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Molecular Endocrinology, doi:10.1210/me.2003-0393
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Molecular Endocrinology 18 (3): 653-665
Copyright © 2004 by The Endocrine Society

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

Sabine Mazerbourg, Cynthia Klein, Jaesook Roh, Noora Kaivo-Oja, David G. Mottershead, Olexander Korchynskyi, Olli Ritvos and Aaron J. W. Hsueh

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.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
OVARIAN GRANULOSA CELL proliferation and differentiation are influenced by gonadotropins as well as paracrine factors secreted by both the oocyte and the surrounding somatic cells (1, 2). These paracrine factors include members of the TGF-ß superfamily such as TGF-ß, activins, bone morphogenetic protein (BMP)2, BMP3, BMP4, BMP6, and BMP7 (3, 4, 5), and the two oocyte-derived growth factors, growth differentiation factor-9 (GDF-9) (6, 7, 8) and GDF-9B/BMP15 (9, 10, 11). In contrast to most TGF-ß family members expressed in ovarian somatic cells, the expression of GDF-9 is confined to the oocyte of primary and larger follicles in rats (8, 12), mice (7, 13), and humans (14). Studies on GDF-9 null mice have demonstrated the important role of this oocyte factor in the stimulation of early follicular growth (7). Subsequent studies demonstrated that treatment with GDF-9 enhances primary and preantral follicle growth in vitro and in vivo (8, 15) and promotes the survival and progression of human follicles in organ culture (16). GDF-9 also has been shown to play a role in granulosa cell differentiation in small antral and preovulatory follicles (17), as well as stimulate inhibin production by granulosa cells (18, 19), enable cumulus expansion (20), and increase thecal cell androgen production (21).

Members of the TGF-ß family initiate signaling by assembling type I and type II serine/threonine kinase receptor complexes that activate Smad transcription factors (22). The type I receptors are also designated as activin receptor-like kinases (ALKs). BMP2 and BMP4 interact with the BMP type II receptor (BMPRII) and the type I receptors, ALK3 and ALK6 (23). In contrast, activin signals through the type II receptors, ActRII or ActRIIB, together with the type I receptor, ALK4, whereas TGF-ß signals through the TGF-ß receptor type II (TGFßRII) and the type I receptors, ALK5 and ALK1 (22, 24).

After ligand binding, the type II receptor phosphorylates the type I ALK receptors, which subsequently initiate two distinct downstream Smad-signaling pathways. The BMP-responsive ALK2, ALK3, and ALK6 phosphorylate the BMP pathway-specific receptor-regulated Smads (R-Smads), Smad1, 5, and 8 (22, 23). In contrast, the TGF-ß/activin-responsive ALK5 and ALK4 phosphorylate the other R-Smads, Smad2 and Smad3 (22). Once phosphorylated, the R-Smads form heteromeric complexes with the common Smad4. This complex, in turn, translocates to the nucleus to regulate gene expression. In addition, the inhibitory Smads (Smad6 and Smad7) antagonize TGF-ß/activin/BMP signaling (25, 26, 27, 28).

GDF-9 is most closely related to GDF-9B/BMP15, both having homology closer to BMP proteins than to the activin and TGF-ß proteins (29, 30). We have shown previously that BMPRII is the type II receptor for GDF-9 in granulosa cells (29), consistent with the close homology between GDF-9 and BMPs. However, GDF-9 activates Smad2 in granulosa cells (18, 19), presumably by activating the TGF-ß/activin pathway. In the present study, permanent cell lines and granulosa cells were used to further elucidate the intracellular mechanisms of GDF-9 action. We identified a cell line (P19) in which a TGF-ß/activin-responsive promoter reporter (CAGA) can be activated by GDF-9, and demonstrated the essential role of Smad3 and Smad2 in GDF-9 signaling. We also identified a GDF-9-nonresponsive cell line (Cos7) and demonstrated that overexpression of ALK5 and Smad3 conferred GDF-9 responsiveness. The essential role of ALK5 and downstream Smad proteins in GDF-9 signaling was further confirmed in cultured rat granulosa cells by overexpression studies using small interfering RNA (siRNA) for ALK5 and inhibitory Smads.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Treatment with GDF-9, Similar to TGF-ß and Activin, Stimulates the CAGA Promoter in P19 Cells
GDF-9 secreted by the oocyte is known to regulate the proliferation and differentiation of granulosa cells (17). We hypothesized that GDF-9 shares receptors and downstream signaling molecules with related TGF-ß/activin/BMP ligands and took advantage of the availability of different promoter-luciferase constructs for these hormones to elucidate the GDF-9 signaling pathway. As shown in Table 1Go, the CAGA promoter is known to be activated by the TGF-ß/activin pathway mediated by Smad3 (31), whereas the BMP-responsive element (BRE) promoter is activated by BMP6 through the BMP pathway mediated by Smad1 and 5 (32), and the GCCG promoter is activated by BMP7 also through the BMP pathway mediated by Smad1, 5, and 8 (33).


