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. 2012 Mar 27;153(6):2831–2838. doi: 10.1210/en.2011-1967

AMPK Activation by Dihydrotestosterone Reduces FSH-Stimulated Cell Proliferation in Rat Granulosa Cells by Inhibiting ERK Signaling Pathway

Pradeep P Kayampilly 1, K M J Menon 1,✉
PMCID: PMC3359611  PMID: 22454147

Abstract

We have previously reported that 5α-dihydrotestosterone (DHT) inhibits FSH-mediated granulosa cell proliferation by reducing cyclin D2 mRNA expression and blocking cell cycle progression at G1/S phase. The present study investigated the role of AMP activated protein kinase (AMPK) in DHT-mediated inhibition of granulosa cell proliferation. Granulosa cells harvested from 3-d estradiol primed immature rats were exposed to different concentrations of DHT (0, 45, and 90 ng/ml) for 24 h. Western blot analysis of immunoprecipitated AMPK showed a dose-dependent activation (P < 0.05) as evidenced by the increased phosphorylation at thr 172. In addition, time-courses studies (0, 6, 12, and 24 h) using DHT (90 ng/ml) showed a time-dependent increase in AMPK activation with maximum effect at 24 h. FSH inhibited AMPK phosphorylation and promoted granulosa cell proliferation, but pretreatment with DHT (90 ng/ml) for 24 h prior to FSH treatment reduced this effect. Pharmacological activation of AMPK with 5-aminoimidazole-4-carboxamide-1-β4-ribofuranoside abolished FSH-mediated ERK phosphorylation, indicating that AMPK is a negative upstream regulator of ERK. Furthermore, inhibition of AMPK activation by compound C reversed the DHT-mediated reduction in positive cell cycle regulator, cyclin D2, and 5-bromo-2′-deoxyuridine incorporation. These results suggest that elevated levels of DHT activate AMPK, which in turn inhibits ERK phosphorylation. Thus, inhibition of ERK phosphorylation by activated AMPK in response to DHT might contribute to decreased granulosa cell mitogenesis and ovulatory dysfunction seen in hyperandrogenic states.

The optimum growth of somatic cell types in the ovarian follicle is necessary for the normal ovulatory process (1). Gonadotropic hormones and other growth factors regulate both steroidogenesis and the growth and proliferation of these cells, which are critical for normal ovulation (2–4). In pathophysiological conditions such as polycystic ovarian syndrome (PCOS), these highly synchronized processes of growth and proliferation are disrupted, leading to ovulatory failure. It is now well established that hyperandrogenism is one of the main diagnostic features of PCOS (5). Furthermore, it has been reported that in PCOS patients androgens are converted to 5α-reduced metabolites at higher levels compared with control patients (6–10). Higher levels of insulin due to insulin resistance, which often coexists with hyperandrogenism, augment the expression of 5α-reductase, the enzyme that converts androgens to their 5α-reduced metabolites (11). We have shown that 5α-reduced metabolites of androgens such as 5α-dihydrotestosterone (DHT) can reduce FSH-mediated granulosa cell mitogenesis (12).

Our previous reports and studies from other laboratories have established that FSH uses multiple signaling pathways to increase granulosa cell mitogenesis (13–18). Recently we have shown that FSH promotes granulosa cell mitogenesis by inhibiting the AMP activated protein kinase (AMPK). FSH-treatment inhibited AMPK activation, which in turn reduced the expression of the cell cycle inhibitor molecule p27kip. Activation AMPK, on the other hand, resulted in increased p27 kip expression (18).

In the present study we have examined the role of AMPK in DHT-mediated inhibition of granulosa cell mitogenesis. Our results show that DHT activates AMPK in a time- and dose-dependent manner and reduces FSH-mediated mitogenic signaling, leading to the inhibition of granulosa cell proliferation.

