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Inhibition of Extracellular Signal-Regulated Protein Kinase-2 Phosphorylation by Dihydrotestosterone Reduces Follicle-Stimulating Hormone-Mediated Cyclin D2 Messenger Ribonucleic Acid Expression in Rat Granulosa Cells

Departments of Obstetrics and Gynecology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: Dr. K.M.J. Menon, 6428 Medical Science I, 1150 West Medical Center Drive, University of Michigan, Ann Arbor, Michigan 48109. E-mail: kmjmenon{at}umich.edu.

	Abstract

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Granulosa cell mitogenesis is critical for the development of normal ovarian follicles. FSH and other mitogenic stimuli play a crucial role in this process. We have shown that exposing granulosa cells to 5-dihydrotestosterone (DHT) reduces forskolin-stimulated cyclin D2 mRNA expression, which leads to cell cycle arrest resulting in reduced cell proliferation. The present study investigated the signaling molecules upstream of cyclin D2 in FSH-mediated, cAMP-dependent signaling pathway that may be negatively affected by DHT, leading to inhibition of cell cycle progression. Because ERK is an important molecule in mitogenic signaling, the possible effect of DHT on its phosphorylation was examined. Granulosa cells from 3-d estradiol-primed immature rats were treated with DHT (90 ng/ml) for 24 h and subsequently stimulated with forskolin. DHT treatment reduced forskolin stimulation of ERK phosphorylation. Although DHT exposure did not affect cellular cAMP production in response to forskolin, treating the cells with DHT for 24 h significantly reduced protein kinase A activity. DHT also caused a reduction in ERK-2 phosphorylation in response to FSH similar to that seen with forskolin. Furthermore, blocking ERK phosphorylation as well as DHT treatment resulted in a reduction in FSH-stimulated cyclin D2 mRNA expression. From these results, we conclude that DHT treatment reduces the FSH-mediated ERK phosphorylation in granulosa cells, leading to reduced cyclin D2 mRNA expression that culminates in cell cycle arrest.

	Introduction

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PITUITARY HORMONES AND growth factors stimulate granulosa cell proliferation in the primordial follicle (1). The growing follicle consists of an oocyte surrounded by a single layer of granulosa cells. An increase in the rate of cell proliferation in response to mitogenic stimuli results in the formation of the large preovulatory follicle with many layers of granulosa cells and outer layers of thecal cell (1, 2, 3, 4). Thecal cells synthesize androgens, and these androgens are aromatized into estrogens in granulosa cells (5, 6, 7). Estrogens have been known to be a positive regulator of follicular growth by enhancing the response to FSH and other mitogenic stimuli (8). Elevated levels of androgens, however, impair follicular function, resulting in anovulation as evidenced in certain pathophysiological conditions (9).

There are several reports in the literature showing the inhibitory effect of androgens on ovarian follicular function. For example, FSH-mediated induction of LH receptors (10) has been shown to be blocked by androgens in granulosa cells (11, 12). Some of the earlier studies implicated 5-reduced metabolites of androgens for this deleterious effect (13, 14, 15). Administration of dihydrotestosterone (DHT), a 5-reduced metabolite of testosterone, has been shown to reduce ovulation in rats (16). More recently, we have demonstrated that DHT inhibits granulosa cell proliferation by reducing the cyclin D2 mRNA expression and arrests cell cycle progression at the G1/S interphase (17).

Although the 5-reduced metabolites of androgens are known to antagonize FSH action, the site at which DHT blocks the FSH signaling pathway is not understood. In this study, we have examined the effect of DHT in the FSH- and cAMP-dependent signaling pathway, which leads to cyclin D2 mRNA expression in rat granulosa cells. The ability of growth factors to stimulate cell proliferation requires ERK (or MAPK) (18). FSH has been known to activate ERK through a cAMP-dependent signaling pathway in granulosa cells (19, 20). The mammalian ERK family includes ERK-1 and ERK-2 (MAPK-1 and MAPK-2), p38 MAPK, c-Jun N-terminal kinase and stress-activated protein kinases, ERK-3, ERK-4, ERK-5, and ERK-7. Among these kinases, ERK-1 and ERK-2 are the most commonly activated kinases in signal transduction during mitogenesis (21).

Because FSH has been shown to act through the cAMP-dependent protein kinase (PKA)-dependent signaling pathway leading to the activation of MAPK (19, 20), we examined these steps as possible targets of DHT action. The present study examines the effects of DHT treatment on PKA activity and ERK-2 phosphorylation. We show that DHT causes a reduction in forskolin and FSH-stimulated ERK-2 activation. Reduced ERK activation, in turn, leads to reduced cyclin D2 mRNA expression. Thus, the present study shows that DHT reduces cyclin D2 mRNA expression by inhibiting ERK activation.

