title: Bone morphogenetic protein-3b (BMP-3b) inhibits osteoblast differentiation via Smad2/3 pathway by counteracting Smad1/5/8 signaling.
abstract: 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.
authors.affiliation: Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kitaku, Okayama 700-8558, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Fumio
authors.initials: F
authors.lastname: Otsuka
authors.forename: Jun
authors.initials: J
authors.lastname: Hino
authors.forename: Tomoko
authors.initials: T
authors.lastname: Miyoshi
authors.forename: Mariko
authors.initials: M
authors.lastname: Takano
authors.forename: Mikiya
authors.initials: M
authors.lastname: Miyazato
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Makino
authors.forename: Kenji
authors.initials: K
authors.lastname: Kangawa
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology / Q000502:physiology; D002454:Cell Differentiation; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D055413:Growth Differentiation Factor 10 / Q000494:pharmacology / Q000502:physiology*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D010006:Osteoblasts / Q000502:physiology*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D058258:Serpin E2 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism*; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000502:physiology
chemical_list: D000943:Antigens, Differentiation; D019485:Bone Morphogenetic Proteins; D024042:Collagen Type I; D050659:Core Binding Factor Alpha 1 Subunit; D055413:Growth Differentiation Factor 10; C493716:Runx2 protein, mouse; D058258:Serpin E2; C511869:Serpine2 protein, mouse; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; D015675:Osteocalcin; D008156:Luciferases