Materials and Methods

The phenol red free DME-F12 medium and Trizol reagent were the products of Life Technologies Inc. (Gaithersburg, MD). Ovine FSH (NIDDK-oFSH-20) was purchased from Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA). DHT (5α-androstan-17β-3-one) and AMPK activator 5-aminoimidazole-4-carboxamide-1-β4-ribofuranoside (AICAR), inhibitor compound C, [6-(4-[2-piperidn-1-ylethoxy] phenyle)-3-pyridin-4-ylpyrazolo (1,5-a)pyrimidine] and β-tubulin antibody were purchased form Sigma (St. Louis, MO). AMPK as well as ERK antibodies and antigoat IgG horseradish peroxidase conjugates were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phosphorylated AMPK, Akt, and ERK as well as antimouse and antirabbit IgG horseradish peroxidase conjugates were from Cell Signaling Technology Inc. (Beverly, MA). The 5-bromo-2′-deoxyuridine (BrdU) cell proliferation kit and phosphatase inhibitor cocktail set II were from Calbiochem (La Jolla, CA). Protein G agarose beads were obtained from Upstate Cell Signaling Solutions (Lake Placid, NY). Reagents as well as the primers and probes for the cyclin D2 real-time PCR were from Applied Biosystems (Foster City, CA). Western blot chemiluminiscent detection kit (SuperSignal West Femto maximum sensitivity substrate) was from Thermo Scientific (Rockford, IL).

Animals and treatments

Immature female rats (22–23 d old, Sprague Dawley strain) were purchased from Charles River Laboratories (Wilmington, MA). The animals were kept and used under the guidelines from the University Committee on the Use and Care of Animals. They were housed in a temperature-controlled room with the proper dark-light cycles (12 h light, 12 h dark) 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 serum free, phenol red-free DME-F12 medium.

Granulosa cell isolation and culture

Granulosa cells from immature female rats were harvested as described previously (12). 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 at 37 C. After incubation, the suspension was diluted with 3 vol DMEM-F12, centrifuged at 250 × g, and treated sequentially with trypsin (20 μg/ml) for 1 min, 300 μg/ml soybean trypsin inhibitor for 5 min, and deoxyribonuclease I (100 μg /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 cell number was determined. Cell viability was examined by the trypan blue exclusion method. Cells were cultured in serum free DME-F12 media supplemented with 20 mm HEPES (pH 7.4), 4 mm glutamine, 100 IU penicillin/ml, and 100 μg /ml streptomycin. Before seeding, the culture dishes were coated with 10% fetal calf serum for 2 h at 37 C and washed with DMEM-F12.

Western blot analysis

To examine the dose- and time-dependent effects of DHT on AMPK, granulosa cells, after overnight attachment, were treated with different doses of DHT (0, 45, and 90 ng/ml) for 24 h or treated with 90 ng/ml DHT for 0, 6, 12, and 24 h. In those studies in which AMPK activation or inhibition was studied, AMPK activation was achieved by pretreating the cells with AICAR (0.5 mm) for 1 h and AMPK inhibition by incubating cells with compound C (20 μm) for 1 h before hormone treatment. These preincubations were followed by FSH treatment for the time intervals indicated in each experiment. Reactions were stopped by removing the media, and total protein was solubilized using radioimmunoprecipitation assay buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 50 mm NaF, and 0.1% sodium dodecyl sulfate) or AMPK buffer (15 mm HEPES, 137 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 10 mm sodium pyrophosphate, 2 mm EDTA, 50 mm NAF, 1% Nonidet P-40, 10% glycerol, phosphatase inhibitor cocktail, and protease inhibitors). For AMPK immunoprecipitation, 1.5 μg of AMPK antibody was added to equal amount of total protein and incubated overnight at 4 C. After the overnight incubation, 30 μl of protein G agarose beads was added to lysate and incubated for additional 3 h. At the end of incubation, the agarose beads were pelleted by centrifugation at 120,000 × g for 3 min. Thirty microliters of SDS-PAGE sample buffer were added to the beads and heated for 5 min at 100 C. Proteins were separated using SDS-PAGE (4–20%) and transferred to nitrocellulose membrane and probed with specific antibodies. Protein loading was normalized by reprobing the same blots with either total AMPK or total ERK as indicated in each experiment. Detection of signals was performed with SuperSignal West Femto maximum sensitivity Western blotting detection system (Thermo Scientific).

In coimmunoprecipitation experiments, granulosa cells were treated with DHT (90 ng/ml) for different time intervals (0, 12, and 24 h). After incubation, total cell lysates were prepared using AMPK lysis buffer as described earlier. AMPK immunoprecipitates as well as total lysates from each experimental group were separated on a 4–20% gel. The blots were initially probed with phosphorylated AMPK (pAMPK) followed by ERK and total AMPK. The purity of immunoprecipitated samples was confirmed by probing the same blot with antibody against β-tubulin.

