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

Li,Hai; Zhao,Dahang; Wang,Shengjing; Ding,Jing; Zhao,Li

doi:10.3892/mmr.2016.5803

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

Authors:
- Hai Li
- Dahang Zhao
- Shengjing Wang
- Jing Ding
- Li Zhao
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Affiliations: Department of Pediatric Orthopaedics, Xin‑Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, P.R. China
Published online on: October 5, 2016 https://doi.org/10.3892/mmr.2016.5803
Pages: 4545-4550
Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

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.

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