	Materials and Methods

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Materials
Ovine FSH (NIDDK-oFSH-20) was kindly provided by Dr. A.F. Parlow of the National Hormone and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Diseases (Torrence, CA). DHT (5-androstan-17 ß-ol-3-one), 17ß estradiol (1,3,5 [10]-estratriene-3, 17ß-diol), 3-isobutyl-1-methylxanthine (IBMX), cAMP, ATP, and Kemptide were purchased form Sigma Chemical Co. (St. Louis, MO). Forskolin was from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). [³² P] ATP (4500 Ci/mmol) and [³² P] deoxy-CTP (3000 Ci/mmol) were purchased from ICN Biomedicals, Inc. (Irvine, CA). Antibodies for total ERK, PKA catalytic subunit , and ß-tubulin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody against phosphorylated ERK-2 was purchased from Cell Signaling Technology Inc. (Beverly, MA). Antimouse and antirabbit IgG horseradish peroxidase conjugate, enhanced chemiluminescence Western blotting detection reagents, and BIOTRAK cAMP¹²⁵ I assay system were from Amersham Pharmacia Biotech (Piscataway, NJ). Phenol red-free DMEM-F12 medium, TRIzol reagent, and the RTS RadPrime DNA labeling system were the products of Life Technologies Inc. (Gaithersburg, MD). Whatman ion exchange cellulose papers (grade P81) were purchased from Fisher Scientific (Pittsburgh, PA). The MAPK kinase inhibitor U0126 was obtained from Promega (Madison, WI).

Animals and treatments
Twenty-two-day-old female Sprague Dawley rats (Harlan) were housed in a temperature-controlled room with proper dark-light cycles and were under the care of the University of Michigan Unit of Laboratory Animal Medicine. The animals were primed with estradiol (1.5 mg/d) for 3 d to stimulate the development of large preantral follicles (22) and killed 24 h after the last estradiol administration by CO₂ asphyxiation, and their ovaries were collected. Granulosa cells were harvested and cultured in DMEM-F12 medium containing 90 ng/ml DHT or vehicle alone for 24 h.

Granulosa cell isolation and culture
Granulosa cells were isolated as previously described (17, 23). In brief, the ovaries were cleared from the surrounding fat and punctured with 27-gauge needles. Cells were collected in phenol red-free DMEM-F12 containing 0.2% BSA, 10 mM HEPES, and 6.8 mM EGTA, incubated for 15 min at 37 C under 95% O₂-5% CO₂, and centrifuged for 5 min at 250 x g. The pellets were suspended in a solution containing 0.5 M sucrose, 0.2% BSA, and 1.8 mM EGTA in DMEM-F12 and incubated for 5 min. After the incubation, the suspension was diluted with 3 vol DMEM-F12, centrifuged for 5 min at 250 x g, and treated sequentially with trypsin (20 µg/ml) for 1 min, 300 µg/ml soybean trypsin inhibitor for 6 min, and deoxyribonuclease (DNAse I) (100 µg/ml) for 6 min at 37 C to remove dead cells. The cells were then rinsed twice with serum-free media and suspended in DMEM-F12, and cell number was determined. Cell viability was examined by the trypan blue exclusion technique, and the cells were cultured in serum-free DMEM-F12 supplemented with 20 mM HEPES (pH 7.4), 4 mM glutamine, 100 IU penicillin/ml, and 100 µg/ml streptomycin. Before plating, the culture dishes were coated with 10% fetal calf serum for 2 h at 37 C and washed with DMEM-F12.

cAMP assay
After a 24-h incubation with DHT (90 ng/ml), cells were treated with 0.5 mM of IBMX for 15 min and, thereafter, stimulated with forskolin (10 µM) when indicated. The reaction was stopped by removing the media, and cells were washed with PBS. Cells were harvested using PBS-EDTA, pelleted by centrifugation at 2500 x g for 8 min at 4 C, and dissolved in 100% ethanol. After 21 h, ethanol was evaporated under vacuum. cAMP production was measured using the Biotrak cAMP [¹²⁵I] assay system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.

