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.

Physiological effects of major genes affecting ovulation rate in sheep

“…In rat granulosa cells, GDF9 is known to bind to BMPR-II [32] and to stimulate inhibin A and B production via the Smad2 pathway [26] inferring that the type 1 receptor might be an activin or TGFβ receptor rather than ALK3 or ALK6 [24]. Indeed a recent report shows that ALK5 (TGFβRI) is the type 1 receptor for GDF9 [19]. This finding suggests that GDF9 could have autocrine effects on the oocyte from the type 1 stage and paracrine effects on both the granulosa and theca cells from the type 4 (range [20].…”

Single-cell expression analysis of BMP15 and GDF9 in mature oocytes and BMPR2 in cumulus cells of women with polycystic ovary syndrome undergoing controlled ovarian hyperstimulation

“…The TGFβ superfamily of ligands interact with heterotetrameric complexes of type 1 and type 2 receptors (BMPR2). Thus, GDF9 activates the type 1 receptor (TGFβRI or activin-like receptor 5(ALK5) [25] and BMP15 activates the BMPRIB receptor or activin-like receptor 6 (ALK6). Following ligand binding, TGFβRI or BMPRIB recruits the BMPR2 that results in phosporylation of the type 1 receptor, which in turn activates the intracellular-signal-mediated (Smad) proteins [16,26,27].…”

Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells

“…We have shown that mGDF9 induces Smad2 phosphorylation and inhibin production in rat diethylstilbestrol-treated granulosa cells (Roh et al 2003) and in human granulosa-luteal (hGL) cells , not requiring any purification steps to reveal these activities. Further, we have shown that the downstream signaling actions of mGDF9 are mediated by the type I receptor, ALK5, initiating the subsequent activation of Smad2 and Smad3, both in cultured rat granulosa cells (Mazerbourg et al 2004) and in hGL cells Kaivo-Oja et al 2005). It has also been shown that mGDF9 uses the BMP type II receptor (BMPRII) as its other signaling receptor in rat granulosa cells (Vitt et al 2002).…”