Growth differentiation factor-9 signaling is mediated by the type I receptor, activin receptor-like kinase 5

Mol Endocrinol. 2004 Mar;18(3):653-65. doi: 10.1210/me.2003-0393. Epub 2003 Dec 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Activin Receptors / drug effects
  • Activin Receptors / genetics
  • Activin Receptors / metabolism*
  • Activin Receptors, Type I / drug effects
  • Activin Receptors, Type I / genetics
  • Activin Receptors, Type I / metabolism*
  • Activins / metabolism
  • Activins / pharmacology
  • Animals
  • Bone Morphogenetic Protein 15
  • Bone Morphogenetic Proteins / metabolism
  • COS Cells / drug effects
  • COS Cells / metabolism
  • Cells, Cultured
  • DNA-Binding Proteins / drug effects
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Female
  • Granulosa Cells / drug effects
  • Granulosa Cells / metabolism
  • Growth Differentiation Factor 9
  • Intercellular Signaling Peptides and Proteins / metabolism*
  • Intercellular Signaling Peptides and Proteins / pharmacology
  • Phosphorylation
  • Promoter Regions, Genetic
  • Protein-Serine-Threonine Kinases
  • RNA, Small Interfering
  • Rats
  • Rats, Sprague-Dawley
  • Receptor, Transforming Growth Factor-beta Type I
  • Receptors, Transforming Growth Factor beta / drug effects
  • Receptors, Transforming Growth Factor beta / genetics
  • Receptors, Transforming Growth Factor beta / metabolism*
  • Signal Transduction
  • Smad Proteins
  • Smad2 Protein*
  • Smad3 Protein
  • Smad6 Protein
  • Smad7 Protein
  • Trans-Activators / drug effects
  • Trans-Activators / genetics
  • Trans-Activators / metabolism
  • Transforming Growth Factor beta / metabolism
  • Transforming Growth Factor beta / pharmacology

Substances

  • Bmp15 protein, rat
  • Bone Morphogenetic Protein 15
  • Bone Morphogenetic Proteins
  • DNA-Binding Proteins
  • Gdf9 protein, rat
  • Growth Differentiation Factor 9
  • Intercellular Signaling Peptides and Proteins
  • RNA, Small Interfering
  • Receptors, Transforming Growth Factor beta
  • Smad Proteins
  • Smad2 Protein
  • Smad2 protein, rat
  • Smad3 Protein
  • Smad3 protein, rat
  • Smad6 Protein
  • Smad6 protein, rat
  • Smad7 Protein
  • Smad7 protein, rat
  • Trans-Activators
  • Transforming Growth Factor beta
  • Activins
  • Protein-Serine-Threonine Kinases
  • Activin Receptors
  • Activin Receptors, Type I
  • Receptor, Transforming Growth Factor-beta Type I
  • Tgfbr1 protein, rat