Western blot analysis
Granulosa cells, after 24 h of exposure to DHT (90 ng/ml) or vehicle, were stimulated with forskolin (10 µM) or ovine FSH (50 ng/ml) as indicated for each experiment. The cells were washed with PBS, lysed in modified radioimmunoprecipitation assay buffer (RIPA), and subjected to SDS-PAGE. Western blot analysis for phosphorylated ERK-2 was performed with specific antibody against phosphorylated form of ERK-2. Protein loading was normalized by reprobing the same blot with antibody against nonphosphorylated form of the ERK-2. The PKA protein level was examined by Western blot analysis using an antibody against PKA catalytic subunit . The protein loading was normalized on the basis of tubulin. Detection was performed with an enhanced chemiluminescence Western blotting detection system (ECL; Amersham Pharmacia Biotech). The signals were measured using an Arcus II scanner (Agfa, Wilmington, MA) and quantitated using the NIH Image 1.61 program (Bethesda, MD).

Inhibition of ERK pathway
The inhibitor U0126 (1,4,-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio] butadiene) was used to block ERK phosphorylation as previously described (24). Briefly, granulosa cells after overnight attachment were incubated with vehicle or U0126 (10 µM) for 15 min followed by forskolin (10 µM) stimulation for 5 min. The cells were then lysed, and Western blot analysis was performed for phosphorylated ERK-2. The blot was stripped and reprobed for total ERK-2 to normalize for protein loading.

PKA activity assay
PKA activity was assayed by measuring the transfer of ³²P from [³²P] ATP to Kemptide, a protein kinase substrate, as reported previously, with some modifications (25). In brief, granulosa cells from 3-d estradiol-primed rats were incubated with 90 ng/ml DHT or vehicle. After 24 h of incubation, media was removed, and cells were washed once with PBS. Cells were then harvested in ice-cold phosphate buffer (pH 7.4) [10 mM NaHPO₄, 1 mM EDTA, 1 mM dithiothreitol, and 250 mM sucrose containing 0.2 mM phenylmethylsulfonylfluoride and leupeptin (10 µg/ml)] and homogenized. The homogenate was centrifuged at 14000 x g for 5 min at 4 C, and supernatant was collected. Ten microliters of the supernatant were then added to a 40-µl reaction mixture containing 30 µM Kemptide, 20 mM Tris-HCl (pH 7.4), 10 mM magnesium acetate, 0.5 mM IBMX, 10 mM dithiothreitol, 5 mM NaF, and 200 µM [³² P] ATP (200 cpm/pmol), in the absence or presence of 5 µM exogenous cAMP, and incubated at 30 C for 0, 3, and 5 min. At the end of the incubation period, 10 µl of the reaction mixture was spotted onto Whatman ion exchange cellulose filter papers (P81) and washed three times with 75 mM phosphoric acid and once with 95% ethanol. Filter papers were then dried under a heat lamp and placed in vials, and radioactivity was determined after the addition of scintillation fluid using a liquid scintillation counter.

Northern blot analysis of cyclin D2
Granulosa cells from 3-d estradiol-primed rats were harvested. After overnight attachment, one group of cultures was treated with DHT (90 ng/ml) for 24 h followed by FSH (50 ng/ml) for 2 h. In experiments in which the effect of MAPK kinase inhibitor (U0126) on FSH-stimulated cyclin D2 mRNA expression was tested, one group of cultures was pretreated with the inhibitor (10 µM) for 15 min, and a second group was pretreated with vehicle. After 15 min, one group of cultures from both control and inhibitor-treated cells was incubated with FSH (50 ng/ml) for 2 h. At the end of the incubation with FSH, the medium was decanted, and cells were washed once with PBS; the cells were harvested, and total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions (Life Technologies Inc.). The cyclin D2 cDNA (1.1 kb) probe was radiolabeled using [³² P] deoxy-CTP and the RTS RadPrime DNA labeling system and was hybridized to blots overnight at 42 C using a 2 x 10⁷ cpm-labeled probe. The hybridized blots were washed and exposed at -70 C to XAR film (Eastman Kodak Co., Rochester, NY). The films were developed, and the signals were measured using the National Institutes of Health Image 1.61 program. Blots were stripped and rehybridized with a radiolabeled cDNA probe corresponding to 18S rRNA to monitor total RNA loading.

