CREB coactivator CRTC2/TORC2 and its regulator calcineurin crucially mediate follicle-stimulating hormone and transforming growth factor ß1 upregulation of steroidogenesis

In vitro and in vivo studies implicate that follicle-stimulating hormone (FSH) and transforming growth factor beta 1 (TGF beta 1) play crucial physiological roles in regulating ovarian granulosa cell function essential to fertility control in females. FSH induces cAMP and calcium signaling, thereby activating transcription factor CREB to upregulate steroidogenic gene expression, and TGF beta 1 greatly enhances FSH-stimulated steroidogenesis. A CREB coactivator CRTC2/TORC2 was identified to function as a cAMP and calcium-sensitive coincidence sensor. This led us to explore the role of CRTC2 and its regulator calcineurin in FSH and TGF beta 1-stimulated steroidogenesis. Primary culture of granulosa cells from gonadotropin-primed immature rats was used. Immunoblotting analysis shows that FSH rapidly and transiently induced dephosphorylation/activation of CRTC2, and FSH+TGF beta 1 additionally induced late-phase CRTC2 dephosphorylation. Immunofluorescence analysis further confirms FSH +/- TGF beta 1 promoted CRTC2 nuclear translocation. Using selective inhibitors, we demonstrate that FSH activated CRTC2 in a PKA- and calcineurin-dependent manner, and TGF beta 1 acting through its type I receptor (TGF beta RI)-modulated FSH action in a calcineurin-mediated and PKA-independent fashion. Next, we investigated the involvement of calcineurin and CRTC2 in FSH and TGF beta 1-stimulated steroidogenesis. Calcineurin and TGF beta RI inhibitor dramatically reduced the FSH +/- TGF beta 1-increased progesterone synthesis and protein levels of StAR, P450scc, and 3 beta-HSD enzyme. Furthermore, chromatin-immunoprecipitation and immunoprecipitation analyses demonstrate that FSH +/- TGF beta 1 differentially increased CRTC2, CREB, and CBP binding to these steroidogenic genes, and CREB nuclear association with CRTC2 and CBP. In all, this study reveals for the first time that CRTC2 and calcineurin are critical signaling mediators in FSH and TGF beta 1-stimulated steroidogenesis in ovarian granulosa cells. J. Cell. Physiol. 227: 24302440, 2012. (c) 2011 Wiley Periodicals, Inc.