CRTC2 and Nedd4 ligase involvement in FSH and TGFß1 upregulation of connexin43 gap junction.

The major mission of the ovarian follicle is the timely production of the mature fertilizable oocyte, and this is achieved by gonadotropin-regulated, gap junction-mediated cell-cell communication between the oocyte and surrounding nurturing granulosa cells. We have demonstrated that FSH and transforming growth factor beta 1 (TGFß1) stimulate Gja1 gene-encoded connexin43 (Cx43) gap junction formation/function in rat ovarian granulosa cells is important for their induction of steroidogenesis; additionally, cAMP-protein kinase A (PKA)- and calcium-calcineurin-sensitive cAMP response element-binding (CREB) coactivator CRTC2 plays a crucial role during steroidogenesis. This study was to explore the potential molecular mechanism whereby FSH and TGFß1 regulate Cx43 synthesis and degradation, particularly the involvement of CRTC2 and ubiquitin ligase Nedd4. Primary culture of granulosa cells from ovarian antral follicles of gonadotropin-primed immature rats was used. At 48 h post-treatment, FSH plus TGFß1 increased Cx43 level and gap junction function in a PKA- and calcineurin-dependent manner, and TGFß1 acting through its type I receptor modulated FSH action. Chromatin-immunoprecipitation analysis reveals FSH induced an early-phase (45 min) and FSH+TGFß1 further elicited a late-phase (24 h) increase in CRTC2, CREB and CBP binding to the Gja1 promoter. Additionally, FSH+TGFß1 increased the half-life of hyper-phosphorylated Cx43 (Cx43-P2). Also, the proteasome inhibitor MG132 prevented the brefeldin A (blocker of protein transport through Golgi)-reduced Cx43-P2 level and membrane Cx43 gap junction plaque. This is associated with FSH+TGFß1-attenuated Cx43 interaction with Nedd4 and Cx43 ubiquitination. In all, this study uncovers that FSH and TGFß1 upregulation of Cx43 gap junctions in ovarian granulosa cells critically involves enhancing CRTC2/CREB/CBP-mediated Cx43 expression and attenuating ubiquitin ligase Nedd4-mediated proteosomal degradation of Cx43 protein.

Calcineurin and CRTC2 mediate FSH and TGFß1 upregulation of Cyp19a1 and Nr5a in ovary granulosa cells.

Estrogens are essential for female reproduction and overall well-being, and estrogens in the circulation are largely synthesized in ovarian granulosa cells. Using primary cultures of ovarian granulosa cells from gonadotropin-primed immature rats, we have recently discovered that pituitary FSH and ovarian cytokine transforming growth factor beta 1 (TGFß1) induce calcineurin-mediated dephosphorylation-activation of cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC2) to modulate the expression of Star, Cyp11a1, and Hsd3b leading to increased production of progesterone. This study explored the role of calcineurin and CRTC2 in FSH and TGFß1 regulation of Cyp19a1 expression in granulosa cells. Ovarian granulosa cells treated with FSH displayed increased aromatase protein at 24  h post-treatment, which subsided by 48  h, while TGFß1 acting through its type 1 receptor augmented the action of FSH with a greater and longer effects. It is known that the ovary-specific Cyp19a1 PII-promoter contains crucial response elements for CREB and nuclear receptor NR5A subfamily liver receptor homolog 1 (LRH1/NR5A2) and steroidogenic factor 1 (SF1/NR5A1), and that the Nr5a2 promoter also has a potential CREB-binding site. Herein, we demonstrate that FSH+TGFß1 increased LRH1 and SF1 protein levels, and their binding to the Cyp19a1 PII-promoter evidenced, determined by chromatin immunoprecipitation analysis. Moreover, pretreatment with calcineurin auto-inhibitory peptide (CNI) abolished the FSH+TGFß1-upregulated but not FSH-upregulated aromatase activity at 48  h, and the corresponding mRNA changes in Cyp19a1, and Nr5a2 and Nr5a1 at 24  h. In addition, FSH and TGFß1 increased CRTC2 binding to the Cyp19a1 PII-promoter and Nr5a2 promoter at 24  h, with CREB bound constitutively. In summary, the results of this study indicate that calcineurin and CRTC2 have important roles in mediating FSH and TGFß1 collateral upregulation of Cyp19a1 expression together with its transcription regulators Nr5a2 and Nr5a1 in ovarian granulosa cells.

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 ß1 (TGFß1) 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 TGFß1 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 TGFß1-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 + TGFß1 additionally induced late-phase CRTC2 dephosphorylation. Immunofluorescence analysis further confirms FSH ± TGFß1 promoted CRTC2 nuclear translocation. Using selective inhibitors, we demonstrate that FSH activated CRTC2 in a PKA- and calcineurin-dependent manner, and TGFß1 acting through its type I receptor (TGFßRI)-modulated FSH action in a calcineurin-mediated and PKA-independent fashion. Next, we investigated the involvement of calcineurin and CRTC2 in FSH and TGFß1-stimulated steroidogenesis. Calcineurin and TGFßRI inhibitor dramatically reduced the FSH ± TGFß1-increased progesterone synthesis and protein levels of StAR, P450scc, and 3ß-HSD enzyme. Furthermore, chromatin-immunoprecipitation and immunoprecipitation analyses demonstrate that FSH ± TGFß1 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 TGFß1-stimulated steroidogenesis in ovarian granulosa cells.