Statistical analysis
Statistical analysis was carried out using the unpaired t test using Sigma Stat computer software (version 2.0; SPSS Inc., Chicago, IL). Each experiment was repeated at least three times with similar results. Blots are representative of one experiment, and graphs represent the mean ± SE of three experiments.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of DHT on forskolin-mediated stimulation of ERK-2 phosphorylation
Recent studies have shown that FSH signaling pathway involves ERK activation in granulosa cells (19). Forskolin, a diterpene, mimics the actions of FSH by increasing intracellular levels of cAMP (19, 26, 27). Therefore, we first tested whether DHT had any effect on forskolin-mediated activation of ERK. The results of Western blot analysis showed that control cells stimulated with forskolin for 5 min produced a significant increase (P < 0.05) in ERK-2 phosphorylation (Fig. 1A), whereas DHT treatment significantly reduced (P < 0.05) this stimulation (Fig. 1C). The antibody used in this experiment specifically recognizes the phosphorylated (activated) form of ERK-2. The total ERK-2 protein remained identical in all experimental groups (Fig. 1B), suggesting that forskolin induces the phosphorylation of ERK-2. DHT specifically inhibited forskolin-mediated induction of ERK-2 phosphorylation.

View larger version (38K): [in this window] [in a new window]   FIG. 1. Inhibitory effect of DHT on forskolin (FSK)-stimulated ERK-2 phosphorylation in rat granulosa cells. Granulosa cells from 3-d estradiol-primed immature rats (1.5 mg/d) were cultured in the presence or absence of DHT (90 ng/ml) for 24 h. After the incubation, cells were stimulated with FSK (10 µM) or vehicle for 5 min. The reaction was stopped by removing the media, and cells were washed with PBS. Cell lysate was prepared using RIPA buffer. Equal amounts of protein were subjected to SDS-PAGE and transferred onto nitrocellulose membrane. A, The membrane was probed with specific antibody for phosphorylated ERK-2 (pERK-2). B, The same blot was stripped and reprobed with antibody for total ERK-2. C, Mean fold increase in pERK-2 expression normalized for total ERK-2 when compared with control. Error bar represents ± SEM; a, significant difference (P < 0.05) compared with control; b, significant difference (P < 0.05) when compared with FSK treatment.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Effect of DHT on forskolin (FSK)-stimulated cAMP production in rat granulosa cells. Granulosa cells from 3-d estradiol-primed immature rats (1.5 mg/d) were cultured in the presence or absence of DHT (90 ng/ml) for 24 h. After the incubation, cells were treated with 0.5 mM IBMX for 15 min and stimulated with FSK (10 µM) or vehicle for 0, 3, and 5 min. The reaction was stopped by removing the media. Cells were washed with PBS and harvested using PBS-EDTA. cAMP production was measured using Biotrak cAMP [125I] assay system as described in Materials and Methods. Each experiment was done in triplicate. The experiment was repeated at least three times. Error bars represents ± SEM. Bars with same superscript letters are significantly different (P < 0.001).

View larger version (14K): [in this window] [in a new window]   FIG. 3. Effect of DHT on PKA activity in rat granulosa cells in the absence of exogenous cAMP. Granulosa cells from 3-d estradiol-primed immature rats (1.5 mg/d) were cultured in the presence or absence of DHT (90 ng/ml). After 24 h of incubation, the media was removed, and cells were washed with PBS. PKA activity of control and DHT-treated cells were measured as described in Materials and Methods. Each experiment was done in triplicate. The experiment was repeated at least three times. Error bar represents ± SEM. Bars with same superscript letters are significantly different (P < 0.001).

View larger version (15K): [in this window] [in a new window]   FIG. 4. Effect of DHT on PKA activity in rat granulosa cells in presence of exogenous cAMP. Granulosa cells from 3-d estradiol-primed immature rats (1.5 mg/d) were cultured in the presence or absence of DHT (90 ng/ml). After 24 h of incubation, media was removed, and cells were washed with PBS. PKA activity of control and DHT-treated cells in presence of exogenous cAMP was measured as described in Materials and Methods. Each experiment was done in triplicate and repeated at least three times. Error bar represents ± SEM. Bars with same superscript letters are significantly different (P < 0.01).

View larger version (39K): [in this window] [in a new window]   FIG. 5. Effect of DHT on PKA protein level in rat granulosa cells. Granulosa cells from 3-d estradiol-primed immature rats (1.5 mg/d) were cultured in the presence or absence of DHT (90 ng/ml) for 24 h. Afterward, the incubation cells were washed with PBS, and cell lysate was prepared using RIPA buffer. Equal amounts of protein were separated by SDS-PAGE and transblotted onto a nitrocellulose membrane. The blot was probed with an antibody for PKA catalytic subunit (PKA cat) (top panel). Protein loading was normalized by stripping and reprobing the same blot with antibody for ß-tubulin (middle panel). Fold difference in PKA cat protein expression normalized for ß-tubulin compared with control is shown in the lower panel. Error bar represents ± SEM.

