Department of Orthopedics, The Fifth People's Hospital of Shanghai, Fudan University, Shanghai, People's Republic of China.
Department of Orthopedics, The Fifth People's Hospital of Shanghai, Fudan University, Shanghai, People's Republic of China..
Life Sci. 2018 Nov 1;212:129-137. doi: 10.1016/j.lfs.2018.09.053. Epub 2018 Oct 2.
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.
成骨细胞分化和增殖的确切机制仍有待进一步阐明。昼夜节律钟已被普遍认为控制着哺乳动物的行为活动和生物过程。隐花色素 1（Cry1）是核心生物钟基因之一，与骨代谢有关。然而，Cry1 调节成骨作用的确切作用和潜在机制尚不清楚。
采用 Western blot 和 qRT-PCR 检测 Cry1 表达水平、成骨相关信号通路分子和成骨转录标志物。进行碱性磷酸酶（ALP）染色和茜素红 S 染色来衡量成骨状态，同时使用 CCK8 测定法检测细胞生长速率。通过异位骨形成测定来确定成骨细胞的成骨能力。
Cry1 在成骨细胞分化过程中上调，同时伴随着成骨转录因子。然后，建立了 Cry1 上调和敲低细胞系，我们发现 Cry1 过表达在体外和体内均促进成骨细胞的成骨和增殖。此外，Cry1 过表达可增强经典 Wnt/β-连环蛋白信号，而抑制 β-连环蛋白则抑制了 Cry1 上调的成骨细胞增强的成骨能力。
综上所述，这些结果表明 Cry1 通过经典 Wnt/β-连环蛋白信号促进人成骨细胞的成骨分化。
