Signalling pathways mediating specific synergistic interactions between GDF9 and BMP15

Abstract

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are two proteins selectively expressed in the oocyte which are essential for normal fertility. Both of these proteins are members of the transforming growth factor beta (TGF-β) superfamily and as such are produced as pre-proproteins, existing after proteolytic processing as a complex of the respective pro and mature regions. Previous work has shown that these two proteins interact both at the genetic and cellular signalling levels. In this study, our aim was to determine if the purified mature regions of GDF9 and BMP15 exhibit synergistic interactions on granulosa cells and to determine if such interactions are specific to these two proteins. We have used primary cultures of murine granulosa cells and [(3)H]-thymidine incorporation or transcriptional reporter assays as our readouts. We observed clear synergistic interactions between the mature regions of GDF9 and BMP15 when either DNA synthesis or SMAD3 signalling were examined. GDF9/BMP15 synergistic interactions were specific such that neither factor could be replaced by an analogous TGF-β superfamily member. The GDF9/BMP15 synergistic signalling response was inhibited by the SMAD2/3 phosphorylation inhibitor SB431542, as well as inhibition of the mitogen-activated protein kinase or rous sarcoma oncogene (SRC) signalling pathways, but not the nuclear factor kappa B pathway. In this study, we show that purified mature regions of GDF9 and BMP15 synergistically interact in a specific manner which is not dependent on the presence of a pro-region. This synergistic interaction is targeted at the SMAD3 pathway, and is dependent on ERK1/2 and SRC kinase signalling.

Figure 1

Synergistic activity of GDF9 and BMP15. Mouse granulosa cells (A) were either left untreated (control) or treated with one of the following: (i) an increasing dose of GDF9 (3.1–12.5 ng/ml), (ii) an increasing dose of BMP15 (3.1–12.5 ng/ml) or (iii) a combination of both GDF9 and BMP15 at a 1:1 ratio (3.1–12.5 ng/ml each). Mouse cumulus cells (B) were similarly left untreated (control) or treated with either 12.5 ng/ml of GDF9, 12.5 ng/ml of BMP15 or a combination of both GDF9 and BMP15 (both at 12.5 ng/ml). [³H]-thymidine incorporation was measured following 24 h of culture. Bars represent means ± SEM from three replicate experiments expressed as fold change relative to the control. Data were analysed by multivariate ANOVA and only the GDF9 × BMP15 interaction term is reported in the figure.

Figure 2

GDF9/BMP15 synergistic activity stimulates the SMAD3, but not the SMAD1/5/8 pathway. Mouse granulosa cells were transiently transfected during culture with either a SMAD3 responsive CAGA-luciferase plasmid (A) or a SMAD1 responsive BRE-luciferase plasmid (B). In both experiments cells were either left untreated (control) or treated with one of the following; TGF-β1 (2 ng/ml), BMP6 (50 ng/ml), GDF9 (3.1–12.5 ng/ml), BMP15 (3.1–12.5 ng/ml) or a combination of both GDF9 and BMP15 at a 1:1 ratio (3.1–12.5 ng/ml each). Luciferase activity was measured following a 48 h treatment period. Bars represent means ± SEM from four replicate experiments expressed as fold change relative to the control. Data were analysed by multivariate ANOVA and only the GDF9 × BMP15 interaction term is reported in the figure.

Figure 3

GDF9/BMP15 synergistic activity is inhibited by the Alk4/5/7 inhibitor SB431542. Mouse granulosa cells were transiently transfected during culture with a SMAD3 responsive CAGA-luciferase plasmid (A) or radio-labelled with [³H]-thymidine (B). In both experiments, cells were left untreated (control) or treated with GDF9 (12.5 ng/ml), BMP15 (12.5 ng/ml), GDF9 + BMP15 (12.5 ng/ml each) or GDF9 + BMP15 + SB431542 (0.13–5 µM). Bars represent means ± SEM from three replicate experiments expressed as fold change relative to the control. Data were analysed by one-way ANOVA; *P < 0.01, **P < 0.001 designate significant differences relative to the GDF9 + BMP15 positive control.

Figure 4

GDF9/BMP15 synergism is specific. Mouse granulosa cells were left untreated (control), or treated with the following growth factors; activin A, activin B, BMP6, BMP7 (all at 50 ng/ml) or TGF-β1 (2 ng/ml), each either alone (open bars), or in combination with GDF9 (12.5 ng/ml) or BMP15 (12.5 ng/ml). [³H]-thymidine incorporation was measured following 24 h of culture. Bars represent means ± SEM from four replicate experiments expressed as fold change relative to the control. Potential synergistic interactions between growth factors were tested by two-way ANOVA and the P values for the respective interaction terms only are presented in the figure.

Figure 5

ERK1/2 and SRC but not NF-κB are required for GDF9/BMP15 synergistic activity. [³H]-thymidine incorporation was measured in granulosa cells either left untreated (control) or treated with GDF9 (12.5 ng/ml), BMP15 (12.5 ng/ml), GDF9 + BMP15 (12.5 ng/ml each) or GDF9 + BMP15 plus one of the following inhibitors: U0126 (0.13–5 µM) or its inactive analogue U0124 (5 µM) (A), PP2 (0.15–5 µM) (B), or SN50 or its inactive analogue SN50M (both at 0.3–5 µg/ml) (C). Bars represent means ± SEM from three to four replicate experiments expressed as fold change relative to the control. Data were analysed by one-way ANOVA; *P < 0.05, **P < 0.001 designate significant differences relative to the GDF9 + BMP15 positive control.