Follicle-Stimulating Hormone Increases Tuberin Phosphorylation and ...

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Endocrinology 148(8):3950 –3957 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2007-0202

Follicle-Stimulating Hormone Increases Tuberin Phosphorylation and Mammalian Target of Rapamycin Signaling through an Extracellular Signal-Regulated Kinase-Dependent Pathway in Rat Granulosa Cells Pradeep P. Kayampilly and K. M. J. Menon Departments of Obstetrics and Gynecology and Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109 FSH-mediated regulation of mammalian target of rapamycin (mTOR) signaling in proliferating granulosa cells and the effect of dihydrotestosterone (DHT) on this pathway were examined. Inhibiting mTOR activation using rapamycin significantly reduced the FSH-mediated increase in cyclin D2 mRNA expression, suggesting that mTOR plays a role in the FSH-mediated increase in granulosa cell proliferation. FSH treatment of granulosa cells showed a 2-fold increase in phosphorylation of p70S6 kinase (p70S6K), the downstream target of mTOR. The increase in p70S6K phosphorylation by FSH treatment was abolished by prior exposure to DHT, suggesting that DHT inhibits FSH-mediated activation of mTOR signaling in cultured granulosa cells. The effect of FSH and DHT treatment on tuberin (TSC2), the upstream regulator of mTOR, was then examined. FSH treatment increased TSC2 phosphorylation, and pretreatment with DHT for 24 h re-

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HE HORMONAL CONTROL of follicular development uses a number of distinct and sometimes interconnected signaling pathways. FSH exerts its effect on steroidogenesis as well as growth and proliferation of granulosa cells through divergent signaling pathways initiated by the activation of cAMP production. The subsequent activation of downstream pathways leads to the stimulation of different physiological end points. For example, we have reported that FSH increases granulosa cell proliferation by increasing cyclin D2 mRNA expression through a protein kinase A (PKA) and ERK-mediated pathway (1). Because the normal process of ovulation depends on the proliferation and growth of granulosa cells, any abnormality in these well-coordinated signaling processes disrupts cell proliferation and leads to anovulation. We have shown that dihydrotestosterone (DHT), a potent 5␣-reduced derivative of testosterone, arrests granulosa cell cycle progression at G1 phase by reducing cyclin D2 mRNA expression (2, 3). We also have found that DHT inhibits FSH- and insulin-mediated granulosa cell proliferation by reducing PKA activity (1) and insulin reFirst Published Online May 17, 2007 Abbreviations: DHT, Dihydrotestosterone; mTOR, mammalian target of rapamycin; PI3-kinase, phosphatidylinositol-3-kinase; PKA, protein kinase A; p70S6K, p70S6 kinase; TSC, tuberous sclerosis complex. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

duced this stimulation. These results indicate that reduced p70S6K phosphorylation observed in DHT-treated cells might be the result of reduced TSC2 phosphorylation. Because Akt is the upstream activator of TSC2 phosphorylation, the effect of Akt inhibition was examined to test whether FSH-mediated TSC2 phosphorylation proceeds through an Akt-dependent pathway. Our results show that inhibiting Akt phosphorylation did not block FSH-stimulated TSC2 phosphorylation, whereas ERK inhibition reduced FSH-mediated stimulation. These results demonstrate the involvement of ERK rather than Akt in FSH-mediated TSC2 phosphorylation in granulosa cells. Based on these observations, we conclude that in granulosa cells, FSH uses a protein kinase A-/ERK-dependent pathway to stimulate TSC2 phosphorylation and mTOR signaling, and DHT treatment significantly reduces this response. (Endocrinology 148: 3950 –3957, 2007)

ceptor substrate-1 phosphorylation, respectively (3). The levels of 5␣-reductase, the enzyme that converts testosterone to DHT, have been shown to be 100-fold higher in patients with hyperandrogenism (4). Hyperandrogenism is now considered as one of the main diagnostic features of the disease polycystic ovarian syndrome (5). In our ongoing efforts to understand the signaling pathways that regulate growth and proliferation of granulosa cells, we have examined the FSH-mediated mammalian target of rapamycin (mTOR) signaling in the granulosa cell. The mTOR signaling is one of the signaling pathways that controls growth and proliferation of cells from yeast to mammals in response to growth factors, mitogenic factors, hormones, and nutrient availability (6). Fingar et al. (7) proposed that the mTOR pathway can regulate proliferation by controlling cell growth or acting directly on the phosphorylation cascade that controls cell cycle regulatory proteins. Inhibition of mTOR has been shown to cause G1 phase arrest of the cell cycle (8 –11). Phosphorylation of S6 protein kinase (S6K1) and eukaryotic initiation factor 4E (elF4E)-binding protein (4EBP1) are the two well known functions of the activated mTOR complex (12). A number of studies have placed tuberous sclerosis complex (TSC), which consists of hamartin (TSC1) and tuberin (TSC2), as an upstream regulator of the mTOR signaling pathway (13–17). The TSC complex possesses GTPase activity toward a small GTP-binding protein, Rheb, which is required for mTOR activation (18). Alam et al.