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  		Table 1. Sequences for Each Reporter Construct

 
In preliminary studies, we transfected these three luciferase-reporter constructs into different cell lines and identified P19 cells as a GDF-9-responsive cell line. As shown in Fig. 1AGo, treatment of P19 cells with GDF-9, similar to TGF-ß and activin, stimulated the luciferase reporter driven by the CAGA promoter. Although BMP2 was ineffective on the CAGA promoter (Fig. 1AGo), treatment with BMP2, but not GDF-9, TGF-ß, or activin, stimulated the reporters driven by the BRE or the GCCG promoter (Fig. 1Go, B and C). Furthermore, GDF-9 stimulation of the CAGA promoter was found to be dose-dependent with an ED50 value of 3 nM (Fig. 1DGo). These findings suggest that P19 cells derived from mouse teratocarcinoma likely express receptors and downstream genes necessary for GDF-9 signaling, thus providing a useful in vitro model to elucidate proteins essential for GDF-9 signal transduction.



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  		Fig. 1. Treatment with GDF-9 Stimulates the CAGA, But Not the BRE or the GCCG Promoter, in Mouse Carcinoma P19 Cells

P19 cells were transiently transfected with the TGF-ß/activin-responsive CAGA reporter (A), the BMP-responsive reporter BRE (B), or the BMP-responsive GCCG reporter (C). Cells were incubated for 24 h in the absence (Ct) or presence of GDF-9 (10 nM), TGF-ß (1 nM), activin (5 nM), or BMP2 (5 nM). D, P19 cells were transfected with the CAGA reporter and treated with increasing doses of GDF-9. The relative luciferase activity was normalized based on the ß-galactosidase activity to correct for variations in transfection efficiency.

 
GDF-9 Treatment Stimulates the Phosphorylation of Smad3 and Smad2, But Not Smad1, in P19 Cells
Two R-Smad proteins, Smad2 and Smad3, are downstream mediators for TGF-ß and activin (22). Because GDF-9, like TGF-ß and activin, is capable of stimulating the CAGA promoter, a known target of Smad3, we tested whether treatment with GDF-9 could induce the phosphorylation of different Smad proteins in P19 cells. As shown in Fig. 2Go, A and B, treatment with GDF-9, like activin, increased the level of phospho-Smad3 and phospho-Smad2. Unlike BMP2, GDF9 did not increase the level of phospho-Smad1 (Fig. 2CGo). These data suggested the GDF-9 stimulation of the CAGA promoter is correlated with its activation of Smad3, as previously found for TGF-ß (31).



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  		Fig. 2. Stimulatory Effects of GDF-9 on the Phosphorylation of Smad2 and Smad3, But Not Smad1, in P19 Cells

A, Stimulation of Smad3 phosphorylation by GDF-9 and activin. B, Stimulation of Smad2 phosphorylation by GDF-9 and activin. C, Lack of stimulation of Smad1 phosphorylation by GDF-9 in P19 cells. Immunoblot analysis of phosphorylated Smad proteins was performed using cell extracts after treatment of P19 cells with GDF-9 (10 nM), activin (5 nM), or BMP2 (2.5 nM) for 60 min. A nonspecific higher molecular weight band was found in the Smad2 blot.

 
GDF-9 Stimulation of the CAGA Promoter Is Blocked by the Inhibitory Smad7 But Not by Smad6
Because the transcriptional activities of all R-Smad proteins (Smad1, 2, 3, 5, and 8) are blocked by the inhibitory Smad7 (25, 26), whereas those of Smad1, 5, and 8 are blocked by the inhibitory Smad6 (27, 28), we tested the inhibitory activities of the two inhibitory Smad proteins in P19 cells treated with GDF-9. As shown in Fig. 3AGo, cotransfection with Smad7, but not Smad6, led to a dose-dependent suppression of the GDF-9 stimulation of the CAGA promoter, together with a minor inhibition of the basal promoter activity. Consistent with the role of Smad1, 5, and 8 in BMP2 signaling, treatment with either Smad6 or Smad7 blocked the BMP2 stimulation of the BRE promoter in the same cells (Fig. 3BGo). These results suggest that GDF-9 signaling does not involve the BMP-responsive pathway mediated by Smad1, 5, and 8 and likely involve Smad2 and Smad3 activated by ALK4, 5, and 7.



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  		Fig. 3. Antagonistic Effect of Inhibitory Smad Proteins on the GDF-9 Stimulation of the CAGA Promoter and on the BMP2 Stimulation of the BRE Promoter

A, P19 cells were transfected with 500 ng of the CAGA reporter and increasing amounts of plasmids encoding the inhibitory Smad7 or Smad6. After an overnight culture, cells were incubated for 24 h with or without GDF-9 (10 nM) before determination of luciferase activity. B, P19 cells were transfected with 250 ng of the BRE reporter and increasing amounts of plasmid encoding the inhibitory Smad7 or Smad6 before treatment with BMP2 (5 nM). The relative luciferase activity was normalized based on the ß-galactosidase activity to correct for variations in transfection efficiency.

