title: Growth differentiation factor-9 signaling is mediated by the type I receptor, activin receptor-like kinase 5.
abstract: Growth differentiation factor-9 (GDF-9) is an oocyte-derived growth factor and a member of the TGF-beta superfamily that includes TGF-beta, activin, and bone morphogenetic proteins (BMPs). GDF-9 is indispensable for the development of ovarian follicles from the primary stage, and treatment with GDF-9 enhances the progression of early follicles into small preantral follicles. Similar to other TGF-beta family ligands, GDF-9 likely initiates signaling mediated by type I and type II receptors with serine/threonine kinase activity, followed by the phosphorylation of intracellular transcription factors named Smads. We have shown previously that GDF-9 interacts with the BMP type II receptor (BMPRII) in granulosa cells, but the type I receptor involved is unknown. Using P19 cells, we now report that GDF-9 treatment stimulated the CAGA-luciferase reporter known to be responsive to TGF-beta mediated by the type I receptor, activin receptor-like kinase (ALK)5. In contrast, GDF-9 did not stimulate BMP-responsive reporters. In addition, treatment with GDF-9 induced the phosphorylation of Smad2 and Smad3 in P19 cells, and the stimulatory effect of GDF-9 on the CAGA-luciferase reporter was blocked by the inhibitory Smad7, but not Smad6. We further reconstructed the GDF-9 signaling pathway using Cos7 cells that are not responsive to GDF-9. After overexpression of ALK5, with or without exogenous Smad3, the Cos7 cells gained GDF-9 responsiveness based on the CAGA-luciferase reporter assay. The roles of ALK5 and downstream pathway genes in mediating GDF-9 actions were further tested in ovarian cells. In cultured rat granulosa cells from early antral follicles, treatment with GDF-9 stimulated the CAGA-luciferase reporter activity and induced the phosphorylation of Smad3. Furthermore, transfection with small interfering RNA for ALK5 or overexpression of the inhibitory Smad7 resulted in dose-dependent suppression of GDF-9 actions. In conclusion, although GDF-9 binds to the BMP-activated type II receptor, its downstream actions are mediated by the type I receptor, ALK5, and the Smad2 and Smad3 proteins. Because ALK5 is a known receptor for TGF-beta, diverse members of the TGF-beta family of ligands appear to interact with a limited number of receptors in a combinatorial manner to activate two downstream Smad pathways.
authors.affiliation: Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305-5317, USA.
authors.forename: Sabine
authors.initials: S
authors.lastname: Mazerbourg
authors.forename: Cynthia
authors.initials: C
authors.lastname: Klein
authors.forename: Jaesook
authors.initials: J
authors.lastname: Roh
authors.forename: Noora
authors.initials: N
authors.lastname: Kaivo-Oja
authors.forename: David G
authors.initials: DG
authors.lastname: Mottershead
authors.forename: Olexander
authors.initials: O
authors.lastname: Korchynskyi
authors.forename: Olli
authors.initials: O
authors.lastname: Ritvos
authors.forename: Aaron J W
authors.initials: AJ
authors.lastname: Hsueh
mesh_terms: D029404:Activin Receptors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D030201:Activin Receptors, Type I / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D028341:Activins / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D055430:Bone Morphogenetic Protein 15; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D019556:COS Cells / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism; D055429:Growth Differentiation Factor 9; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D051381:Rats; D017207:Rats, Sprague-Dawley; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D051785:Smad Proteins; D051899:Smad2 Protein; D051900:Smad3 Protein; D051905:Smad6 Protein; D051906:Smad7 Protein; D015534:Trans-Activators / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology
chemical_list: C525904:Bmp15 protein, rat; D055430:Bone Morphogenetic Protein 15; D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; C525899:Gdf9 protein, rat; D055429:Growth Differentiation Factor 9; D036341:Intercellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; D051785:Smad Proteins; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D051905:Smad6 Protein; C497804:Smad6 protein, rat; D051906:Smad7 Protein; C497808:Smad7 protein, rat; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D028341:Activins; D017346:Protein Serine-Threonine Kinases; D029404:Activin Receptors; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625843:Tgfbr1 protein, rat