Real-time PCR

Aliquots of total RNA (50 ng) extracted from different experimental groups were reverse transcribed in a reaction volume of 20 μl using 2.5 μm random hexamer, 500 μm deoxynucleotide triphosphate, 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 real-time PCR quantification was then performed using 5 μl of the diluted cDNA in triplicates and predesigned primers and probes for cyclin D2 (TaqMan Assay on Demand gene expression product, Applied Biosystems, Foster City, CA). Reactions were carried out in a final volume of 25 μl using an Applied Biosystems 7300 real-time PCR system for 40 cycles (95 C for 15 sec and 60 C for 1 min) after the initial incubation for 10 min at 95 C. The fold change in cyclin D2 expression was calculated using the 2-ΔΔCT method (19) with 18S rRNA as the internal control.

BrdU cell proliferation assay

The role of AMPK in DHT-mediated inhibition of cell proliferation was evaluated by measuring the incorporation of BrdU using a BrdU immunoassay kit (Calbiochem) following the manufacturer's protocol. In brief, granulosa cells were plated at a density of 20,000/well in 96-well plates. After overnight attachment they were treated with DHT (90 ng/ml) or vehicle for 24 h followed by AMPK inhibitor (compound C, 20 mm) for an additional 1 h. After incubation, cells were treated with FSH (75 ng/ml) for 6 h along with the BrdU label. The reactions were terminated by removing the media, and cells were incubated with fixative/denaturing solution followed by BrdU antibody for 1 h at room temperature. Unbound antibody was washed away, and then horseradish peroxidase-conjugated goat antimouse IgG was added for 30 min at room temperature. After washing three times, substrate was added and incubated in the dark for 15 min. The plates were read using a spectrophotometric plate reader.

Statistical analysis

Statistical analysis was carried out using one-way ANOVA followed by Tukey multiple comparisons 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. Each blot is a representative of one experiment and the graphs are mean ± se of three experiments. Values were considered statistically significant at P < 0.05.

Results

DHT activates AMPK in a time- and dose-dependent manner

The effect of DHT on AMPK regulation was studied by examining the expression of AMPK phosphorylated at Thr 172. Phosphorylation of this residue in the α-subunit has been shown to be necessary for AMPK activation (20, 21). AMPK immunoprecipitated from total cell lysates of cell cultures treated with different doses of DHT (0, 45, and 90 ng/ml) for 24 h showed a dose-dependent increase in AMPK phosphorylation at thr 172 (Fig. 1A) with a significant increase at 90 ng/ml (P < 0.05). To examine the time-dependent effect of DHT on AMPK, incubations were carried out for 0, 6, 12, or 24 h using 90 ng/ml DHT, and total AMPK was immunoprecipitated from total cell lysates. The results showed that AMPK activation was significantly (P < 0.05) increased by 12 h of DHT treatment, and maximum activation was observed at 24 h (Fig. 1B). Together these results indicate that DHT activates AMPK in a dose- and time-dependent manner.

Fig. 1.

Fig. 1.

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A and B, Dose- and time-dependent effect of DHT on AMPK phosphorylation in rat granulosa cells. Granulosa cells from 3-d estradiol-primed immature rats were harvested and allowed to attach overnight in serum-free, phenol red-free medium. To study the dose-dependent effect of DHT on AMPK (Fig. 1A), cells were treated with different doses of DHT (0, 45, and 90 ng/ml) for 24 h. To examine the time-dependent effect of DHT on AMPK (B), cells were treated with 90 ng/ml DHT for different time periods (0, 6, 12, and 24 h). AMPK protein was immunoprecipitated from equal amounts of total protein, separated using SDS-PAGE on a 4–20% gradient gel, and transferred to nitrocellulose for Western blot analysis. Panel A, Expression of pAMPK. Panel B, Expression of total AMPK. Panel C, Densitometric scanning of pAMPK protein expression normalized to total AMPK and representing the fold difference compared with 0. Error bars represent mean ± se. *, Significant difference (P < 0.05) when compared with 0. Letters a and b represent significant differences between them.

DHT abolishes FSH-mediated inhibition of AMPK

Previous studies from our laboratory show that the mitogenic effect of FSH is also mediated by inhibiting AMPK activation (18). DHT, on the other hand, is known to reverse the effects of FSH in mitogenic signaling pathways (15, 22). Thus, the effect of DHT on FSH-mediated inhibition of AMPK was examined. Granulosa cells after overnight attachment were incubated with DHT (90 ng/ml) for 24 h followed by FSH (75 ng/ml) for 15 min. AMPK was immunoprecipitated from total cell lysates and Western blot analysis for pAMPK was carried out. As expected, FSH treatment reduced AMPK phosphorylation compared with control; however, pretreatment with DHT significantly reversed this response (Fig. 2).

