Bmal1 induces osteoblast differentiation via regulation of BMP2 expression in MC3T3-E1 cells

Aims: Mammalian circadian rhythms regulate many metabolic processes. Recent studies suggest that brain and muscle Arnt-like 1 (BMAL1), an important component of mammalian circadian rhythm, is associated with insulin signaling. Several studies have shown that insulin is associated with bone metabolism; however, the relationship between BMAL1 and osteoblasts remains unclear. Main methods: Expression of osteogenic markers and Bmal1 in MC3T3-E1 cells was measured by RT-PCR and Western blotting. Alizarin red S staining was performed to assess matrix mineralization in MC3T3-E1 cells. Key findings: mRNA levels of osteogenic genes and Bmall were up-regulated in MC3T3-E1 cells upon insulin treatment. In addition, Bmall overexpression increased the expression of osteogenic genes including inhibitor of DNA binding (1d1), Runt-related transcription factor 2 (Runx2), and osteocalcin (OC). Interestingly, expression of Bone morphogenetic protein-2 (BMP2), an important upstream factor of Id1, Runx2, and OC, was markedly increased by Bmall. Finally, we confirmed that insulin-induced BMP2 expression was attenuated in Bmall knockout (KO) cells. PCR analysis and alizarin red S staining showed that insulin-mediated increases gene expression and calcium deposition were reduced in Bmall KO cells compared to wild-type cells. Significance: Taken together, these results demonstrate that Bmall promotes osteoblast differentiation by regulating BMP2 expression in MC3T3-E1 cells. (C) 2016 Published by Elsevier Inc.