View larger version (41K): [in this window] [in a new window]   FIG. 6. Effect of DHT on FSH-stimulated ERK-2 phosphorylation in rat granulosa cells. Granulosa cells from 3-d estradiol-primed immature rats (1.5 mg/d) were cultured in the presence or absence of DHT (90 ng/ml) for 24 h. After the incubation, cells were stimulated with ovine FSH (50 ng/ml) for 5 min. The reaction was stopped by removing the media, and cells were washed with PBS. Cell lysate was prepared using RIPA buffer. Equal amounts of protein were subjected to SDS-PAGE and transferred onto nitrocellulose membranes. A, The membranes were probed with antibody for phosphorylated ERK-2 (pERK-2). B, The blots were then stripped and reprobed with antibody for total ERK-2. C, Mean fold increase in pERK-2 expression normalized for total ERK-2 when compared with control. Error bar represents ± SEM; a, significant difference (P < 0.05) when compared with control; b, significant difference (P < 0.05) when compared with FSH treatment.

View larger version (31K): [in this window] [in a new window]   FIG. 7. Effect of inhibitor U0126 on forskolin (FSK)-stimulated ERK-2 phosphorylation in rat granulosa cells. Granulosa cells from 3-d estradiol-primed rats were incubated with U0126 (10 µM) for 15 min and then stimulated with FSK (10 µM) for 5 min. The reaction was stopped by removing the media, and cell lysate was prepared using RIPA buffer. Equal amounts of total protein were separated by SDS-PAGE and transblotted onto nitrocellulose. A, The blot was probed with antibody for phosphorylated ERK-2 (pERK-2). B, The same blot was stripped and reprobed with antibody for total ERK-2. C, Mean fold increase in pERK-2 expression normalized for total ERK-2 when compared with control. Error bar represents ± SEM; a, significant difference (P < 0.05) when compared with control; b, significant difference (P < 0.05) when compared with FSK treatment.

View larger version (34K): [in this window] [in a new window]   FIG. 8. Effect of ERK inhibition on FSH-stimulated cyclin D2 mRNA expression in rat granulosa cells. Granulosa cells from 3-d estradiol-primed rats were harvested. After overnight attachment, one group of cultures was preincubated with U0126 (10 µM) for 15 min, and the other was preincubated with vehicle. After the incubation, one set of cultures from both the control and inhibitor-treated groups was stimulated with FSH (50 ng/ml) for 2 h. A, Total RNA was extracted, and Northern blot analysis was performed using [32P] cyclin D2 cDNA probe. B, [32P] cDNA for 18S rRNA was used to monitor RNA loading. C, Mean fold increase in cyclin D2 mRNA expression normalized for 18S rRNA when compared with control. Error bar represents ± SEM; a, significant difference (P < 0.05) when compared with control; b, significant difference (P < 0.05) when compared with FSH treatment.

View larger version (46K): [in this window] [in a new window]   FIG. 9. Effect of DHT on FSH-stimulated cyclin D2 mRNA expression in rat granulosa cells. Granulosa cells from 3-d estradiol-primed rats were harvested. After overnight attachment, one group of cultures was incubated with DHT (90 ng/ml) for 24 h, and the other was incubated with vehicle. After the incubation, one set of cultures from both the control and DHT-treated groups was stimulated with FSH (50 ng/ml) for 2 h. A, Total RNA was extracted, and Northern blot analysis was performed using [32P] cyclin D2 cDNA probe. B, [32P] cDNA for 18S rRNA was used to monitor RNA loading. C, Mean fold increase in cyclin D2 mRNA expression normalized for 18S rRNA when compared with control. Error bar represents ± SEM; a, significant difference (P < 0.05) when compared with control; b, significant difference (P < 0.05) when compared with FSH.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Because FSH exerts its effects mainly through the elevation of intracellular cAMP (37, 38), a potential site for inhibitory effect of DHT could lie at the level of adenyl cyclase. Because we have observed an inhibition in the phosphorylation of ERK in DHT-treated cells, we suspected that a similar inhibition might also occur on cAMP production. However, forskolin stimulated cAMP production in both control and DHT-treated cells without any significant difference between them (Fig. 2). This result shows that DHT does not affect adenylate cyclase activity in rat granulosa cells and that its inhibitory effect on ERK-2 lies at a step after the cAMP production. It has already been established that, in the ovarian tissue, gonadotropin stimulation increases the cAMP accumulation and the activation of PKA, leading to increased steroidogenesis (39, 40, 41). Although DHT did not inhibit the cAMP levels, it significantly reduced the PKA activity both in the absence and presence of exogenous cAMP (Figs. 3 and 4). Because both DHT-treated and control cells expressed similar levels of PKA protein (Fig. 5), it is clear that the reduced activity seen in response to DHT is not due to reduced PKA protein expression in the cells. The mechanism by which DHT inhibits PKA activity is not currently known.