Fig. 2.

Fig. 2.

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Effect of DHT treatment on FSH-mediated inhibition of AMPK phosphorylation. Granulosa cells were isolated from 3-d estradiol-primed immature rats as described in Materials and Methods. After overnight attachment, cells were treated with DHT (90 ng/ml) for 24 h, after which they were incubated with FSH (75 ng/ml) for an additional 15 min. Immunoprecipitation of AMPK was carried out from equal amounts of total protein and were separated on 4–20% gel. The blots were probed with antibody against pAMPK (A) and protein loading was normalized by reprobing with antibody against total AMPK (B). C, Densitometric scanning of pAMPK protein expression normalized to total AMPK. Error bars represent mean ± se. *, Significant difference (P < 0.05) when compared with control, and different letters (a and b) represent significant differences (P < 0.05) between them. CONT, Control; D, DHT.

DHT-mediated inhibition of ERK occurs through AMPK-dependent pathway

We next examined whether AMPK activation affects FSH-mediated ERK phosphorylation because DHT is known to inhibit cell cycle progression by reducing ERK activation (15). AICAR is a pharmacological activator of AMPK, which has been extensively used to activate AMPK in various cell types (23). As shown in Fig. 3, pretreatment with 0.5 mm AICAR for 1 h significantly reduced the FSH-mediated ERK phosphorylation, whereas FSH-mediated Akt phosphorylation was not affected, suggesting that ERK is a specific downstream target of AMPK. The role of AMPK as a regulator of ERK was strengthened by the interaction between these two molecules observed in coimmunoprecipitation studies. Total AMPK was immunoprecipitated, using a monoclonal antibody against AMPK α1/2-subunits, from cells treated with DHT for different time intervals. Western blot analysis of immunoprecipitated samples demonstrated the presence of ERK protein (Fig. 4) in immunoprecipitates. The presence of ERK protein was further confirmed by running total cell lysates along with the immunoprecipitated samples. As shown in Fig. 4C, total cell lysates from the three experimental time intervals also showed ERK protein at the same position. Collectively these observations suggest that AMPK is an upstream negative regulator of ERK and AMPK activation reduces FSH-mediated ERK phosphorylation.

Fig. 3.

Fig. 3.

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Effect of AMPK activation on FSH-mediated ERK and Akt phosphorylation. Three-day estradiol-primed immature rats were used for the experiments. Cells were harvested as described in Materials and Methods. After overnight attachment, one group of cultures was incubated with AMPK activator (AICAR, 0.5 mm) for 1 h, and the second group served as control. After the treatments, one set of cultures from both the control and activator-treated groups was stimulated with FSH (75 ng/ml) for 15 min, and the other received vehicle. Equal amounts of protein were separated on a 4–20% gel, and Western blot analysis was carried out using phosphorylated ERK (pERK) antibody (A). The same blot was stripped and reprobed using an antibody against phosphorylated Akt (pAkt; B). Protein loading was monitored by reprobing the same blot with total ERK antibody (C). Densitometric scanning of pERK (hatched bar) and pAkt (solid bar) protein expression normalized to total ERK. Error bars represent mean ± se. *, Significant difference (P < 0.05) when compared with control, and different letters (a and b) represent significant differences (P < 0.05) between them. CONT, Control.

Fig. 4.

Fig. 4.

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ERK coimmunoprecipitates with AMPK. Rat granulosa cells after overnight attachment were treated with DHT (90 ng/ml) for different time intervals (0, 12, and 24 h). After incubation total AMPK was immunoprecipitated with AMPK antibody (monoclonal) from equal amounts of total protein. Fifty micrograms of total protein without immunoprecipitation from each sample were also used as input to examine the purity of immunoprecipitates (β-tubulin) as well as the consistency of size for the ERK protein. Western blot analysis was carried out for pAMPK (A), total AMPK (B), total ERK (C), β-tubulin (D) and IgG (E) respectively. ip, Immunoprecipitated sample; Ly, total cell lysate without immunoprecipitation.