 
Overexpression of ALK5 and Smad3 Confers GDF-9 Responsiveness in the Nonresponsive Cos7 Cells
In preliminary tests, we found that GDF-9 treatment was ineffective in activating the CAGA and BRE promoters in the Cos7 cells (Fig. 4AGo). However, these cells not only responded to TGF-ß and activin based on the stimulation of the CAGA promoter (Fig. 4AGo), but also responded to BMP2 and BMP7 as revealed by the stimulation of the BRE promoter-reporter (Fig. 4BGo). These results suggested that the Cos7 cells express BMPRII to mediate BMP actions as well as ALK4, ALK5, and Smad3 to mediate the actions of TGF-ß and activin. We performed an exhaustive search of the human genome and could not identify novel receptors with sequence similarity to the known type I receptors and thus hypothesized that GDF-9 likely acts through a known type I receptor. Because ALK4 (for activin), ALK5 (for TGF-ß), and ALK7 (for nodal) are likely upstream type I receptors for Smad3 (31, 34), they could mediate GDF-9 actions. We further hypothesized that the endogenous levels of one or more of the type I receptors, the type II BMPRII receptor, and/or Smad2 or Smad3 are too low in Cos7 cells to allow efficient GDF-9 signaling.



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  		Fig. 4. Treatment with GDF-9 Does Not Stimulate the CAGA Promoter in Cos7 Cells

Cos7 cells were transfected with 500 ng of the TGF-ß/activin-responsive CAGA reporter (A); or the BMP-responsive reporter BRE (B). Cells were incubated for 24 h in the absence (Ct) or presence of GDF-9 (10 nM), activin (5 nM), TGF-ß (1 nM), BMP2 (5 nM), or BMP7 (5 nM). The relative luciferase activity was normalized based on the ß-galactosidase activity to correct for variations in transfection efficiency.

 
We performed overexpression tests in Cos7 cells in an attempt to gain GDF-9 responsiveness. Although overexpression of BMPRII did not confer GDF-9 responsiveness (data not shown), transfection of these cells with increasing amounts of the ALK5 expression plasmid led to a dose-dependent stimulation of the CAGA promoter activity by GDF-9 (Fig. 5AGo). In these cells, transfection with the ALK5 plasmid also increased TGF-ß, but not activin signaling. In contrast, transfection of increasing amounts of the ALK4 plasmid alone led to a dose-dependent increase in luciferase activity (Fig. 5BGo, left panel). In these ALK4-transfected cells, treatment with GDF-9 and TGF-ß, unlike activin, was ineffective in further stimulating the CAGA promoter. In addition, overexpression of ALK7 did not confer hormonal responsiveness to GDF-9, activin, or TGF-ß (Fig. 5CGo).



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  		Fig. 5. Overexpression of ALK5 Confers GDF-9 Responsiveness in Cos7 Cells

A, Cells were transfected with 500 ng of the CAGA reporter and increasing amounts of the ALK5 plasmid. After 5 h of incubation, cells were incubated for 24 h without or with GDF-9 (10 nM), activin (5 nM), or TGF-ß (1 nM). Similar experiments were performed by using plasmids encoding ALK4 (B) or ALK7 (C). D, Cells were transfected with 500 ng of the CAGA reporter and 30 ng of the plasmids encoding ALK5, 1, 2, 3, or 6. Cells were incubated for 24 h without or with GDF-9 (10 nM). The relative luciferase activity was normalized based on the ß-galactosidase activity.

 
To completely rule out the possible involvement of other type I receptors in GDF9 signaling, we further overexpressed the remaining known type I receptors in Cos7 cells. As shown in Fig. 5DGo, overexpression of ALK1, 2, 3, or 6 did not confer GDF9 activation of the CAGA promoter.

Because the CAGA promoter is responsive to Smad3, but not Smad2, activation (Table 1Go) (31), we further tested whether transfection with Smad3, alone or together with ALK5, could confer GDF-9 responsiveness to Cos7 cells. As shown in Fig. 6Go, cells overexpressing Smad3 did not show increased GDF-9 responsiveness. However, cotransfection of the Smad3 plasmid with a submaximal level (10 ng) of the ALK5 plasmid further augmented the GDF-9 stimulation of the CAGA promoter. These data suggested that Smad3 and ALK5 could act together to confer GDF-9 responsiveness to Cos7 cells.



