Lindane, a gap junction blocker, suppresses FSH and transforming growth factor beta1-induced connexin43 gap junction formation and steroidogenesis in rat granulosa cells.

The present study was designed to explore the role of gap junctions in follicle-stimulating hormone (FSH) and transforming growth factor beta1 (TGF beta1)-stimulated steroidogenesis in ovarian granulosa cells of gonadotropin-primed immature rats. There were three specific aims. First, we investigated the effect of FSH and TGF beta1 as well as lindane (a general gap junction blocker) on the level of connexin43 (Cx43), the major gap junction constituent in granulosa cells, and on gap junction function. The second aim was to determine the effect of lindane on FSH and TGF beta1-stimulated progesterone production and the levels of two critical players, cytochrome P450 side-chain cleavage (P450scc) enzyme and steroidogenic acute regulatory (StAR) protein. The third aim was to further investigate the specific involvement of Cx43 gap junctions in FSH and TGF beta1-stimulated steroidogenesis using a Cx43 mimetic peptide blocker. Immunoblotting analysis showed that FSH plus TGF beta1 dramatically increased the levels of phosphorylated Cx43 without significantly influencing the level of nonphosphorylated Cx43, and this stimulatory effect was completely suppressed by lindane. Also, immunofluorescence analysis showed that Cx43 immuno-reactivity increased in the FSH plus TGF beta1-treated group and predominantly appeared in a punctate pattern at cell-cell contact sites, and lindane reduced such cell periphery immunostaining. Furthermore, TGF beta1 enhanced the FSH-induced gap junction intercellular communication and lindane completely suppressed this effect. In addition, lindane suppressed the FSH and TGF beta1-stimulated increases in progesterone production and the levels of P450scc enzyme and StAR protein. This study demonstrates a clear temporal association between the Cx43 protein level/gap junction communication and progesterone production in rat ovarian granulosa cells in response to FSH and TGF beta1 as well as lindane. Furthermore, a specific Cx43 gap junction blocker suppressed FSH plus TGF beta1-stimulated progesterone production. In conclusion, this study suggests that Cx43 gap junctions may play a critical role in FSH plus TGF beta1-stimulated progesterone production in rat ovarian granulosa cells.

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.