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Kayampilly and Menon • Granulosa Cell FSH-Mediated TSC2 Phosphorylation

(19) reported the stimulation of mTOR signaling in granulosa cells in response to FSH. They have shown that FSH activation of hypoxia inducible factor-1, a molecule necessary for the induction of protein markers of granulosa cell differentiation, is mediated by the phosphatidylinositol-3-kinase (PI3-kinase)/Akt/Rheb/mTOR pathway. Because mTOR signaling controls growth and proliferation (7), the present study examined the involvement of mTOR in FSH-mediated granulosa cell proliferation and the effect DHT has on this pathway. We show that FSH activates mTOR signaling through an ERK-mediated pathway, and this signaling mechanism is independent of Akt activation. Our studies also show that pretreatment with DHT reduces the stimulatory effect of FSH on mTOR signaling. Materials and Methods DHT (5␣-androstan-17␤-ol-3-one) and 17␤-estradiol (1,3,5, [10]-estratriene-3,17␤-diol) were purchased form Sigma Chemical Co. (St. Louis, MO). Ovine FSH (NIDDK-oFSH-20) was from Dr. A. F. Parlow (National Hormone and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA). Antibodies for total tuberin and phosphorylated and total ERK were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). mTOR inhibitor, rapamycin, and antibodies against phosphorylated tuberin, Akt, p70S6 kinase (p70S6K) were from Cell Signaling Technology Inc. (Beverly, MA). Antimouse and antirabbit IgG horseradish peroxidase conjugates and enhanced chemiluminescence Western blotting detection reagents were from Amersham Pharmacia Biotech (Piscataway, NJ). The phenol red free DMEM-F12, Trizol reagent, and the Radprime DNA labeling system were the products of Life Technologies Inc. (Gaithersburg, MD). MAPK kinase inhibitor PD 98059 and U0126 were obtained from Promega (Madison, WI), and Akt inhibitor VIII was from Calbiochem (La Jolla, CA). [␣-32P]Deoxy-CTP 3000 Ci/mmol was purchased from MP Biomedicals (Irvine, CA). Reagents as well as the primers and probes for the cyclin D2 real-time PCR were from Applied Biosystems (Foster City, CA).

Animals and treatments Immature female rats (22 d old, Sprague Dawley strain) were purchased from Harlan (Indianapolis, IN) and Charles River Laboratories (Wilmington, MA). Animals were kept and used under University Committee on the Use and Care of Animals guidelines. They were housed in a temperature-controlled room with proper dark-light cycles 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 and were killed 24 h after the last estradiol administration by CO2 asphyxiation, and ovaries were collected. Granulosa cells were harvested and cultured in DMEM-F12.

Granulosa cell isolation and culture Granulosa cells from immature female rats were harvested as described previously (2). Briefly, ovaries were cleared from the surrounding fat and punctured with 25-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 ⫻ 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 incubation, the suspension was diluted with 3 vol DMEM-F12, centrifuged at 250 ⫻ g, and treated sequentially with trypsin (20 ␮g/ml) for 1 min, 300 ␮g/ml soybean trypsin inhibitor for 5 min, and DNase I (100 ␮g /ml) for 5 min at 37 C to remove dead cells. The cells were then rinsed twice with serum free media and suspended in DMEM-F12, and the cell number was determined. Cell viability was examined by trypan blue exclusion method. 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 ␮g/ml strep-

Endocrinology, August 2007, 148(8):3950 –3957

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tomycin. Before seeding, the culture dishes were coated with 10% fetal calf serum for 2 h at 37 C and washed with DMEM-F12 (20). After allowing cells to attach overnight, they were treated with DHT (90 ng/ml) or vehicle alone for 24 h.

