Bmal1- and Per2-Mediated Regulation of the Osteogenic Differentiation and Proliferation of Mouse BMSCs by Modulating the Wnt/β-catenin Pathway

Abstract BackgroundBmal1 and Per2 are the core components of the circadian clock genes(CCGs). Bmal1-/- mice exhibit premature aging, as indicated by hypotrichosis and osteoporosis, with a loss of proliferation ability. The same occurs in Per2-/- mice, albeit to a less severe degree. However, whether the effects of Bmal1 and Per2 on proliferation and osteogenic differentiation are synergistic or antagonistic remains unclear. Thus, our study aimed to explore the effects and specific mechanism.Materials and methodsLentiviral and adenoviral vectors were constructed to silence or overexpress Bmal1 or Per2 and MTT, flow cytometry, RT-qPCR, WB, immunohistochemistry, alizarin red staining and ChIP-Seq analyses were applied to identify the possible mechanism. ResultsThe successful knockdown and overexpression of Bmal1/Per2 were detected by fluorescence microcopy. Flow cytometry found out that Bmal1 or Per2 knockdown resulted in G1-phase cell cycle arrest. RT-qPCR showed the different expression levels of Wnt-3a, c-myc1 and axin2 in the Wnt/β-catenin signaling pathway as well as the gene expression change of Rorα and Rev-erbα. Meanwhile, Related proteins such as β-catenin, TCF-1, and P-GSK-3β were detected. ALP activity and the amount of mineral nodules were compared. ChIP-Seq results showed the possible mechanism.ConclusionsBmal1 and Per2, as primary canonical clock genes, showed synergistic effects on the proliferation and differentiation of BMSCs. They would inhibit the Wnt/β-catenin signaling pathway by downregulating Rorα expression or upregulating Rev-erbα expression, both of which were also key elements of CCGs. And this may be the mechanism by which they negatively regulate the osteogenic differentiation of BMSCs.