CRTC2 and Nedd4 ligase involvement in FSH and TGFβ1 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 (TGFβ1) 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 TGFβ1 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 TGFβ1 increased Cx43 level and gap junction function in a PKA- and calcineurin-dependent manner, and TGFβ1 acting through its type I receptor modulated FSH action. Chromatin-immunoprecipitation analysis reveals FSH induced an early-phase (45 min) and FSH+TGFβ1 further elicited a late-phase (24 h) increase in CRTC2, CREB and CBP binding to the Gja1 promoter. Additionally, FSH+TGFβ1 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+TGFβ1-attenuated Cx43 interaction with Nedd4 and Cx43 ubiquitination. In all, this study uncovers that FSH and TGFβ1 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.
School of MedicineInstitute of Physiology, National Yang-Ming University, 155 Linong Street, Section 2, Taipei 112, TaiwanDepartment of NursingHsin-Sheng College of Medical Care and Management, Taoyuan, TaiwanInstitute of Biological ChemistryInstitute of Cellular and Organismic BiologyAcademia Sinica, Taipei, TaiwanCollege of Life ScienceInstitute of Molecular and Cellular Biology, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan School of MedicineInstitute of Physiology, National Yang-Ming University, 155 Linong Street, Section 2, Taipei 112, TaiwanDepartment of NursingHsin-Sheng College of Medical Care and Management, Taoyuan, TaiwanInstitute of Biological ChemistryInstitute of Cellular and Organismic BiologyAcademia Sinica, Taipei, TaiwanCollege of Life ScienceInstitute of Molecular and Cellular Biology, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan.
Wei-Ling
WL
Fang
School of MedicineInstitute of Physiology, National Yang-Ming University, 155 Linong Street, Section 2, Taipei 112, TaiwanDepartment of NursingHsin-Sheng College of Medical Care and Management, Taoyuan, TaiwanInstitute of Biological ChemistryInstitute of Cellular and Organismic BiologyAcademia Sinica, Taipei, TaiwanCollege of Life ScienceInstitute of Molecular and Cellular Biology, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan.
Si-Yi
SY
Lai
School of MedicineInstitute of Physiology, National Yang-Ming University, 155 Linong Street, Section 2, Taipei 112, TaiwanDepartment of NursingHsin-Sheng College of Medical Care and Management, Taoyuan, TaiwanInstitute of Biological ChemistryInstitute of Cellular and Organismic BiologyAcademia Sinica, Taipei, TaiwanCollege of Life ScienceInstitute of Molecular and Cellular Biology, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan.
Wei-An
WA
Lai
School of MedicineInstitute of Physiology, National Yang-Ming University, 155 Linong Street, Section 2, Taipei 112, TaiwanDepartment of NursingHsin-Sheng College of Medical Care and Management, Taoyuan, TaiwanInstitute of Biological ChemistryInstitute of Cellular and Organismic BiologyAcademia Sinica, Taipei, TaiwanCollege of Life ScienceInstitute of Molecular and Cellular Biology, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan.
Ming-Ting
MT
Lee
School of MedicineInstitute of Physiology, National Yang-Ming University, 155 Linong Street, Section 2, Taipei 112, TaiwanDepartment of NursingHsin-Sheng College of Medical Care and Management, Taoyuan, TaiwanInstitute of Biological ChemistryInstitute of Cellular and Organismic BiologyAcademia Sinica, Taipei, TaiwanCollege of Life ScienceInstitute of Molecular and Cellular Biology, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan.
Ching-Fong
CF
Liao
School of MedicineInstitute of Physiology, National Yang-Ming University, 155 Linong Street, Section 2, Taipei 112, TaiwanDepartment of NursingHsin-Sheng College of Medical Care and Management, Taoyuan, TaiwanInstitute of Biological ChemistryInstitute of Cellular and Organismic BiologyAcademia Sinica, Taipei, TaiwanCollege of Life ScienceInstitute of Molecular and Cellular Biology, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan jiuanh@ym.edu.tw fck@ccms.ntu.edu.tw.
Ferng-Chun
FC
Ke
School of MedicineInstitute of Physiology, National Yang-Ming University, 155 Linong Street, Section 2, Taipei 112, TaiwanDepartment of NursingHsin-Sheng College of Medical Care and Management, Taoyuan, TaiwanInstitute of Biological ChemistryInstitute of Cellular and Organismic BiologyAcademia Sinica, Taipei, TaiwanCollege of Life ScienceInstitute of Molecular and Cellular Biology, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan jiuanh@ym.edu.tw fck@ccms.ntu.edu.tw.
Jiuan-Jiuan
JJ
Hwang
D000818:Animals; D002450:Cell Communication; D018031:Connexin 43 / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D005640:Follicle Stimulating Hormone / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism*; D000075702:Nedd4 Ubiquitin Protein Ligases; D011485:Protein Binding; D055550:Protein Stability; D021381:Protein Transport; D051381:Rats; D015534:Trans-Activators / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
C545700:CRTC2 protein, rat; D018031:Connexin 43; D056827:Endosomal Sorting Complexes Required for Transport; D015534:Trans-Activators; D053773:Transforming Growth Factor beta1; D005640:Follicle Stimulating Hormone; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618484:Nedd4 protein, rat; D044767:Ubiquitin-Protein Ligases
CREB; CRTC2; FSH; Nedd4 ubiquitin ligase; TGFβ1; calcineurin; connexin43; gap junction; ovary granulosa cell