AMPK inhibitor reverses DHT-mediated reduction of cyclin D2 expression and BrdU labeling in granulosa cells

Because DHT-mediated inhibition of cell proliferation appears to proceed through AMPK activation, we tested whether reversing this effect would reduce DHT's inhibitory effect on proliferation, using cyclin D2 mRNA as a proliferation marker. Granulosa cells after overnight attachment were incubated with DHT (90 ng/ml) or vehicle for 24 h. After incubation, the DHT treated cultures were pretreated with compound C (20 μm) for 1 h followed by FSH treatment for 2 h. Compound C is a reversible competitive inhibitor of AMPK and is widely used to inhibit AMPK (24). Control groups received either compound C or FSH. Real-time PCR analysis showed that DHT exposure significantly (P < 0.05) reduced FSH mediated increases in cyclin D2 expression (Fig. 5). However, inhibiting AMPK activation by pretreatment with compound C significantly (P < 0.05) overcame DHT's inhibitory effect on FSH-stimulated cyclin D2 expression. These findings were further confirmed using BrdU incorporation assay. As in the case of cyclin D2 mRNA, DHT treatment reduced FSH-mediated stimulation of BrdU incorporation, whereas inhibition of AMPK by compound C reversed this effect (Fig. 6).

Fig. 5.

Fig. 5.

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Effect of compound C on DHT-mediated inhibition of cyclin D2 mRNA expression. After overnight attachment, granulosa cells harvested from immature rats were treated with DHT (90 ng/ml) for 24 h followed by AMPK inhibitor compound C (20 mm) for 1 h. At the end of incubation, the cells were treated with FSH (75 ng/ml) for 2 h. The reaction was stopped by removing the media, and total RNA was reverse transcribed as described in Materials and Methods. Real-time PCR of the cDNA was conducted using predesigned primers and probes for rat cyclin D2 and normalized with 18S rRNA. Each bar represents the fold change in cyclin D2 mRNA expression compared with control. Error bars represent the mean ± se of three experiments, each one of which had three determinants per treatment. *, Significant difference (P < 0.05) when compared with control. Different letters (a, b, and c) represent significant differences (P < 0.05) between them. CONT, Control; D, DHT; F, FSH; C, compound C.

Fig. 6.

Fig. 6.

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Effect of compound C on DHT-mediated inhibition of BrdU incorporation. Granulosa cells (20,000/well) from estradiol-treated immature rats were plated in 96-well plates and allowed to attach overnight in serum free-phenol red-free media. After attachment, the media were removed and cells received fresh media with (treatment group) or without (control group) DHT (90 ng/ml) for 24 h followed by compound C (20 μm) for an hour. The cells were then treated with FSH (75 ng/ml) and BrdU label (20 μl) for 6 h. A BrdU cell proliferation assay was carried out as described in Materials and Methods. Error bars represent the mean ± se of three experiments, each one of which had three determinants per treatment. *, Significant difference (P < 0.05) when compared with control, and different letters (a, b, and c) represent significant differences (P < 0.05) between them. CONT, Control; F, FSH; D, DHT; C, compound C.

Discussion

AMPK signaling is an energy-sensitive mechanism that maintains the optimum energy levels in the cell by balancing the supply and demand for ATP (25). Lower energy levels activate AMPK to curtail energy-consuming anabolic processes such as protein synthesis and cell proliferation while promoting energy generating catabolic processes (26). Dysregulation of AMPK signaling pathway has been implicated in disease states such as metabolic syndrome (27). Recent reports suggest that, in addition to monitoring the energy levels, AMPK could also mediate hormonal responsiveness in normal cells (28, 29). For example, we have reported that FSH stimulates granulosa cell proliferation by inhibiting AMPK activity, indicating that AMPK signaling also serve as part of the regulatory mechanism controlling cell function (18).

Our earlier studies showed that under hyperandrogenic states DHT, a 5α-reduced androgen, exerts an inhibitory effect on FSH-stimulated granulosa cell proliferation by reducing cyclin D2 mRNA expression (12). Cyclin D2 is a positive regulator of the cell cycle at G1-S phase, and therefore, its expression is considered as a marker of granulosa cell proliferation (30, 31). The present study shows that the inhibitory effect of DHT in FSH-stimulated granulosa cell proliferation is mediated through the activation of AMPK.