Western blot analysis To examine the effect of DHT on FSH-mediated phosphorylation of p70S6K, the cells, after 24 h exposure to DHT (90 ng/ml) or vehicle, were stimulated with FSH (50 ng/ml) for the time intervals described for each experiment. ERK activation was blocked using the MAPK kinase inhibitors PD 98059 and U0126. In experiments where the PD 98059 (20 ␮m) and PKA inhibitor (10 ␮m, H89) were used, cells were pretreated with inhibitors for 1 h. Cells were preincubated with 30 ␮m U0126 for 30 min to attain ERK inhibition. For Akt inhibition (5 ␮m, Akt inhibitor VIII), the preincubation time was 15 min. These preincubations were followed by FSH or insulin treatments for the time intervals mentioned in each experiment. Reactions were stopped by removing the media, and total protein was solubilized using RIPA buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS). Tuberin from the cells was immunoprecipitated using an antibody against total tuberin in a buffer containing 10 mm Tris-HCl (pH 7.5), 100 mm sodium chloride, 1% Nonidet P-40, 50 mm sodium fluoride, 2 mm EDTA, and protease inhibitors. Proteins were separated using SDS-PAGE (proteins ⬍ 100 kDa were separated on 10% gel and transferred to nitrocellulose membrane whereas those above 100 kDa were separated on 7.5% gel and transferred to polyvinylidene difluoride membrane). Protein loading was normalized by reprobing the same blots with antibodies against the nonphosphorylated form of each protein probed or total ERK. Detection was performed with an enhanced chemiluminescence Western blotting detection system.

Northern blot analysis The effect of mTOR inhibition on FSH-mediated cyclin D2 mRNA expression was examined by treating the cells with rapamycin, a specific inhibitor for mTOR. Cultured cells were incubated with rapamycin (100 nm) or vehicle for 15 min. One group of cells from both control and rapamycin treatment were incubated with FSH (50 ng/ml) for 2 h. At the end of incubation, the cells were harvested, and total RNA was extracted using TRIzol reagent following the manufacturer’s protocol (Life Technologies). Cyclin D2 cDNA (1.1 kb) probe was radiolabeled using [␣-32 P]Deoxy-CTP and RadPrime labeling system and was hybridized to blots overnight at 42 C using 2 ⫻ 107 cpm labeled probe. The hybridized blots were washed and exposed at ⫺70 C to XAR film (Eastman Kodak Co., Rochester, NY). The blots were stripped and rehybridized with cDNA probe corresponding to 18S rRNA to monitor total RNA loading.

Real-time PCR Aliquots of total RNA (50 ng) extracted from the control and experimental groups (rapamycin 100 nm treatment) of granulosa cells were reverse-transcribed in a reaction volume of 20 ␮l using 2.5 ␮m random hexamer, 500 ␮m dNTPs, 5.5 mm MgCl2, 8 U ribonuclease inhibitor, and 25 U multiscribe reverse transcriptase. The reactions were carried out in a PTC-100 (MJ Research, Watertown, MA) thermal controller (25 C for 10 min, 48 C for 30 min, and 95 C for 5 min). The resulting cDNAs were diluted with water. The real-time PCR quantification was then performed using 5 ␮l of the diluted cDNAs in triplicate using predesigned primers and probes for rat cyclin D2 (TaqMan Assay on Demand Gene Expression product). Reactions were carried out in a final volume of 25 ␮l using Applied Biosystems 7300 RealTime PCR system for 40 cycles (95 C for 15 sec, 60 C for 1 min) after initial incubation for 10 min at 95 C. The fold change in cyclin D2 expression was calculated using the standard curve method with 18S rRNA as the internal control.

Statistical analysis Statistical analysis was carried out by unpaired t test using Graphpad Prism computer software (version 3.0 cx; Graphpad Inc., San Diego, CA). Each experiment was repeated at least three times with similar results. Blots are representative of one experiment, and graphs are mean ⫾ se of three experiments.



