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Growth Differentiation Factor-9 Signaling Is Mediated by the Type I Receptor, Activin Receptor-Like Kinase 5

Division of Reproductive Biology (S.M., C.K., J.R., A.J.W.H.), Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305-5317; Developmental and Reproductive Biology Program (N.K.-O., D.G.M., O.R.), Biomedicum Helsinki and Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, 00014 Helsinki, Finland; and Department of Cellular Biochemistry (O.K.), Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands

Address all correspondence and requests for reprints to: Aaron J. W. Hsueh, Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305-5317. E-mail: aaron.hsueh{at}stanford.edu.

Growth differentiation factor-9 (GDF-9) is an oocyte-derived growth factor and a member of the TGF-ß superfamily that includes TGF-ß, 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-ß 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-ß 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-ß, diverse members of the TGF-ß family of ligands appear to interact with a limited number of receptors in a combinatorial manner to activate two downstream Smad pathways.

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