Cryptochrome 1 promotes osteogenic differentiation of human osteoblastic cells via Wnt/β-Catenin signaling

The exact mechanism underlying osteoblast differentiation and proliferation remains to be further elucidated. The circadian clock has been universally acknowledged controls behavioral activities and biological process in mammals. Cryptochrome 1 (Cry1), one of the core circadian genes, is associated with bone metabolism. However, the exact role and potential mechanism of Cry1 in regulating osteogenesis are still unclear.

Western blotting and qRT-PCR were applied to detect Cry1 expression levels, molecules in osteogenesis related signaling pathways and osteogenic transcriptional markers. The ALP staining and Alizarin red S staining were performed to weigh osteogenic state, while CCK8 assay was used to detect cell growth rates. Osteogenic capability of osteoblasts was determined using an ectopic bone formation assay.

Cry1 was upregulated in the process of osteoblast differentiation, along with osteogenic transcriptional factors. Then, Cry1 upregulation and knockdown cell lines were established and we found Cry1 overexpression promoted osteogenesis and proliferation of osteoblasts both in vitro and in vivo. Besides, the canonical Wnt/β-Catenin signaling was increasingly activated by Cry1 overexpression, whereas inhibition of β-Catenin restrained enhanced osteogenic capability of Cry1 upregulated osteoblasts.

In conclusion, these results suggest that Cry1 promotes osteogenic differentiation of human osteoblasts through the canonical Wnt/β-Catenin signaling.