Abstract
Bone morphogenetic proteins (BMPs) are important mediators of osteoclast differentiation. Although accumulating evidence has implicated BMPs in osteoblastogenesis, the mechanisms by which BMPs regulate osteoclastogenesis remain unclear. Activin A receptor type 1 (ACVR1) is a BMP type 1 receptor essential for skeletal development. Here, we observed that BMP-7, which preferentially binds to ACVR1, promotes osteoclast differentiation, suggesting ACVR1 is involved in osteoclastogenesis. To investigate this further, we isolated osteoclasts from either Acvr1-floxed mice or mice with constitutively-activated Acvr1 (caAcvr1) carrying tamoxifen-inducible Cre driven by a ubiquitin promotor and induced Cre activity in culture. Osteoclasts from the Acvr1-floxed mice had reduced osteoclast numbers and demineralization activity, whereas those from the caAcvr1-mutant mice formed large osteoclasts and demineralized pits, suggesting that BMP signaling through ACVR1 regulates osteoclast fusion and activity. It is reported that BMP-2 binds to BMPR1A, another BMP type 1 receptor, whereas BMP-7 binds to ACVR1 to activate SMAD1/5/9 signaling. Here, Bmpr1a-disrupted osteoclasts displayed reduced phospho-SMAD1/5/9 (pSMAD1/5/9) levels when induced by BMP-2, whereas no impacts on pSMAD1/5/9 were observed when induced by BMP-7. In contract, Acvr1-disrupted osteoclasts displayed reduced pSMAD1/5/9 levels when induced either by BMP-2 or BMP-7, suggesting that ACVR1 is the major receptor for transducing BMP-7 signals in osteoclasts. Indeed, LDN-193189 and LDN-212854, which specifically block SMAD1/5/9 phosphorylation, inhibited osteoclastogenesis of caAcvr1-mutant cells. Moreover, increased BMP signaling promoted nuclear translocation of nuclear factor-activated T-cells 1 (NFATc1), which was inhibited by LDN treatments. Taken together, ACVR1-mediated BMP-SMAD signaling activates NFATc1, a regulatory protein crucial for receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis.
Highlights
Bone morphogenetic proteins (BMPs) are important mediators of osteoclast differentiation
These results indicate that Acvr1 is highly expressed both in osteoblasts and osteoclasts, whereas Bmpr1a is expressed at higher levels only in osteoblasts
Our findings demonstrate that ACVR1-mediated BMP signaling regulates RANKL-dependent osteoclastogenesis via the canonical SMAD-signaling pathway
Summary
M-CSF, macrophage colony–stimulating factor; BMP, bone morphogenetic protein; ACVR1, activin type 1 receptor; RANK, receptor activator of NF-B; RANKL, receptor activator of nuclear factor-B ligand; M-CSF, macrophage colony–stimulating factor, BMM, bone marrow mononuclear cells; TRAP, tartrate-resistant acid phosphatase; NFATc1, nuclear factor–activated T-cell 1; ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PI3K, phosphatidylinositol 3-kinase; OB, osteoblast; qRT-PCR, quantitative reverse-transcribed PCR; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; BMSC, bone marrow stromal cell; BMOC, bone marrow osteoclast; SPOC, spleen osteoclast; TNF, tumor necrosis factor; TM, tamoxifen; IL, interleukin; TGF, transforming growth factor-; 4-OHT, (Z)-4-hydroxytamoxifen; LDN-19, LDN-193189; LDN-21, LDN-212854; rh, recombinant human; DAPI, 4Ј,6-diamidino-2-phenylindole. Bmpr1b KO mice displayed no changes in osteoclast numbers in vivo, whereas osteoclasts from Bmpr1b KO mice displayed an increase in osteoclast formation and a decrease in bone-resorbing activity in vitro [17] These results suggest BMPR1A and BMPR1B play a distinct role in the differentiation and function of osteoclasts. These data suggest that ACVR1-mediated BMP signaling regulates RANKL-induced osteoclastogenesis via the canonical SMAD-signaling pathway
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