Bone morphogenetic protein-3b (BMP-3b) inhibits osteoblast differentiation via Smad2/3 pathway by counteracting Smad1/5/8 signaling

Despite the involvement of BMP-3b (also called GDF-10) in osteogenesis, embryogenesis and adipogenesis, the functional receptors and intracellular signaling of BMP-3b have yet to be elucidated. In the present study, we investigated the cellular mechanism of BMP-3b in osteoblast differentiation using mouse myoblastic C2C12 cells. BMP-3b stimulated activin/TGF-β-responsive promoter activities. The stimulatory actions of BMP-3b on activin/TGF-β-responsive activities were suppressed by co-treatment with BMP-2. BMP-responsive promoter activities stimulated by BMP-2 were significantly inhibited by treatment with BMP-3b. BMP-3b suppressed the expression of osteoblastic markers including Runx2, osteocalcin and type-1 collagen induced by BMP-2, -4, -6 and -7. BMP-2-induced Smad1/5/8 phosphorylation and mRNA levels of the BMP target gene Id-1 were suppressed by co-treatment with BMP-3b, although BMP-3b failed to activate Smad1/5/8 signaling. Of interest, the BMP-3b suppression of BMP-2-induced Id-1 expression was not observed in cells overexpressing Smad4 molecules. On the other hand, BMP-3b directly activated Smad2/3 phosphorylation and activin/TGF-β target gene PAI-1 mRNA expression, while BMP-2 suppressed BMP-3b-induced Smad2/3 signal activation. BMP-2 inhibition of BMP-3b-induced PAI-1 expression was also reversed by overexpression of Smad4. Analysis using inhibitors for BMP–Smad1/5/8 pathways revealed that these BMP-3b effects were mediated via receptors other than ALK-2, -3 and -6. Furthermore, results of inhibitory studies using extracellular domains for BMP receptor constructs showed that the activity of BMP-3b was functionally facilitated by a combination of ALK-4 and ActRIIA. Collectively, BMP-3b plays an inhibitory role in the process of osteoblast differentiation, in which BMP-3b and BMP-2 are mutually antagonistic possibly by competing with the availability of Smad4.