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  		Fig. 6. Overexpression of Smad3 Augments GDF-9 Responsiveness Conferred by ALK5 in Cos7 Cells

Cells were transfected with 500 ng of the CAGA reporter, with or without the ALK5 receptor (10 ng), and increasing amounts of Smad3. Four hours after transfection, cells were incubated for 24 h without or with GDF-9 (10 nM). The relative luciferase activity was normalized based on the ß-galactosidase activity to correct for variations in transfection efficiency.

 
Cultured Granulosa Cells Respond to GDF-9 in the Stimulation of CAGA Promoter Activity and the GDF-9 Stimulation of Smad3 Phosphorylation
Based on findings using P19 and Cos7 cells, we tested the ability of GDF-9 to stimulate the CAGA promoter in its known target cells. Granulosa cells were obtained from early antral follicles of immature rats pretreated with estrogens to stimulate follicle growth, followed by transfection with the CAGA promoter and hormonal treatment. As shown in Fig. 7Go, A and B, treatment with GDF-9, similar to activin and TGF-ß, increased CAGA promoter activity in a dose-dependent manner with an ED50 value of 2 nM.



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  		Fig. 7. Treatment with GDF-9 Stimulates the CAGA Promoter and Induces the Phosphorylation of Smad3 in Cultured Granulosa Cells

A, Granulosa cells from early antral follicles were transiently transfected with 250 ng of the CAGA reporter, followed by incubation for 24 h in the absence (Ct) or presence of GDF-9 (10 nM), TGF-ß (0.1 nM), or activin (5 nM). B, Granulosa cells were transfected with the CAGA reporter and stimulated with increasing doses of GDF-9. The relative luciferase activity was normalized based on the ß-galactosidase activity to correct for variations in transfection efficiency. C, Stimulatory effects of GDF-9 on the phosphorylation of Smad3 in cultured granulosa cells. Immunoblot analysis of the granulosa cell extracts showed the phosphorylation of Smad3 after stimulation by GDF-9 (10 nM) and activin (5 nM) for 60 min. A nonspecific higher molecular weight band was found.

 
Our earlier data demonstrated the ability of GDF-9 to stimulate Smad2 phosphorylation in granulosa cells (18, 19). Because studies using P19 cells demonstrated the ability of GDF-9 to stimulate Smad3 phosphorylation, we tested whether treatment with GDF-9 could induce the phosphorylation of Smad3 proteins in granulosa cells. As shown in Fig. 7CGo, treatment with GDF-9, like activin, increased the level of phospho-Smad3. The lower molecular weight band corresponded to recombinant Smad3 after transfection of granulosa cells with the FLAG-Smad3 plasmid and blotting with the M2 antibody. The higher molecular weight band represents a nonspecific signal. These data suggested that the GDF-9 stimulation of the CAGA promoter is correlated with its stimulation of Smad3 phosphorylation in granulosa cells.

We further tested the effect of inhibitory Smad proteins on GDF-9 signaling in granulosa cells. As shown in Fig. 8Go, transfection of these cells with the inhibitory Smad7 suppressed GDF-9 stimulation of the CAGA promoter, consistent with findings derived from P19 cells. In contrast, only minimal suppression of GDF-9 effects was observed when cells were transfected with the highest dose (150 ng) of Smad6.



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  		Fig. 8. Antagonistic Effects of Smad7, But Not Smad6, on the GDF-9 Stimulation of the CAGA Promoter in Cultured Granulosa Cells

Granulosa cells were transiently transfected with 250 ng of the CAGA reporter and increasing amounts (10, 50, 150 ng/500 µl) of the plasmid encoding the inhibitory Smads. At 4 h after transfection, cells were incubated for 24 h with or without GDF-9 (10 nM). The relative luciferase activity was normalized based on the ß-galactosidase activity to correct for variations in transfection efficiency.

 
Inhibitory Effects of ALK5 siRNA on GDF-9 Signaling in Granulosa Cells
We further tested whether granulosa cells, like Cos7 cells, require ALK5 for GDF-9 signaling. Control and ALK5 siRNAs were transfected into cultured granulosa cells before testing the ability of GDF-9 to activate the CAGA promoter. As shown in Fig. 9AGo, transfection with increasing amounts of ALK5 siRNA, but not the control siRNA, led to dose-dependent decreases in CAGA promoter activity stimulated by GDF-9. Although treatment with ALK5 siRNA also suppressed CAGA promoter activity stimulated by TGF-ß (Fig. 9BGo), no suppression of CAGA promoter activity by activin was observed (Fig. 9CGo). These findings demonstrated the important role of ALK5 in GDF-9 actions in granulosa cells.



