Evidence That Activation of AMPK by DHT Inhibits Granulosa Cell Proliferation by Reducing ERK Phosphorylation.

The proliferation and growth of granulosa cells, processes crucial for normal ovulation, are controlled by follicle stimulating hormone (FSH) and other paracrine factors through an array of diverse signaling pathways. This orderly progression of granulosa cell growth is disrupted in hyperandrogenic states such as polycystic ovarian syndrome (PCOS). The increased expression of 5-α reductase in the ovarian follicles of PCOS patients implicates a role for 5-α reduced androgens in ovulatory dysfunction associated with this syndrome. We have previously reported that 5-α dihydrotestosterone (DHT) inhibits FSH-mediated granulosa cell proliferation by reducing cyclin D2 mRNA expression and blocking cell cycle progression at G1/S phase. In the present study we investigated the role of AMP Kinase, an inhibitory signal for cell proliferation, in mediating DHT's inhibition of granulosa cell proliferation. Granulosa cells harvested from 3 day estradiol primed immature rats were cultured in a serum-free, phenol red-free media. After overnight attachment, cultures were exposed to different concentrations of DHT (0, 45, 90 and 180 ng/ml) for 24 hours. At the end of this treatment period, cells were lysed using an AMPK lysis buffer, and subjected to immunoprecipitation using AMPK specific antibody. AMPK activation was analyzed by Western blot using an antibody against AMPK phosphorylated at thr 172, since phosphorylation of this residue is necessary for AMPK activation. The results showed a significant increase (p<0.05) in AMPK phosphorylation by DHT treatment in a dose dependent manner with maximum effect seen at 180 ng/ml. Since previous studies have shown that ERK plays an important role in FSH mediated granulosa cell mitogenesis, we examined whether the inhibitory effect of DHT on proliferation occurs through AMPK-mediated inhibition of ERK phosphorylation. Granulosa cells, after overnight attachment, were treated with the pharmacological activator of AMPK (AICAR) for 1 hour followed by FSH (75ng/ml) for 15 minutes, and ERK phosphorylation was determined by Western blot analysis. The results showed, as expected, a significant increase (p<0.05) in ERK phosphorylation in response to FSH treatment. Interestingly, pretreatment with AICAR abolished this stimulation, indicating that AMPK is a negative upstream regulator of ERK. The inhibitory effect of AMPK on ERK phosphorylation was further confirmed by demonstrating physical interaction between these two proteins in a co-immunoprecipitation assay. 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 be responsible for the observed decrease in granulosa cell mitogenesis and anovulation seen in hyperandrogenic states. (Supported by NIH Grant HD-38424).

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