Interplay of PI3K and cAMP/PKA signaling, and rapamycin-hypersensitivity in TGFbeta1 enhancement of FSH-stimulated steroidogenesis in rat ovarian granulosa cells

Transforming growth factor (TGF) beta1 facilitates FSH-induced differentiation of rat ovarian granulosa cells. The signaling crosstalk between follicle stimulating hormone (FSH) and TGFbeta receptors remains unclear. This study was to investigate the interplay of cAMP/protein kinase A (PKA) and phosphatidylinositol-3-kinase (PI3K) signaling including mammalian target of rapamycin (mTOR)C1 dependence in FSH- and TGFbeta1-stimulated steroidogenesis in rat granulosa cells. To achieve this aim, inhibitors of PKA (PKAI), PI3K (wortmannin), and mTORC1 (rapamycin) were employed. PKAI and wortmannin suppressions of the FSH-increased progesterone production were partly attributed to decreased level of 3beta-HSD, and their suppression of the FSH plus TGFbeta1 effect was attributed to the reduction of all the three key players, steroidogenic acute regulatory (StAR) protein, P450scc, and 3beta-HSD. Further, FSH activated the PI3K pathway including increased integrin-linked kinase (ILK) activity and phosphorylation of Akt(S473), mTOR(S2481), S6K(T389), and transcription factors particularly FoxO1(S256) and FoxO3a(S253), which were reduced by wortmannin treatment but not by PKAI. Interestingly, PKAI suppression of FSH-induced phosphorylation of cAMP regulatory element-binding protein (CREB(S133)) disappeared in the presence of wortmannin, suggesting that wortmannin may affect intracellular compartmentalization of signaling molecule(s). In addition, TGFbeta1 had no effect on FSH-activated CREB and PI3K signaling mediators. We further found that rapamycin reduced the TGFbeta1-enhancing effect of FSH-stimulated steroidogenesis, yet it exhibited no effect on FSH action. Surprisingly, rapamycin displayed a suppressive effect at concentrations that had no effect on mTORC1 activity. Together, this study demonstrates a delicate interplay between cAMP/PKA and PI3K signaling in FSH and TGFbeta1 regulation of steroidogenesis in rat granulosa cells. Furthermore, we demonstrate for the first time that TGFbeta1 acts in a rapamycin-hypersensitive and mTORC1-independent manner in augmenting FSH-stimulated steroidogenesis in rat granulosa cells.