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  		Fig. 9. Treatment with ALK5 siRNA Blocked the GDF-9 Stimulation of the CAGA Promoter Activity in Cultured Granulosa Cells

A, Granulosa cells were transfected with 250 ng of the CAGA reporter and increasing amounts (0.3, 1, and 10 nM) of the control or ALK5 siRNA. One day after transfection, cells were incubated for 20 h with or without GDF-9 (10 nM). Similar tests were performed using TGF-ß (0.1 nM) (B) or activin (5 nM) (C). The relative luciferase activity was normalized based on the ß-galactosidase activity to correct for variations in transfection efficiency.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Based on overexpression and siRNA analyses, the present results demonstrate the role of ALK5 and Smad3 in the mediation of GDF-9 signaling in two cell lines and in ovarian granulosa cells. We showed that GDF-9 action is mediated by ALK5 and Smad2/3, proteins previously found to be essential for TGF-ß/activin signaling. The TGF-ß/activin/BMP superfamily proteins appear to use a limited set of intracellular receptors and Smad proteins for ligand signaling; GDF-9 binds to the BMPRII in the BMP pathway and activates downstream responses mediated by ALK5 and Smad2/3 in the TGF-ß/activin pathway. The combinatorial expression and interactions of these signal transduction genes in a tissue- and development-specific manner allow the manifestation of differential hormonal responsiveness and transcriptional activation or repression of target genes. For GDF-9, highly restricted expression of this ligand in the oocyte further refines its ovary-specific role in follicle development.

As shown in Fig. 10Go, TGF-ß and activin interact with their respective type II receptors, followed by the activation of the type I receptors, ALK5 and ALK4, respectively. This, in turn, leads to the phosphorylation of the downstream Smad3 and Smad2 proteins (35, 36, 37). Stimulation of the CAGA promoter by these ligands is mediated by the Smad3 and Smad4 proteins (31). In contrast, BMP2 binds to the specific type II receptor, BMPRII, and the type I receptors, ALK3 and ALK6, leading to the activation of Smad1, 5, and 8 (22). This is followed by the subsequent activation of the BRE and GCCG promoters (32, 33). Furthermore, BMP7 binds to the type II receptors, BMPRII, ActRII, and ActIIB to activate ALK2, 3, and 6. This is also followed by the phosphorylation of Smad1, 5, and 8 (22).



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  		Fig. 10. Diagrammatic Representation of the Intracellular Signaling Pathway for GDF-9 and Other TGF-ß Family Proteins

GDF-9 and related TGF-ß, activin, BMP2, and BMP7 bind to respective type II and type I receptors in target cells to activate downstream R-Smad proteins. Although GDF-9 interacts with the type II receptor for BMPs (BMPRII), its downstream actions are mediated by the type I receptor ALK5, and by Smad2 and Smad3 known to be essential for TGF-ß/activin signaling.

 
As shown in Table 1Go, Smad-binding elements in the promoters of target genes (22) are useful for constructing reporter genes found to be activated by diverse TGF-ß family members (31, 32, 33, 38, 39). In P19 cells, treatment with GDF-9 stimulates the CAGA promoter. This construct consists of 12 tandem repeats of the Smad-binding element present in the plasminogen activator inhibitor type I promoter (31) and is specifically activated by the Smad3 protein downstream of ALK4, 5, and 7 (31, 38, 39). Using immunoblots, we demonstrated the GDF-9 induction of Smad3 phosphorylation in P19 cells and granulosa cells. In P19 cells, the observed lower phosphorylation of Smad3 by GDF9 as compared with activin is consistent with the lower stimulatory effect of GDF9 on the CAGA promoter. Furthermore, GDF-9 activates Smad2, but not Smad1, in P19 cells, as previously found in rat and human granulosa cells (18, 19). The essential role of Smad2 and Smad3 in GDF-9 signaling is further confirmed by the selective blocking effect of inhibitory Smad7, but not Smad6, in both P19 cells and granulosa cells. Smad7 stably interacts with all activated type I receptors to prevent R-Smad activation and downstream transcriptional modulation (25, 26), whereas Smad6 specifically competes with R-Smad1 for complex formation with Smad4 (27), thus preferentially inhibiting the BMP pathway (28). The lack of effect of the inhibitory Smad6 on GDF-9 signaling is consistent with the conclusion that the downstream BMP pathway genes (Smad1/5/8) are not involved in GDF-9 actions.

Because Smad2 and Smad3 are activated by ALK4, 5, and 7 (22, 34), we overexpressed these type I receptors in the nonresponsive Cos7 cells and identified ALK5 as a type I receptor for GDF-9 based on the stimulation of the CAGA promoter. Furthermore, overexpression of Smad3 augmented the stimulatory effects of coexpressed ALK5. ALK5 is a known receptor for TGF-ß isoforms (40, 41), and the present demonstration of ALK5 as the type I receptor for GDF-9 indicated that this receptor is shared by TGF-ß and GDF-9. A recent study further indicated that ALK5 serves as a type I receptor for myostatin (42).