In the present study, we have shown that the inhibitory effect of DHT on forskolin-mediated ERK phosphorylation also occurs on FSH-stimulated ERK phosphorylation (Fig. 6). We have provided evidence to show that DHT inhibition of cyclin D2 mRNA expression is due to its inhibitory effect on ERK phosphorylation. FSH treatment significantly increased (P < 0.05) ERK phosphorylation as well as cyclin D2 mRNA expression in granulosa cells (Figs. 6 and 8). The inhibition of ERK phosphorylation by U0126 led to a reduction in FSH-stimulated cyclin D2 mRNA expression (Fig. 8). We have also shown that DHT pretreatment causes a similar inhibition of FSH-stimulated cyclin D2 mRNA expression (Fig. 9). From these results, it is apparent that DHT-mediated reduction of cyclin D2 mRNA expression occurs, at least partially, through the inhibition of ERK activation. Although U0126 pretreatment caused nearly complete inhibition of ERK2 phosphorylation, this inhibitor evoked only partial inhibition of FSH-stimulated cyclin D2 mRNA expression. A probable reason for this difference could be that ERK signaling may be only one arm of FSH signaling that culminates in the stimulation of cyclin D2 expression. Recent reports suggest that FSH can also activate pathways other than the classical cAMP-mediated PKA-dependent pathway. For example, FSH is known to activate Akt kinase or protein kinase B (Akt/PKB) through the phosphatidylinositol 3-kinase kinase pathway independent of PKA (42). However, the present study clearly shows that cyclin D2 is one of the downstream targets of ERK signaling and that DHT inhibits both forskolin- (17) and FSH-mediated cyclin D2 mRNA expression by blocking, at least partially, signaling through the ERK pathway.

Taken together, our studies show that the inhibition of ERK activation by DHT reduces FSH-stimulated cyclin D2 mRNA expression in granulosa cells. The cell cycle arrest and reduced proliferation of DHT-exposed granulosa cells, as previously reported (17), might be the result of reduced ERK activation. We have also shown for the first time that cyclin D2 expression in rat granulosa cells is sensitive to ERK activation. Furthermore, this inhibitory effect of DHT in FSH-stimulated signaling occurs at a post-cAMP level. The mechanism of inhibition of PKA activity by DHT is not understood at the present time. The involvement of other molecules like protein kinase inhibitors in mediating the DHT response or a direct nongenomic action of DHT on the ERK pathway cannot be ruled out at the present time.

	Acknowledgments

 
The authors express their appreciation to Prof. Michael D. Uhler, Dr. Anil Nair, Dr. Utpal Munshi, Dr. Roberto Towns, Helle Peegel, and Christine Clouser for critical reading of this manuscript.

	Footnotes

 
This work was supported by NIH Grant HD-38424.

Abbreviations: DHT, 5-Dihydrotestosterone; IBMX, 3-isobutyl-1-methylxanthine; PKA, cAMP-dependent protein kinase; RIPA, modified radioimmunoprecipitation assay.

Received August 8, 2003.