Results DHT inhibits mTOR signaling

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To determine the involvement of FSH on mTOR-mediated mitogenic signaling, initial experiments were designed to examine the phosphorylation of p70S6K, one of the downstream targets of mTOR, in response to FSH. Granulosa cells from 3-d estradiol-treated immature female rats were harvested and allowed to attach overnight in serum- and phenol red free DMEM. They were stimulated with FSH (50 ng/ml) for different time intervals, and the phosphorylation of p70S6K was examined by Western blot analysis. Results presented in Fig. 1 show that FSH treatment caused a 2-fold increase in the phosphorylation of S6K by 15 min, which was then reduced to initial levels at 60 min. The intermediary role of mTOR in FSH-mediated mitogenesis was then examined by analyzing the mRNA expression of cyclin D2, a cell cycle regulatory protein that controls the G1/S transition in granulosa cells. We have previously shown that decreased cyclin D2 mRNA expression correlates with reduced granulosa cell proliferation (2). Cultured cells were incubated with the mTOR inhibitor rapamycin (100 nm) or vehicle for 15 min before 2 h FSH treatment. The results show that inhibiting mTOR activation significantly reduced FSH-mediated cyclin D2 mRNA expression (Fig. 2, A and B). Toxicity of rapamycin treatment in cultured cells was ruled out using cell viability assay. Rapamycin treatment at 100 nm concentration did not

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FIG. 1. Time course study of FSH treatment on p70S6K phosphorylation in granulosa cells. Granulosa cells were isolated from 3-d estradiol-primed immature rats as described in Materials and Methods. After overnight attachment, cells were treated with FSH (50 ng/ml) for 0, 5, 15, 30, and 60 min. Total protein was extracted, and Western blot analysis was performed. A, Expression of phosphorylated p70S6K; B, the same blot stripped and reprobed with antibody for total ERK2; C, quantitative expression of phosphorylated p70S6K normalized for total ERK. Blots are representative of one experiment, and the graph represents the mean of three experiments. Error bars represent mean ⫾ SE. *, Significant differences (P ⬍ 0.05) when compared with 0 min. Ph, Phosphorylated.

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FIG. 2. Effect of mTOR inhibition on FSH-mediated cyclin D2 mRNA expression. A, Granulosa cells were harvested as described in Materials and Methods. One group of cultures was incubated with mTOR inhibitor (rapamycin, 100 nM) for 15 min, and the other group was incubated with vehicle. After treatment, one set of cultures from both the control and inhibitor-treated groups was treated with FSH (50 ng/ml) for 2 h, and the other set received vehicle. Total RNA from the cells was reverse-transcribed, and the resulting cDNAs were subjected to real-time PCR using predesigned primers and probes for rat Cyclin D2 as described in Materials and Methods. The graph represents the change in cyclin D2 mRNA expression normalized for 18S rRNA. Error bar represents the mean ⫾ SE of three experiments. *, Significant difference (P ⬍ 0.05) when compared with control; different letters represent significant differences (P ⬍ 0.05) between them. B, FSH-mediated cyclin D2 mRNA expression in control and rapamycin-treated cells. Northern blot analysis was performed using total RNA extracted from the four treatment groups. Top, Northern blot analysis using [␣32P]cyclin D2 cDNA probe; middle, expression of 18S rRNA; bottom, densitometric scanning of cyclin D2 mRNA expression normalized for 18S rRNA. Rapa, Rapamycin.


DHT reduces FSH-mediated tuberin (TSC2) phosphorylation

Because DHT treatment showed a reduction in FSH-mediated p70S6K phosphorylation, we examined the upstream molecules in mTOR signaling that may be targets of DHT inhibition. Tuberin (TSC2), a 200-kDa protein that is a component of the TSC1/TSC2 complex, suppresses the mTOR pathway, but phosphorylation of tuberin has been shown to A

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FIG. 3. Effect of DHT treatment on FSH-mediated p70S6K phosphorylation in granulosa cells. The 22-d-old rats were given estradiol injection sc for 3 d. Granulosa cells were isolated and after overnight attachment were incubated with or without DHT (90 ng/ml) for 24 h. Control groups received equal amounts of vehicle. Subsequently, one set from both control and DHT-treated cells was incubated with FSH (50 ng/ml) for 15 min, and the other set received vehicle. Reaction was stopped by removing the media, and cell lysate was prepared using RIPA buffer. Equal amounts of total protein were separated on 10% SDS-PAGE and transferred to nitrocellulose. A, Expression of phosphorylated p70S6K; B, expression of nonphosphorylated total p70S6K in the same blot; C, phosphorylated p70S6K expression normalized for total p70S6K. Blots are representative of one experiment, and the graph represents the mean of three experiments. Error bars represent mean ⫾ SE. *, Significant difference (P ⬍ 0.05) when compared with control; a and b represent significant difference (P ⬍ 0.05) between them. Ph, Phosphorylated.