To further demonstrate the role of the ALK5 gene in GDF-9 signaling in the ovary, we designed 21-nucleotide duplexes of siRNA homologous to the ALK5 gene to allow sequence-specific posttranscriptional gene silencing (43) in cultured rat granulosa cells. Transfection with siRNA corresponding to the sequence of ALK5 suppressed the GDF-9 stimulation of the CAGA promoter. Because the inhibitory effects of ALK5 siRNA is more pronounced on GDF9 than on TGF-ß signaling, these data suggest that the level of ALK5 required for GDF9 signaling is higher than that for TGF-ß.

Earlier reports demonstrated the expression of ALK5, referred to as TGFßRI, in the granulosa cells of primordial, preantral, and antral follicles (44) as well as the expression of Smad3 in the granulosa cells of preantral, small antral, and large antral follicles (45). Although the fertility status of ALK5 null mice cannot be studied due to embryonic lethality at midgestation (46), the Smad3 null mice are viable but have reduced fertility compared with wild-type mice (47). Consistent with the important role of the GDF-9 pathway in initial follicle recruitment, decreases in Smad3 expression in Smad3 null mice did not affect the size of the primordial follicle pool at birth but did alter the growth of primordial follicles to the antral stage.

Our earlier data demonstrated that BMPRII binds to GDF-9 and transfection of antisense RNA against BMPRII blocks GDF-9 signaling in granulosa cells (29). In association with ALK3 or ALK6, BMPRII has been shown to selectively bind BMPs (48, 49, 50, 51, 52, 53), but the present data suggest this type II receptor is also capable of interacting with ALK5, which was believed to mediate the action of TGF-ß. Our data suggested cross talk between the classic BMP type II receptor, and ALK5 and the downstream Smad3 and Smad2 proteins (Fig. 10Go). Of interest, direct interactions between BMPRII and the cytoplasmic domain of ALK5 have been detected using yeast two-hybrid analyses (48). Furthermore, recent data indicated that ligand-activated TGFßRII interacts with ALK1, in addition to ALK5, to stimulate the Smad1, 5, and 8 pathway (24, 54), providing another example of cross talk between the classic BMP and TGF-ß/activin pathways. Moreover, activation of ALK1 by TGF-ß was shown to involve a receptor complex consisting of ALK1, ALK5, and TGFßRII (55). Indeed, cotransfection and binding experiments suggested the possibility of combinatorial interactions between different type II and type I receptors in transfected cells (51, 56, 57, 58).

Although embryonic cells are unlikely to be exposed to GDF-9, P19 cells of embryonic origin express all the genes needed for GDF-9 stimulation of the CAGA promoter. Identification of the GDF-9-responsive P19 and nonresponsive Cos7 cell lines provides convenient in vitro models with which to investigate signal components for GDF-9. This approach should be useful for studies on the signaling of other TGF-ß family ligands with uncharacterized receptors and Smad proteins. It is interesting to note that endogenous levels of ALK5 in Cos7 cells are sufficient for full TGF-ß signaling but inadequate for GDF-9 signaling. In Cos7 cells, the levels of ALK5 may be too low for the formation of functional GDF9/BMPRII/ALK5 complexes. It is likely that the exact levels (and ratios) of different type I and type II receptors for optimal signal transduction are ligand specific, thus representing another regulatory step for the signaling of a wide array of TGF-ß ligands by a limited number of receptors. Alternatively, the exact level of the receptor requirement may be modified by the expression of accessory receptors such as betaglycan (the TGF-ß receptor type III), endoglin (24, 59), or cripto (39, 60).

GDF-9B/BMP15, with close sequence homology to GDF-9, is also expressed exclusively in the oocyte (9, 10, 14) and a point mutation of this gene in Inverdale sheep is associated with defective follicle development (61). Like GDF-9, GDF-9B stimulates granulosa cell proliferation and progesterone production (11, 62). Unlike GDF-9, GDF-9B was found to bind the extracellular domain of ALK6 and activates the downstream Smad1. Thus, the actions of paralogous GDF-9 and GDF-9B are mediated by distinct receptors and intracellular pathways, suggesting these oocyte ligands could play unique roles in follicle development. In addition to these oocyte ligands, several other TGF-ß and BMP family members are expressed in the ovary. Treatment with BMP7 or BMP4, secreted by theca cells, reduces FSH-induced progesterone production by granulosa cells (4), whereas both TGF-ß and activin regulate proliferation and differentiation of cultured granulosa cells (5, 63). Thus, diverse TGF-ß superfamily members originating from different follicular compartments use a limited set of receptors and Smad proteins to exert distinct but overlapping effects on granulosa cell function, thereby leading to a coordinated regulation of follicle development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents and Hormones
McCoy’s 5a medium, L-15 Leibovitz medium, DMEM, {alpha}-MEM, and OptiMEM were obtained from Life Technologies (Gaithersburg, MD). L-Glutamine, penicillin, and streptomycin were purchased from BioWhittaker (Wakersville, MD). Recombinant activin A, BMP2, BMP7, and human TGF-ß1 were from R&D Systems (Minneapolis, MN).