DHT-mediated AMPK activation in primary granulosa cells was demonstrated by a dose- and time-dependent increase in AMPK phosphorylation. The role of AMPK in inhibiting cell proliferation is now well established because pharmacological activation of AMPK has been shown to cause cell cycle arrest in different cell types including granulosa cells (18, 32–34). Activation of AMPK by DHT as demonstrated in the present study along with its established role in cell cycle arrest suggests that DHT-mediated inhibition of granulosa cell proliferation might be mediated through AMPK activation.

Because it is well established that ERK activation is critical for FSH-mediated granulosa cell mitogenesis (15, 17, 35, 36), the possible communication between ERK and AMPK pathways was investigated by examining whether AMPK is an upstream regulator of ERK. Our results clearly show that it is indeed the case (Fig. 3). This observation is in agreement with those of Kim et al. (37) showing that AICAR treatment reduced ERK activation in NIH3T3 cells. The inhibition of ERK phosphorylation in response to AMPK activation is in agreement with our previous report that DHT exposure inhibits FSH-mediated ERK activation leading to reduced granulosa cell proliferation (15). The blockade of ERK phosphorylation due to AMPK activation further validates the role of AMPK in DHT-mediated inhibition of granulosa cell proliferation. Additionally, a physical interaction between ERK and AMPK was also observed in pull-down assays, but the mechanism by which AMPK inhibits ERK phosphorylation is not currently understood.

Because AMPK is an intermediary molecule in DHT's antagonistic effects on granulosa cell proliferation, it would be expected that inhibition of AMPK should render DHT ineffective in blocking FSH response. Our findings corroborate this notion because blocking DHT-mediated AMPK activation before FSH treatment was able to significantly (P < 0.05) overcome DHT's inhibitory effect on cyclin D2 (Fig. 5). Sustained AMPK activation by DHT treatment, on the other hand, reduced the FSH mediated increase in cyclin D2 expression. These results were further supported by a BrdU cell proliferation assay in which suppressing AMPK activation reversed DHT-mediated inhibitory effect. Collectively these results support our notion that AMPK mediates DHT's adverse effect on granulosa cell proliferation.

Based on these results, a model for the regulation of granulosa cell proliferation is proposed. As shown in Fig. 7, DHT-mediated AMPK activation may reduce cell proliferation through multiple ways. First, DHT reduces ERK phosphorylation and second, it makes AMPK nonresponsive to FSH's inhibitory signals. This is supported by our previous finding that in primary cultures of granulosa cells, FSH-mediated AMPK inhibition reduced the expression of cell cycle inhibitor protein, p27kip, leading to increased proliferation, whereas AMPK activation reverses this effect (18). Thus, the inability of FSH to suppress AMPK phosphorylation in the presence of DHT (Fig. 2) might lead to increased p27 kip expression and reduced granulosa cell proliferation. Taken together, the present results demonstrate that dysregulation of FSH signaling elicited by AMPK activation plays a role in mediating the deleterious effect of DHT in granulosa cell proliferation. In addition to our findings, other recent reports suggest that AMPK activation can also inhibit other proliferative signaling pathways like mammalian target of rapamycin, acting at multiple levels (38). Because in granulosa cells, mammalian target of rapamycin plays a role in mediating FSH-induced proliferation (17), it is likely that in addition to ERK, DHT-mediated AMPK activation might have other targets that regulate proliferation, which are yet to be identified.

Fig. 7.

Fig. 7.

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Schematic representation of the involvement of AMPK in DHT's inhibitory effect in FSH-mediated mitogenesis. Results from the present study suggest that DHT exposure increases AMPK activation, which in turn reduces FSH-mediated inhibition of AMPK. AMPK activation also blocks the mitogenic stimulus of FSH on ERK activation leading reduced cyclin D2 mRNA expression. These effects as well as the reported increase in cell cycle inhibitor p27kip due to AMPK activation might be playing a significant role in DHT-mediated inhibition of granulosa cell proliferation.

Acknowledgments

We express our appreciation to Helle Peegel for the critical reading of the manuscript.

This work was supported by National Institutes of Health Grant HD 38424.

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
AICAR
5-Aminoimidazole-4-carboxamide-1-β4-ribofuranoside
AMPK
AMP activated protein kinase
BrdU
5-bromo-2′-deoxyuridine
compound C
6-(4-[2-piperidn-1-ylethoxy] phenyle)-3-pyridin-4-ylpyrazolo (1,5-a)pyrimidine
DHT
5α-dihydrotestosterone
pAMPK
phosphorylated AMPK
PCOS
polycystic ovarian syndrome.

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