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reduce granulosa cell viability compared with control in a 48-h study (data not shown). After establishing the role of mTOR in FSH-mediated cyclin D2 mRNA expression, the effect of DHT was examined. To this end, granulosa cells were cultured in medium containing DHT (90 ng/ml) or vehicle for 24 h. Subsequently, one set of cells from both control and DHT-treated cultures was stimulated with FSH (50 ng/ml) for 15 min. As expected, FSH treatment increased the expression of phosphorylated p70S6K in control cells, whereas pretreatment with DHT significantly reduced this stimulation as seen in the Western blot (Fig. 3). This result show that DHT exerts an inhibitory effect on FSH-mediated mTOR signaling in granulosa cells.


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FIG. 4. Effect of DHT treatment on FSH-stimulated TSC2 phosphorylation in granulosa cells. Granulosa cells from 3-d estradiol-primed immature rats were harvested. Cells were allowed to attach overnight in serum free, phenol red free DMEM-F12. One group was then incubated with DHT (90 ng/ml) for 24 h and the other with vehicle. After incubation, one set of cultures from both the control and DHTtreated groups was stimulated with FSH (50 ng/ml) for 10 min, whereas the second set received an equal amount of vehicle. Total TSC2 was immunoprecipitated from the lysate as described in Materials and Methods. A, Western blot analysis for phosphorylated TSC2 was performed; B, expression of total TSC2 used to monitor protein loading; C, phosphorylated TSC2 expression normalized for total TSC2 in three separate experiments. Blots are representative of one experiment. Error bars represent mean ⫾ SE. *, Significant differences (P ⬍ 0.05) when compared with control; a and b represent significant difference (P ⬍ 0.05) between them. Ph, Phosphorylated.

abrogate its inhibitory effect on mTOR (21). Cultured cells were pretreated with or without DHT for 24 h and stimulated with FSH for 10 min. The cells were harvested, and Western blot analysis was performed to determine tuberin phosphorylation. The results showed a significant increase in tuberin phosphorylation in response to FSH (Fig. 4). However, in DHT-treated cells, the stimulatory effect of FSH was significantly reduced (Fig. 4). Thus, DHT-mediated inhibition of tuberin phosphorylation might account for the reduced p70S6K activation in DHT-exposed cells. Akt inhibition does not inhibit FSH-mediated TSC2 phosphorylation in granulosa cells

Because it is now well established that TSC2 phosphorylation is dependent on Akt phosphorylation in response to insulin and other growth regulators, the involvement of Akt in response to FSH was examined. To test this, cultured granulosa cells were preincubated with an Akt-specific inhibitor (AKT inhibitor VIII, 5 ␮m) for 15 min followed by stimulation with FSH for 10 min. Total tuberin was immunoprecipitated from the whole-cell lysates, and Western blot

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analysis was performed. The blots were probed with antibody against phosphorylated TSC2. The results show that inhibition of Akt did not elicit any significant reduction in FSH-mediated TSC2 phosphorylation (Fig. 5). The inhibitory potential of the compound was verified by examining the phosphorylation of Akt in insulin-stimulated granulosa cells under the same experimental conditions. Pretreatment with inhibitor significantly reduced insulin-mediated phosphorylation of Akt (Fig. 6). We also confirmed the specificity of the inhibition by examining FSH-mediated ERK phosphorylation in the inhibitor-treated cells. Cultured cells after 15 min preincubation with Akt inhibitor were stimulated with FSH (50 ng/ml) for 15 min. Western blot analysis showed that pretreatment with inhibitor abolished FSH-mediated Akt phosphorylation while producing no effect on FSH-stimulated ERK phosphorylation (Fig. 7). These results clearly show the Akt-independent activation of mTOR in response to FSH treatment. Inhibition of ERK pathway reduces FSH-mediated tuberin phosphorylation

Because Akt inhibition did not abolish FSH-mediated activation of mTOR signaling, the possible role of ERK as an upstream activator of mTOR in FSH signaling was examined. To test this possibility, cultured granulosa cells were pretreated with inhibitors (20 ␮m PD 98059 for 1 h or 30 ␮m U0126 for 30 min) or vehicle followed by treatment with FSH A

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FIG. 6. Effect of Akt inhibition on insulin-stimulated Akt phosphorylation in granulosa cells. Granulosa cells from 3-d estradiol-primed immature rats were cultured in serum free, phenol red free medium. After overnight attachment, one group of cultures was treated with Akt inhibitor (5 ␮M) for 15 min, and the other group received vehicle. After incubation, one set of cultures from both the control and inhibitor-treated groups was stimulated with insulin (100 nM) for 15 min. A, Western blot analysis was performed with antibody for phosphorylated Akt; B, same blot stripped and reprobed with antibody for total ERK2, showing protein loading. AktINH, Akt inhibitor; Ph, Phosphorylated.