Recombinant GDF-9 was generated and characterized as previously described (17). Briefly, expression vectors for wild-type and epitope-tagged GDF-9 were constructed using pcDNA3.1 Zeo (Invitrogen, Carslbad, CA). N-tagged GDF-9 encoded a Flag epitope for the M1 antibody followed by six histidine residues fused to the amino terminus of mature GDF-9. Clonal human embryonic kidney 293T cell lines stably expressing wild-type and tagged GDF-9 were used. Quantitation of N-tagged GDF-9 was done after purification with nickel column and measurement of protein content using Micro BCA protein assay kit (Perstorp Life Science, Rockford, IL). Purified N-tagged GDF-9 was then used as a standard for the quantitation of wild-type GDF-9 in the conditioned medium of 293T cells by immunoblots using specific GDF-9 antibodies. Phospho-Smad1 and phospho-Smad2 antibodies were obtained from Dr. C. H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden). Phospho-Smad3 antibody was provided by Dr. E. Leof (Mayo Clinic, MN). Specificity of the antibodies for phospho-Smads has been previously reported (64, 65, 66).

Expression Plasmids and Reporter Gene Constructs
pcI-ALK4 (ActRIB) was provided by Dr. O. Klibanski (Massachusetts General Hospital, Boston, MA) and subcloned in the pcDNA3 vector (Invitrogen). The pcDNA3-ALK1, pcDNA3-ALK2 (ActRIA), pcDNA3-ALK3 (BMPRIA), pcDNA3-ALK5 (TGF-ß RI), pcDNA3-ALK6 (BMPRIB), pcDNA3-ALK7, pcDNA3-Smad3, pcDNA3-Smad6, and pcDNA3-Smad7 expression plasmids were from Dr. P. Ten Dijke (The Netherlands Cancer Institute, Amsterdam, The Netherlands). The reporter plasmid pGL3(CAGA)12-lux was provided by Dr. C. H. Heldin (Ludwig Institute for Cancer Research) (31). The reporter pGL2 (GCCG)15-lux was provided by Dr. K. Kusanagi (Japanese Foundation for Cancer Research, Tokyo, Japan) (33). The pGL3 (BRE)2-lux has been described previously (32). The sequences for each reporter construct are described in Table 1Go.

siRNA
siRNAs were chemically synthesized by QIAGEN (Valencia, CA). The siRNA sequences targeting ALK5 corresponded to the nucleotides 1399–1421 of the rat ALK5 sequence (GenBank accession no. L26110) 5'-AACAGATGGCAGAGCTGTGAGGC-3'. The control siRNA 5'-AATTCTCCGAACGTGTCACGT-3' was from QIAGEN. For annealing of siRNAs, 20 µM single-strand RNA were incubated in the annealing buffer [100 mM potassium acetate, 30 mM HEPES-KOH (pH 7.4), 2 mM magnesium acetate] for 1 min at 90 C followed by 1 h at 37 C.

Cell Lines and Transfections
P19 cells were cultured in {alpha}-MEM supplemented with 10% fetal bovine serum (FBS), together with 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Cells were seeded at 60% confluency in 24-well plates and transiently transfected in {alpha}-MEM for 4 h with 1.8 µg of DNA per well using Fugene-6 (Roche Diagnostics Corp., Indianapolis, IN). After transfection, cells were treated with the appropriate ligands for 24 h in {alpha}-MEM containing 5% FBS. Cos7 cells were cultured in DMEM/high glucose supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Cos7 cells were seeded at 90% confluency in 24-well plates and transiently transfected in DMEM/high glucose for 4 h with 1 µg of DNA per well using Lipofectamine 2000 (Invitrogen). After transfection, cells were treated with the appropriate ligand for 24 h in DMEM/high glucose containing 1% FBS. Fifty nanograms of the pCMV-ß-galactosidase vector were cotransfected to monitor transfection efficiency.

To harvest cells, lysis buffer (200 µl) (Promega Corp., Madison, WI) was added to each well and 30 µl of the supernatant was used for luciferase determination using a luminometer (Luminark microplate reader, Bio-Rad Laboratories, Inc., Hercules, CA). Fifty microliters of the cell lysate were also used to measure the ß-galactosidase activity. The reporter activity is expressed as the ratio of relative light unit/ß-galactosidase activity. Data are the mean ±SEM of triplicates from representative experiments.

Animals
Immature female rats (Sprague-Dawley, 25 d old, body weight from 50–60 g) were obtained from Charles River Laboratories (Wilmington, MA). Animals were anesthetized and killed using CO2 72 h after insertion of diethylstilbestrol implants. All animals were housed under controlled humidity, temperature, and light regimen and fed ad libitum on a standard rat chow. Animal care was consistent with institutional and National Institutes of Health guidelines.

