AMPK activation by dihydrotestosterone reduces FSH-stimulated cell proliferation in rat granulosa cells by inhibiting ERK signaling pathway.

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-ß4-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.

Stimulatory effect of insulin on 5alpha-reductase type 1 (SRD5A1) expression through an Akt-dependent pathway in ovarian granulosa cells.

Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 µg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P<0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P<0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation.

Follicle-stimulating hormone inhibits adenosine 5'-monophosphate-activated protein kinase activation and promotes cell proliferation of primary granulosa cells in culture through an Akt-dependent pathway.

FSH, acting through multiple signaling pathways, regulates the proliferation and growth of granulosa cells, which are critical for ovulation. The present study investigated whether AMP-activated protein kinase (AMPK), which controls the energy balance of the cell, plays a role in FSH-mediated increase in granulosa cell proliferation. Cells isolated from immature rat ovaries were grown in serum-free, phenol red free DMEM-F12 and were treated with FSH (50 ng/ml) for 0, 5, and 15 min. Western blot analysis showed a significant reduction in AMPK activation as observed by a reduction of phosphorylation at thr 172 in response to FSH treatment at all time points tested. FSH also reduced AMPK phosphorylation in a dose-dependent manner with maximum inhibition at 100 ng/ml. The chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, 0.5 mm) increased the cell cycle inhibitor p27 kip expression significantly, whereas the AMPK inhibitor (compound C, 20 microm) and FSH reduced p27kip expression significantly compared with control. FSH treatment resulted in an increase in the phosphorylation of AMPK at ser 485/491 and a reduction in thr 172 phosphorylation. Inhibition of Akt phosphorylation using Akt inhibitor VIII reversed the inhibitory effect of FSH on thr 172 phosphorylation of AMPK, whereas ERK inhibitor U0126 had no effect. These results show that FSH, through an Akt-dependent pathway, phosphorylates AMPK at ser 481/495 and inhibits its activation by reducing thr 172 phosphorylation. AMPK activation by 5-amino-imidazole-4-carboxamide-1-beta-D-ribofuranoside treatment resulted in a reduction of cell cycle regulatory protein cyclin D2 mRNA expression, whereas FSH increased the expression by 2-fold. These results suggest that FSH promotes granulosa cell proliferation by increasing cyclin D2 mRNA expression and by reducing p27 kip expression by inhibiting AMPK activation through an Akt-dependent pathway.

Follicle-stimulating hormone increases tuberin phosphorylation and mammalian target of rapamycin signaling through an extracellular signal-regulated kinase-dependent pathway in rat granulosa cells.

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 reduced 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.

Dihydrotestosterone inhibits insulin-stimulated cyclin D2 messenger ribonucleic acid expression in rat ovarian granulosa cells by reducing the phosphorylation of insulin receptor substrate-1.

The effect of 5alpha-dihydrotestosterone (DHT) on insulin-stimulated granulosa cell proliferation was examined using cyclin D2 mRNA as a marker. Granulosa cells from 3-d estradiol-treated immature rats showed a concentration-dependent increase in cyclin D2 mRNA expression in response to insulin. Exposure to DHT reduced the insulin-stimulated cyclin D2 mRNA expression. Inhibition of the two insulin-signaling pathways, ERK and phosphatidylinositol 3 kinase (PI3 kinase), by using specific inhibitors, also reduced this insulin-stimulated response. These results suggest that both ERK and PI3 kinase signaling are involved in insulin stimulated granulosa cell proliferation. DHT exposure resulted in reduced insulin-stimulated ERK phosphorylation. DHT treatment also reduced the insulin mediated insulin receptor substrate-1 and Raf-1 phosphorylation, the upstream molecules of ERK in insulin signaling pathway. Additionally, inhibition of insulin stimulated PI3 kinase activation reduced ERK phosphorylation. The present study therefore shows that the inhibitory effect of DHT on insulin-stimulated granulosa cell proliferation occurs early in the signaling pathway at the level of insulin receptor substrate-1 phosphorylation, leading to reduced ERK phosphorylation and subsequent inhibition of cyclin D2 mRNA expression.

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.

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 5alpha-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.