(50 ng/ml) for 10 min. After cell harvest, total tuberin was immunoprecipitated from cell lysates, proteins were separated on 7.5% SDS-PAGE, and Western blot analysis was performed. The results show that ERK inhibition significantly reduced FSH-mediated TSC2 phosphorylation as evidenced by the reduced expression of the 200-kDa band, which corresponds to TSC2 (Fig. 8, A and B). To further confirm the involvement of the ERK pathway in FSH-mediated TSC2 phosphorylation, we used H89 to block PKA activation because PKA inhibition has been previously shown to abrogate the ERK pathway. Cultures treated with PKA inhibitor H89 (10 ␮m) or vehicle for 1 h followed by FSH treatment for 10 min showed an inhibition of FSH-mediated tuberin phosphorylation (Fig. 9). These results demonstrate that FSH stimulates mTOR signaling through a PKA- and ERK-dependent pathway in cultured granulosa cells. Discussion

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FIG. 5. Effect of Akt inhibition on FSH-stimulated TSC2 phosphorylation. The 3-d estradiol-primed immature rats were used for the experiments. Cells were harvested as described in Materials and Methods. One group of cultures after overnight attachment was incubated with Akt inhibitor VIII (5 ␮M) for 15 min, and the other group was incubated with vehicle. After treatment, one set of cultures from both the control and inhibitor-treated groups was stimulated with FSH for 10 min, and the other set received vehicle. TSC2 protein was immunoprecipitated, and Western blot was performed. A, Expression of phosphorylated TSC2; B, total TSC2 expression; C, phospho-TSC2 expression normalized for total TSC2 in three separate experiments. *, Significant differences (P ⬍ 0.05) when compared with control. AktINH, Akt inhibitor; Ph, Phosphorylated.

It is now well known that the high level of 5␣-reduced metabolites of androgen associated with hyperandrogenism is one of the leading causes of anovulation in women of reproductive age. We have established the inhibitory role of DHT in both FSH- and insulin-mediated mitogenesis of granulosa cells (1–3). In the present studies, we examined the effect of DHT exposure on FSH-mediated mTOR signaling. Our results show that FSH stimulates mTOR signaling as evidenced by the increased phosphorylation of p70S6K, the target molecule of mTOR, in a time-dependent manner (Fig. 1). Blocking mTOR activation using rapamycin reduced FSHmediated cyclin D2 mRNA expression (Fig. 2, A and B). Inhibition of FSH-mediated Akt phosphorylation did not elicit any response in tuberin phosphorylation, indicating that FSH-mediated mTOR signaling in granulosa cells is independent of Akt (Fig. 5). On the other hand, ERK inhibition leads to reduced FSH-mediated tuberin phosphorylation (Fig. 8, A and B). These results suggest that FSHmediated mTOR signaling proceeds mainly through the ERK- rather than the Akt-dependent pathway in granulosa cells. Prior exposure of cells to DHT resulted in a reduction of FSH-mediated mTOR signaling as evidenced by the reduced phosphorylation of TSC2 and the downstream target of mTOR, p70S6K (Figs. 3 and 4).



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FIG. 7. Effect of Akt inhibition on FSH-stimulated Akt and ERK phosphorylation in granulosa cells. Granulosa cells from 3-d estradiol-primed immature rats were isolated. After overnight attachment, one group of cultures received Akt inhibitor (5 ␮M) for 15 min, and the other two groups received vehicle. Subsequently, one set of cultures from vehicle- and Akt inhibitor-treated groups were stimulated with FSH (50 ng/ml) for 15 min, and the third group served as control. Equal amounts of total protein were separated by SDS-PAGE and transblotted to nitrocellulose. A, Blot was probed with antibody for phosphorylated Akt; B, same blot stripped and reprobed with antibody for phosphorylated ERK; C, expression of total ERK2. AktINH, Akt inhibitor; Ph, Phosphorylated.