Granulosa Cell Culture and Transfection
Granulosa cells were obtained from small antral follicles of estrogen-treated rats (67). Ovaries were punctured in L-15 Leibovitz medium. Ovarian debris, oocytes, and small follicles were removed, and the remaining medium containing granulosa cells was collected after low-speed centrifugation at 500 x g for 10 min. Granulosa cells were resuspended into the culture medium (McCoy’s 5a supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin).

Granulosa cells (3 x 105 viable cells/well) were cultured in 24-well plates in McCoy’s 5a medium supplemented with 10% FBS for 3 h. After media change, cells were incubated in the serum-free medium and transfected with 300 ng of DNA per well using Lipofectamine 2000 (Invitrogen). The pCMV-ß-galactosidase plasmid was cotransfected to monitor transfection efficiency. After transfection, cells were treated with the appropriate ligand for 24 h in McCoy’s 5a medium/1% FBS. For the siRNA experiments, 250 ng of DNA were transfected with or without increasing amounts of siRNA together with Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were treated with the appropriate ligand for 24 h in McCoy’s 5a medium/1% FBS. To harvest cells, lysis buffer (200 µl) (Promega Corp.) was added into each well and 30 µl of the supernatant was used for luciferase determination using a luminometer (Luminark microplate reader, Bio-Rad Laboratories, Inc.). The ß-galactosidase activity was also determined to monitor transfection efficiency. The reporter activity is expressed as the ratio of relative light unit/ß-galactosidase activity. Data are the mean ± SEM of triplicates from representative experiments.

Western Blotting Analysis of Smad Proteins
To investigate Smad1, Smad2, and Smad3 activation by GDF-9, P19 cells (106 cells) were cultured overnight in six-well plates in {alpha}-MEM containing 10% FBS and starved for 3 h in serum-free media to minimize basal Smad activity. In addition, granulosa cells (3 x 103 cells) were punctured and plated for 3 h in McCoy’s 5a medium. P19 and granulosa cells were treated with different hormones for 60 min and washed once on ice with chilled PBS before cell lysis in the Laemmli buffer containing ß-mercaptoethanol. Cells were gently sonicated on ice for 15 sec with a sonicator (Sanyo Corp., Osaka, Japan) and boiled for 3 min. Cellular proteins were separated on 8% SDS-PAGE gels and electroblotted onto Amersham Hybond-electrochemiluminescence (for Smad2 experiments) and Hybond-P membranes (for Smad1 and Smad3 experiments) (Amersham Bioscience Corp., Piscataway, NJ). For the detection of phosphorylated Smads, membranes were blocked for 1 h at room temperature in Tris-buffered saline-containing 0.1% Tween and 5% fat-free dry milk. After blocking of nonspecific binding, membranes were incubated with antiphospho-Smad2 or antiphospho-Smad1 antibodies diluted at 1:8000 or antiphospho-Smad3 antibodies diluted at 1:3000 at 4 C overnight. The secondary antirabbit antibodies were used, following the manufacturer’s instructions (Amersham Bioscience Corp). Immunoreactive proteins were detected using enhanced chemiluminescence (ECL kit, Amersham Bioscience Corp).


   ACKNOWLEDGMENTS
 
We thank Dr. P. Ten Dijke (The Netherlands Cancer Institute, Amsterdam, The Netherlands) for providing the ALK5, ALK7, and Smad3/6/7 plasmid constructs. We thank Dr. C. H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden) for the CAGA promoter-luciferase construct, and the phospho-Smad1 and phospho-Smad2 antibodies, Dr. K. Kusanagi (Japanese Foundation for Cancer Research, Tokyo, Japan) for the GCCG promoter-luciferase construct, and Dr. Klibanski (Massachusetts General Hospital, Boston, MA) for the pcI-ALK4 construct. We are grateful to Dr. E. Leof (Mayo Clinic, Rochester, MN) for the generous gift of the phospho-Smad3 antibodies. We thank C. Spencer for editorial assistance.


   FOOTNOTES
 
This work was supported by the National Institute of Child Health and Human Development, and the NIH, through Cooperative Agreement U54 HD31398 as part of the Specialized Cooperative Centers Program in Reproduction Research. The work of the lab of O.R. was supported by grants from the Academy of Finland, the Juselius Foundation, the Novo Nordisk Foundation, and the Helsinki University Central Hospital Funds. The work of O.K. was supported by the Dutch Organization for Scientific Research Grant ALW 809.67.024.

Current address for O.K.: Department of Rheumatology and Thurston Arthritis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7820.

Abbreviations: ActRII, Activin receptor type II; ALK, activin receptor-like kinase; BMP, bone morphogenetic protein; BMPRII, BMP receptor type II; BRE, BMP-responsive element; CAGA, a TGF-ß/activin-responsive promoter reporter; FBS, fetal bovine serum; GDF-9, growth differentiation factor-9; siRNA, small interfering RNA; TGFßRII, TGF-ß receptor type II.

Received for publication October 9, 2003. Accepted for publication December 10, 2003.


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