Accepted for publication December 15, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Channing CP, Schaerf FW, Anderson LD, Tsafrri A 1980 Ovarian follicular and luteal physiology. Int Rev Physiol 22:117–201[Medline]
2. Ryle MJ 1969 A quantitative in vitro response to follicle stimulating hormone. J Reprod Fertil 19:87–94[Abstract/Free Full Text]
3. Rao MC, Midgley Jr AR, Richards JS 1978 Hormonal regulation of ovarian cellular proliferation. Cell 14:71–78[CrossRef][Medline]
4. Hirshfield AN 1991 Development of ovarian follicles in mammalian ovary. Int Rev Cytol 124:43–101[Medline]
5. Bjersing L 1967 On the morphology and endocrine function of granulosa cells of ovarian follicles and corpora lutea. Acta Endocrinol (Copenh) 125(Suppl):1–25
6. Monroe SE, Menon KMJ 1977 Changes in reproductive hormone secretion during the climateric and postmenopausal periods. Clin Obstet Gynecol 20:113–122[CrossRef][Medline]
7. Erickson GF, Magoffin DA, Dyer CA, Hofeditz C 1985 The ovarian androgen producing cells: a review of structure/function relationships. Endocr Rev 6:371–399[CrossRef][Medline]
8. Richards JS 1979 Hormonal control of ovarian follicular development: a 1978 perspective. Recent Prog Horm Res 35:343–373
9. Ehrmann DA, Barnes RB, Roselfield RL 1995 Polycystic ovary syndrome as a form of functional ovarian hypoandrogenism due to dysregulation of androgen secretion. Endocr Rev 16:322–351[CrossRef][Medline]
10. Zeleznik AJ, Midgely Jr AR, Rerechert Jr LE 1974 Granulosa cell maturation in rat: increased binding of human chorionic gonadotropin following treatment with follicle stimulating hormone in vivo. Endocrinology 95:818–825[Medline]
11. Farookhi R 1980 Effects of androgen on induction of gonadotropin receptors and gonadotropin stimulated adenosine 3', 5'-monophosphate production in rat ovarian granulosa cells. Endocrinology 106:1216–1223[Medline]
12. Jia CX, Kessel B, Welsh TH, Huesh AJ 1985 Androgen inhibition of follicle stimulating hormone stimulated luteinizing hormone formation in cultured rat granulosa cells. Endocrinology 117:13–22[Abstract]
13. Hillier SG, van den Boogard A, Reichart Jr LE, van Hall E 1980 Alterations in granulosa cell aromatase activity accompanying preovulatory follicular development in the rat ovary with evidence that 5 reduced C19 steroids inhibit aromatase reaction in vitro. J Endocrinol 84:409–419[Abstract/Free Full Text]
14. Lephart ED, Doody KJ, Mc Phaul MJ, Simpson ER 1992 Inverse relationship between ovarian aromatase cytochrome p450 and 5 reductase enzyme activities and mRNA levels during estrous cycle in the rat. J Steroid Biochem Mol Biol 42:439–447[CrossRef][Medline]
15. Jakimiuk AJ, Weitsman SR, Magoffin DA 1999 5 reductase activity in women with polycystic ovarian syndrome. J Clin Endocrinol Metab 84:2144–2148
16. Conway BA, Mahesh VB, Mills TM 1990 Effect of dihydrotestosterone on the growth and function of ovarian follicles in intact immature female rats primed with PMSG. J Reprod Fertil 90:267–277[Abstract/Free Full Text]
17. Pradeep PK, Li X, Peegel H, Menon KMJ 2002 Dihydrotestosterone inhibits granulosa cell proliferation by decreasing the cyclin D2 mRNA expression and cell cycle arrest at G1 phase. Endocrinology 143:2930–2935[Abstract/Free Full Text]
18. Marshall CJ 1995 Specificity of receptor tyrosine kinase signaling: transient versus sustained extra cellular signal regulated kinase activation. Cell 80: 179–185
19. Cottom J, Salvador LM, Maizels ET, Reierstad S, Park Y, Carr DW, Davare MA, Hell JW, Palmer SS, Dent P, Kawakatsu H, Ogata M, Hunzicker-Dunn M 2003 Follicle-stimulating hormone activates extracellular signal-regulated kinase but not extracellular signal-regulated kinase kinase through a 100-kDa phosphotyrosine phosphatase. J Biol Chem 278:7167–7179[Abstract/Free Full Text]
20. Das S, Maizels ET, Demanno D, St. Clair E, Adam SA, Hunzicker-Dunn M 1996 A stimulatory role of cyclic adenosine 3', 5', monophosphate in follicle stimulating hormone activated mitogen-activated protein kinase signaling pathway in rat ovarian granulosa cells. Endocrinology 137:967–974[Abstract]
21. English J, Pearson G, Wilsbacher J, Swantek J, Karandikar M, Xu S, Cobb MH 1999 New insights into the control of MAP kinase pathways. Exp Cell Res 253:255–270[CrossRef][Medline]
22. Sanders MM, Midgley Jr AR 1982 Rat granulosa cell differentiation: an in vitro model. Endocrinology 111:614–624[Medline]