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The mTOR signaling pathway is a critical component of the growth and proliferation machinery of cells (12). Hormones such as insulin and growth factors are known to stimulate growth and mitogenesis by activating mTOR signaling (10). Because this signaling pathway integrates proliferation and growth and responds to both nutrient and mitogenic factors (7), we examined its proliferative function in granulosa cells under normal as well as hyperandrogenic conditions. Mitogens and growth factors stimulate mTOR through a PI3-kinase/Akt-dependent pathway. From the activated mTOR, signals flow to two downstream targets, ribosomal S6 kinase (S6K) and eukaryotic elongation factor 4E binding protein (4E-BP1) to stimulate translation (22). Two S6K proteins S6K1 and S6K2, have been characterized in mammalian cells (23). S6K1 is the major S6K in mammalian cells (24, 25) and has been used for most of the studies on cell growth (12). S6K1 is activated by phosphorylation at multiple residues. One of these residues, Thr 389 is directly phosphorylated by mTOR (26, 27) and is used as a readout of mTOR activity (28). In our studies, incubation with 50 ng/ml FSH for different time intervals showed a significant increase in phosphorylation of p70S6K (⬃2-fold), initiating at 15 min and lasting up to 30 min during the time periods tested (Fig. 1). This result is in agreement with the findings of Alam et al. (19) that FSH stimulates mTOR signaling in granulosa cells. Furthermore, the present study suggests that FSH-mediated mitogenesis of granulosa cells is regulated, at least partially, through the mTOR pathway because treatment with rapamycin reduced FSH-mediated cyclin D2 mRNA expression (Fig. 2, A and B). Cyclin D2 is one of the D-type proteins that regulate the progression of the cell cycle from G1 to S phase in response to extracellular signals (29). In transgenic mice lacking functional cyclin D2 (cyclin D2⫺/⫺), FSH- and forskolin-mediated granulosa cell proliferation was abolished (30). Therefore, the reduction of cyclin D2 mRNA expression in rapamycin-treated cells is suggestive of a role for mTOR signaling in FSH-mediated granulosa cell proliferation. Exposing granulosa cells to DHT for 24 h before FSH treatment significantly reduced

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FIG. 8. Effect of ERK inhibition on FSH-stimulated TSC2 phosphorylation in granulosa cells. Immature female rats (22 d) were administered estradiol for 3 d. Twenty-four hours after the last estradiol administration, granulosa cells were isolated and cultured. After overnight attachment, one group of cultures was incubated with ERK inhibitor PD 98059 (20 ␮M) for 1 h (A) or with 30 ␮M U0126 for 30 min (B). Control groups were treated 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 10 min. Top, Total TSC2 was immunoprecipitated from the cell lysates, and Western blot analysis was performed using phospho-TSC2 antibody; middle, total TSC2 expression was used to monitor protein loading; bottom, phosphoTSC2 expression normalized for total TSC2 in three separate experiments. Blots are representative of one experiment. Error bars represent mean ⫾ SE. *, Significant differences (P ⬍ 0.05) when compared with control, and different letters represent significant differences (P ⬍ 0.05) between them. PD, PD 98059; Ph, Phosphorylated.



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luteal cells through an Akt-independent pathway (33). Furthermore, there is evidence that mTOR signaling can be activated by both PI3-kinase as well as ERK in response to mitogens in certain cell types (34). Our results are in agreement with these findings. Treatment of granulosa cells with FSH stimulated TSC2 (Fig. 4) and Akt phosphorylation (Fig. 7), but the use of an Akt inhibitor did not produce any reduction in FSH-mediated tuberin phosphorylation (Fig. 5), demonstrating that FSH stimulates TSC2 phosphorylation through an Akt-independent pathway. The inhibitory action of this compound was specific toward Akt because it produced no inhibitory effect on FSHmediated ERK phosphorylation (Fig. 7). FSH is known to regulate granulosa cell functions through an ERK-dependent pathway. Additionally, in our previous studies, we have shown that DHT reduces FSH-mediated mitogenesis by reducing ERK phosphorylation (1). Inhibition of ERK phosphorylation demonstrated a significant reduction in FSHmediated TSC2 phosphorylation, confirming the involvement of ERK in FSH-mediated mTOR signaling (Fig. 8, A and B). The contribution of the ERK pathway in FSH-mediated TSC2 phosphorylation was further strengthened by the demonstration that inhibition of PKA resulted in reduced FSH-mediated TSC2 phosphorylation (Fig. 9). Previous studies from our laboratory

