Interplay of P13K and cAMP/PKA signaling, and rapamycin-hypersensitivity in TGFβ1 enchancement of FSH-stimulated steroidogenesis in rat ovarian granulosa cells

Abstract

Transforming growth factor (TGF)β1 facilitates FSH-induced differentiation of rat ovarian granulosa cells. The signaling crosstalk between follicle stimulating hormone (FSH) and TGFβ receptors remains unclear. This study was to investigate the interplay of cAMP /protein kinase A (PKA) and phosphatidylinositol-3-kinase (P13K) signaling including mammalian target of rapamycin (mTOR)C1 dependence in FSH- and TGFβ1-stimulated steroidogenesis in rat granulosa cells. To achieve this aim, inhibitors of PKA (PKAI), P13K (wortmannin), and mTORC1 (rapamycin) were employed. PKA1 and wortmannin suppressions of the FSH-increased progesterone production were partly attributed to decreased level of 3β-HSD, and their suppression of the FSH plus TGFβ1 efFect was attributed to the reduction of all the three key players, steroidogenic acute regulatory (StAR) protein, P450scc, and 3β-HSD. Further, FSH activated the P13K 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 affiect intracellular compartmentalization of signaling molecule(s). In addition, TGFβ1 had no effect on FSH-activated CREB and P13K signaling mediators. We further found that rapamycin reduced the TGFβ1-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 P13K signaling in FSH and TGFβ1 regulation of steroidogenesis in rat granulosa cells. Furthermore, we demonstrate for the first time that TGFβ1 acts in a rapamycin-hypersensitive and mTORC1-independent manner in augmenting FSH-stimulated steroidogenesis in rat granulosa cells. © 2007 Society for Endocrinology.