title: CREB coactivator CRTC2/TORC2 and its regulator calcineurin crucially mediate follicle-stimulating hormone and transforming growth factor β1 upregulation of steroidogenesis.
abstract: In vitro and in vivo studies implicate that follicle-stimulating hormone (FSH) and transforming growth factor β1 (TGFβ1) 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 TGFβ1 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 TGFβ1-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 + TGFβ1 additionally induced late-phase CRTC2 dephosphorylation. Immunofluorescence analysis further confirms FSH ± TGFβ1 promoted CRTC2 nuclear translocation. Using selective inhibitors, we demonstrate that FSH activated CRTC2 in a PKA- and calcineurin-dependent manner, and TGFβ1 acting through its type I receptor (TGFβRI)-modulated FSH action in a calcineurin-mediated and PKA-independent fashion. Next, we investigated the involvement of calcineurin and CRTC2 in FSH and TGFβ1-stimulated steroidogenesis. Calcineurin and TGFβRI inhibitor dramatically reduced the FSH ± TGFβ1-increased progesterone synthesis and protein levels of StAR, P450scc, and 3β-HSD enzyme. Furthermore, chromatin-immunoprecipitation and immunoprecipitation analyses demonstrate that FSH ± TGFβ1 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 TGFβ1-stimulated steroidogenesis in ovarian granulosa cells.
authors.affiliation: Institute of Physiology, School of Medicine, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Wei-Ling
authors.initials: WL
authors.lastname: Fang
authors.forename: Ming-Ting
authors.initials: MT
authors.lastname: Lee
authors.forename: Leang-Shin
authors.initials: LS
authors.lastname: Wu
authors.forename: Yun-Ju
authors.initials: YJ
authors.lastname: Chen
authors.forename: Jian
authors.initials: J
authors.lastname: Mason
authors.forename: Ferng-Chun
authors.initials: FC
authors.lastname: Ke
authors.forename: Jiuan-Jiuan
authors.initials: JJ
authors.lastname: Hwang
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D015153:Blotting, Western; D050882:CREB-Binding Protein / Q000378:metabolism; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005455:Fluorescent Antibody Technique; D005640:Follicle Stimulating Hormone / Q000378:metabolism*; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011374:Progesterone / Q000096:biosynthesis*; D011375:Progesterone Reductase / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism; D012756:Sheep; D015398:Signal Transduction; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: C508137:Creb1 protein, rat; C545700:CRTC2 protein, rat; D065095:Calcineurin Inhibitors; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D010750:Phosphoproteins; D018125:Receptors, Transforming Growth Factor beta; C508960:TGFB1 protein, human; D015534:Trans-Activators; D053773:Transforming Growth Factor beta1; D000098682:Steroidogenic Acute Regulatory Protein; D011374:Progesterone; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D011375:Progesterone Reductase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D050882:CREB-Binding Protein; C494365:Crebbp protein, rat; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625843:Tgfbr1 protein, rat; D019703:Calcineurin