FIG. 9. Effect of PKA inhibition on FSH-stimulated TSC2 phosphorylation. Granulosa cells were isolated from 3-d estradiol-primed immature rats and cultured in serum free, phenol red free DMEM-F12. After overnight attachment, one group of cultures was incubated with PKA inhibitor H89 (20 ␮M) for 60 min, and the other group was incubated with vehicle. After the incubation, one set of cultures from both the control and H89-treated groups was stimulated with FSH (50 ng/ml) for 10 min. Western blot analysis was performed using immunoprecipitated TSC2 as described in Materials and Methods. A, Expression of phosphorylated TSC2; B, expression of total TSC2; C, phospho-TSC2 expression normalized for total TSC2. Blots are representative of one experiment, and the graph represents the mean of three experiments. Error bars represent mean ⫾ SE. *, Significant difference (P ⬍ 0.05) when compared with control, and different letters represent significant differences (P ⬍ 0.05) between them. Ph, Phosphorylated.

p70S6K phosphorylation compared with controls treated with FSH alone (Fig. 3), demonstrating the inhibitory effect of DHT on FSH-mediated mTOR signaling. Activation of mTOR signaling is regulated by TSC, which consists of two parts, hamartin (TSC1) and tuberin (TSC2) (21). TSC1 maintains the stability of the complex, whereas TSC2 is the functional component of the complex (31). Tuberin inhibits mTOR activity by its GTPase-activating protein activity on Rheb, which is required for mTOR activation (18). Phosphorylation of tuberin by mitogens and growth factors reverses its inhibitory effect on Rheb and thereby activates the mTOR pathway (8, 31, 32). Our results showing granulosa cells treated with FSH produced an increase in TSC2 phosphorylation whereas DHT reduced the extent of this stimulation (Fig. 4) underscores the inhibitory effect of DHT on mTOR signaling. Because reduced TSC2 phosphorylation leads to reduced p70S6K activation (16), these results indicate that the inhibitory effect of DHT on p70S6K might be due to reduced TSC2 phosphorylation. Although mitogens as well as nutrients are known to regulate mTOR signaling through PI3-kinase- and Akt-dependent phosphorylation of TSC2, activation of mTOR might also proceed through alternate pathways. For example, prostaglandin F2␣ has been shown to stimulate TSC2 phosphorylation in

FSH

cAMP

PKA

ERK Tuberin

mTOR

Cyclin D2 mRNA

Cell proliferation FIG. 10. Schematic representation of the involvement of mTOR in FSH-mediated granulosa proliferation. Results from our experiments suggest that, in addition to the established cAMP-PKA-ERK pathway, FSH also stimulates cyclin D2 mRNA expression in granulosa cells through the mTOR pathway. The increase in cyclin D2 mRNA expression appears to occur through a PKA-ERK-dependent phosphorylation of tuberin and subsequent activation of mTOR pathway.


as well as by others had established that FSH stimulates the cAMP-PKA-dependent pathway and activates ERK phosphorylation in granulosa cells (1, 35). Because the Akt inhibitor we used did not affect ERK activation, the increased TSC2 phosphorylation observed in FSH-treated cells appears to occur through an ERK-dependent pathway. Our results are in agreement with those of Rolfe et al. (36) and Ma et al. (37), who showed ERK-dependent phosphorylation impairing tuberin’s ability to inhibit mTOR signaling. We conclude that in granulosa cells, FSH regulates cyclin D2 mRNA expression in part through mTOR, the growth and proliferative signaling pathway. This involves a PKA-ERKdependent phosphorylation of tuberin, by regulating its inhibitory effect on mTOR (Fig. 10). DHT exerts an inhibitory effect on mTOR signaling by reducing phosphorylation of tuberin as well as S6K, the key molecules in the mTOR signaling, by inhibiting PKA-ERK activation. Based on these results, we speculate that the inhibitory effect of DHT on granulosa cell proliferation might occur, in part, through the inhibition of FSH-stimulated mTOR signaling. Acknowledgments We express our appreciation to Dr. Anil Nair, Dr. Palaniappan Murugesan, Helle Peegel for critical reading of the manuscript, and Dr. Kun-Liang Guan for helpful discussion. Received February 13, 2007. Accepted May 10, 2007. 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 Medical School, Ann Arbor, Michigan 48109. E-mail: [email protected]. This work was supported by National Institutes of Health Grant HD-38424. Disclosure Statement: The authors have nothing to disclose.

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