CRTC2 and Nedd4 ligase involvement in FSH and TGFβ1 upregulation of connexin43 gap junction

Abstract

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 (TGFb1) 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 TGFb1 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 TGFb1 increased Cx43 level and gap junction function in a PKA-and calcineurin-dependent manner, and TGFb1 acting through its type I receptor modulated FSH action. Chromatin-immunoprecipitation analysis reveals FSH induced an early-phase (45 min) and FSHCTGFb1 further elicited a late-phase (24 h) increase in CRTC2, CREB and CBP binding to the Gja1 promoter. Additionally, FSHCTGFb1 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 FSHCTGFb1-attenuated Cx43 interaction with Nedd4 and Cx43 ubiquitination. In all, this study uncovers that FSH and TGFb1 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.

The role of FSH and TGF-β superfamily in follicle atresia

“…TGF-β factors have been reported to facilitate FSH functions in gap junction formation and steroidogenesis by enhancing the activity of CREB-regulated transcription coactivator (CRTC2, also known as TORC2) [ 121 , 122 ]. TORC2 can sense both cAMP/PKA and calcium signaling induced by FSH [ 122 ].…”

“…FSH’s stimulation of TORC2 may not be a lasting effect. A later rise as well as translocation of TORC2 requires synergia of TFGβ1 [ 121 , 122 ]. And in this process, rather than PKA, calcineurin seems to matter, which requires further study to figure out the molecular mechanism underlying TGF-β affected calcineurin activities.…”

Granulosa cells of prepubertal cattle respond to gonadotropin signaling and upregulate genes that promote follicular growth and prevent cell apoptosis

“…In addition, we observed that transcripts abundance of CX43 and FOXO1 increased with age in the GCs of untreated animals. These genes encode key regulators of GC function, proliferation, and survival, and their expression was shown to be regulated by FSH in different species (Ackert, Gittens, O'Brien, Eppig, & Kidder, 2001;Cunningham, Zhu, Unterman, & Hammond, 2003;Fang et al, 2015;Nuttinck et al, 2000;Santiquet, Robert, & Richard, 2013;Shen et al, 2014;Ting & Zelinski, 2017).…”

Connexins: Synthesis, Post-Translational Modifications, and Trafficking in Health and Disease

“…While classic lysine-based motifs may not be responsible for direct ubiquitin binding, more recent studies have shown that proline-rich regions of the Cx43 C-terminus (xPPxY) bind to ubiquitin ligase. A number of ubiquitin ligases have also been associated with direct binding, internalization, and degradation of Cx43 (e.g., Trim21 [ 291 ], WWP1 [ 292 ], SMURF2 [ 293 ], and NEDD4 [ 287 , 288 , 289 , 294 ]). NEDD4 also has been directly associated with loss of Cx43 at the plasma membrane in experimental models [ 287 ].…”