Bone morphogenetic protein-9 promotes the differentiation of mouse spleen macrophages into osteoclasts via the ALK1 receptor and ERK 1/2 pathways in vitro

It has been confirmed that bone morphogenetic protein-9 (BMP-9) promotes the differentiation of osteoblasts. However, the ways in which BMP-9 exerts its effects on the differentiation of osteoclasts and bone resorption remain to be elucidated. The present study was designed to investigate the roles and the molecular mechanism of BMP-9 on the proliferation and differentiation of osteoclast precursors in vitro. Mouse spleen macrophages (RAW 264.7 cells) were cultured in the presence of receptor activator for nuclear factor-κb ligand (RANKL) in vitro. Following treatment with different concentrations of BMP-9, a number of parameters were quantitatively monitored. Cell proliferation was determined using an MTT assay. The expression levels of cell BMP receptor-IA (BMPR-IA), BMPR-IB, BMPR-II and anaplastic lymphoma kinase 1 (ALK1) receptor were detected by ELISA, the small mothers against decapentaplegic pathway, extracellular signal-regulated kinase (ERK)1/2 pathways and markers of osteoclast differentiation were detected by western blotting. The results showed that treatment with BMP-9 alone promoted mouse spleen macrophage proliferation, and the differentiation into osteoclasts occurred only in the presence of RANK. The promoting effect of BMP-9 on cell proliferation and osteoclast differentiation occurred in dose-dependent manner. In addition, BMP-9 significantly upregulated the expression of the ALK1 receptor and inhibited the ERK1/2 pathway. The inhibition of the ERK1/2 pathways was ameliorated by transfection with small interfering (si)RNA ALK1. The effect of BMP-9 on osteoclast differentiation was reduced by transfection with siRNA ALK1, however, the effect was enhanced by the ERK1/2 pathway inhibitor, U0126. The results of the present study demonstrated that BMP-9 promoted the osteoclast differentiation of osteoclast precursors via binding to the ALK1 receptor on the cell surface, and inhibiting the ERK1/2 signaling pathways in the cell.