23. El-Hefnawy T, Zeleznik AJ 2001 Synergism between FSH and activin in the regulation of proliferating cell nuclear antigen and cyclin D2 expression in undifferentiated rat granulosa cells. Endocrinology 142:4357–4362[Abstract/Free Full Text]
24. Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copelan RA, Magolda RL, Scherle PA, Trzaskos JM 1998 Identification of a novel inhibitor of mitogen activated protein kinase kinase. J Biol Chem 273:18623–18632[Abstract/Free Full Text]
25. Witt-Enderby PA, Masana MI, Dubocovich ML 1998 Physiological exposure to melatonin supersensitizes the cyclic adenosine 3', 5'-monophosphate-dependent signal transduction cascade in Chinese hamster ovary cells expressing the human mt1 melatonin receptor. Endocrinology 139:3064–3071[Abstract/Free Full Text]
26. Schimmer B, Schulz P 1985 The roles of cAMP and cAMP-dependent protein kinase in forskolin’s actions on Y1 adrenocortical tumor cells. Endocr Res 11:199–209[Medline]
27. Fitzpatrick SL, Richards JS 1991 Regulation of cytochrome P450 aromatase mRNA and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology 129:1452–1462[Abstract]
28. Kolena J, Channing CP 1972 Stimulatory effects of LH, FSH and prostaglandins upon cyclic 3', 5'-AMP levels in porcine granulosa cells. Endocrinology 90:1543–1550[Medline]
29. Channing CP, Tsafriri A 1977 Mechanism of action of luteinizing hormone and follicle stimulating hormone on the ovary in vitro. Metabolism 26:413–468[Medline]
30. Carr DW, Demanno DA, Atwood A, Hunzicker-Dunn M, Scott JD 1993 Follicle stimulating hormone regulation of A-kinase anchoring proteins in granulosa cells. J Biol Chem 268:20729–20732[Abstract/Free Full Text]
31. Meloche S, Seuwen K, Pages G, Pouyssegur J 1992 Biphasic and synergistic activation of p44^mapk (ERK1) by growth factors: correlation between late phase activation and mitogenicity. Mol Endocr 638:845–854
32. Kahan C, Seuwen K, Meloche S, Pouyssegur J 1992 Coordinate, biphasic activation of p44 mitogen activated protein kinase and S6 kinase by growth factors in hamster fibroblasts. Evidence for thrombin-induced signals different from phosphoinositide turnover and adenyl cyclase inhibition. J Biol Chem 267:13369–13375[Abstract/Free Full Text]
33. Heasley LE, Johnson GL 1992 The ß PDGF receptor induces neuronal differentiation of PC12 cells. Mol Cell Biol 3:545–553
34. Nguyen TT, Scimeca JC, Filloux C, Peraldi P, Carpentier J-L, van Obberghen E 1993 Co-regulation of the mitogen activated protein kinase, extracellular signal regulated kinase 1 and the 90 kDa ribosomal S6 kinase in PC12 cells. J Biol Chem 268:9803–9810[Abstract/Free Full Text]
35. Choi K-C, Kang SK, Tai C-J, Auersperg N, Leung PCK 2002 Follicle stimulating hormone activates mitogen activated protein kinase in preneoplastic and neoplastic ovarian surface epithelial cells. J Clin Endocrinol Metab 87:2245–2253[Abstract/Free Full Text]
36. Graves LM, Guy HI, Kozlowski P, Huang M, Lazarowski E, Pope RM, Collins MA, Dahistrand EN, Earp III HS, Evans DR 2000 Regulation of carbamoyl phosphate synthetase by MAP kinase. Nature 403:328–331[CrossRef][Medline]
37. Hsueh AJW, Adashi EY, Jones PBP, Elsh Jr TH 1984 Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev 5:76–127[CrossRef][Medline]
38. Amsterdam A, Rotmensch S 1987 Structure-function relationships during granulosa cell differentiation. Endocr Rev 8:309–337[CrossRef][Medline]
39. Clark MR, Azar S, Menon KMJ 1976 Ovarian adenosine 3', 5' cyclic monophosphate-dependent protein kinase. Regulation by choriogonadotropin and lutropin in rat ovarian cells. Biochem J 158:175–182[Medline]
40. Ling WY, Marsh JM 1977 Reevaluation of the role of cyclic adenosine 3', 5'-monophosphate and protein kinase in the stimulation of steroidogenesis by luteinizing hormone in bovine corpus luteum slices. Endocrinology 100:1571–1578[Abstract]
41. Richards JS, Haddox M, Tash JS, Walter U, Lohmann S 1984 Adenosine 3', 5'-monophosphate-dependent protein kinase and granulosa cell responsiveness to gonadotropins. Endocrinology 114:2190–2198[Abstract]
42. Gonzalez-Robayna IJ, Falender AE, Ochsner S, Firestone GL, Richards JS 2000 Follicle-stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-induced kinase (Sgk): evidence for A kinase-independent signaling by FSH in granulosa cells. Mol Endocrinol 14:1283–1300[Abstract/Free Full Text]

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