinase and glucose transport. With respect to the ovary, although it is well recognized that LH-, FSH-, and insulin receptor sensitivity are increased in ovarian cells isolated from PCOS patients (5, 38, 39, 40, 60, 61, 62), there are few data that describe defects in cellular signaling in PCOS ovarian cells other than those demonstrating decreased abundance of insulin receptor substrate-2 mRNA and protein in PCOS granulosa and theca cells (63).

The data presented in this report are the first to suggest that MAPK signaling in normal and PCOS cells is distinct. These studies have demonstrated that decreased MEK/ERK phosphorylation are directly associated with increased thecal CYP17 gene expression and androgen biosynthesis. Moreover, our findings reveal that in the absence of insulin treatment, phosphorylation state of ERK1/2 is decreased, and CYP17 mRNA accumulation and DHEA biosynthesis are increased in PCOS cells as compared with normal cells. We are currently characterizing the extent to which other components of the MAPK signaling pathway regulate increased CYP17 gene expression and androgen excess in the PCOS ovary. As a consequence of future studies we hope to further our understanding of the underlying cause of increased CYP17 gene expression and possibly other abnormalities in PCOS, e.g. insulin resistance, obesity, arrested follicular development, and endometrial cancer.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 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 3–5 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) (64). 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, twelve 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. 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 (2, 65, 66, 67), including hyperandrogenemia, oligoovulation, and the exclusion of 21{alpha}-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 previously described (2, 9). 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). Other compounds were purchased from Sigma.

Western Blot Analysis
Fourth-passage normal and PCOS theca cells were grown until subconfluent and transferred into serum-free medium with and without forskolin for 24 h. After treatment, theca cells were harvested in ice cold modified RIPA buffer (30 mM Tris, 150 mM NaCl, 50 mM Na F, 0.5 mM EDTA, 0.5% deoxycholic acid, 1.0% Nonidet P-40, 0.1% sodium dodecyl sulfate) containing 1 mM sodium orthovanadate, 0.1 mM phenymethylsufonyl fluoride, 1 mM dithiothreitol, 0.2 mM benzamidine, 1 µM microcystin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. Protein concentration was determined using a bicinchoninic acid protein assay (Pierce, Rockford, IL). Whole cell lysates (35 µg/lane) were separated on a 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane, and Western analysis was performed as previously described (68). Total and phosphospecific MEK1/2 and ERK1/2 used in Western analysis were obtained from Cell Signaling Technology (Beverly, MA).

Replication-Deficient Adenovirus Infections
Adenoviruses expressing constitutively active MEK1 (Ser218 and Ser222 to Glu, CA-MEK1), and/or dominant-negative MEK1 (Ser222 to Ala, DN-MEK1) were used as previously described (29, 30, 69). All recombinant adenoviral vectors were plaque purified, expanded, and titered by detection of formation of visible plaques in a HEK293 monolayer of cells using the agarose gel overlay method. Adenoviral infections involved growing fourth-passage theca cells to 95% confluence, rinsing the cells with PBS, and layering of the adenovirus on the cells in 50% of the normal treatment volume of serum-free medium for 1 h. Subsequently, the cells were cultured in serum-free media with and without treatment as indicated. Using these conditions, >98% of cells showed positive ß-Gal staining after infection with the LacZ adenovirus.

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 adenovirus. At 72 h, the media was collected and RIAs for DHEA were performed without organic solvent extraction using Diagnostic Products (Los Angeles, CA) as previously described (68).

Quantitation of CYP17 mRNA
For quantitative real-time PCR, total mRNA was isolated (9) 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 deoxythymidine, and 200 units Stratascript Reverse Transcriptase (Stratagene, Cedar Creek, TX), and CYP17 mRNA abundance was determined by quantitative real-time PCR as previously described (68). The gene-specific two-step PCR was carried out in triplicate for each cDNA sample and for a series of serial dilutions in an Mx4000 Thermocycler (Stratagene, Cedar Creek, TX) using the Mx4000 Multiplex Quantitative PCR system according to manufacturer’s instructions. TATA-box binding protein (TBP) mRNA abundance was used for data normalization.

17{alpha}-Hydroxylase Enzyme Activity
For evaluation of 17{alpha}-hydroxylase enzyme activity, normal theca cells were grown until subconfluent, then infected with adenovirus and transferred to serum-free medium and treated as indicated for 72 h. The cells were then transferred into medium containing a saturating concentration of 1.0 µM [1,2,6,7-3H]progesterone and the rate of conversion to 17{alpha}-hydroxyprogesterone and 17{alpha}, 20{alpha}-dihydroprogesterone was determined as we have previously reported (9, 10). Cell number was estimated using a Coulter counter (Coulter Electronics, Hialeah, FL) after dispersal of cells with trypsin. Steroidogenic enzyme activities are expressed as picomoles per 106 cells/hour.

Transient Transfection Assay
Subconfluent cultures of theca cells were transfected with reporter gene constructs as we have previously described (11, 12) using the modified calcium-phosphate method of Graham and Vander Eb (70). 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 of reporter plasmid, and 1 µg of pSV-ß-Gal/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. Seventy-two hours after forskolin treatment, the cells were harvested using trypsin/EDTA, pelleted, and resuspended in reporter lysis buffer for LUC assays. LUC assays were performed using the LUC assay system (Promega, Madison, WI). ß-Gal activities were determined by Galacton Light Plus chemiluminescent assay (Tropix, Bedford, MA) and used to normalize LUC activities.

Statistical Analysis
Each experiment was performed using triplicate dishes. After combining the results from individual experiments, nonparametric Mann Whitney tests were performed using PRISM 4.0 from SAS Institute, Inc. (Cary, NC). P < 0.05 was considered statistically significant.


    ACKNOWLEDGMENTS
 
We thank Dr. Jeffery D. Molkentin (Department of Pediatrics, Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, OH) for providing adenoviruses encoding constitutively active and dominant-negative MEK1. We are indebted to Dr. David Spector (Department of Microbiology, Pennsylvania State College of Medicine, Hershey, PA) for his expertise in adenoviral propagation, purification, and plaque titration.


    FOOTNOTES
 
This work was supported by National Institutes of Health Grants HD33852 (to J.M.M. and R.S.L.), HD34449 (to J.M.M., R.S.L., and J.F.S.).

First Published Online October 28, 2004

Abbreviations: CA-MEK1, Constitutively active MEK1; CYP17, gene encoding cytochrome P450c17; DHEA, dehydroepiandrosterone; DN-MEK1, dominant-negative MEK1 molecule; FBS, fetal bovine serum; ß-Gal, ß-galactosidase; LUC, luciferase; MEK1/2, MAPK kinase; MKP-1, MAPK phosphatase-1; P450c17, P450 17{alpha}-hydroxylase/17,20 lyase; PCOS, polycystic ovary syndrome; PD, PD98059; pfu, plaque forming-units; PKB, protein kinase B; TBP, TATA-box binding protein.

Received for publication April 28, 2004. Accepted for publication October 20, 2004.


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