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

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.

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.

mRNA levels of osteogenic genes and Bmal1 were up-regulated in MC3T3-E1 cells upon insulin treatment. In addition, Bmal1 overexpression increased the expression of osteogenic genes including inhibitor of DNA binding (Id1), 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 Bmal1. Finally, we confirmed that insulin-induced BMP2 expression was attenuated in Bmal1 knockout (KO) cells. PCR analysis and alizarin red S staining showed that insulin-mediated increases gene expression and calcium deposition were reduced in Bmal1 KO cells compared to wild-type cells.

Taken together, these results demonstrate that Bmal1 promotes osteoblast differentiation by regulating BMP2 expression in MC3T3-E1 cells.