Wang Hailong, Li Jinqiao, Xu Zihan, Wu Feng, Zhang Hongyu, Yang Chao, Chen Jian, Ding Bai, Sui Xiukun, Guo Zhifeng, Li Yinghui, Dai Zhongquan
State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, Beijing, China.
Space Engineering University, Beijing, China.
PeerJ. 2021 Mar 2;9:e10898. doi: 10.7717/peerj.10898. eCollection 2021.
Osteocalcin (OCN) was the most abundant noncollagen protein and considered as an endocrine factor. However, the functions of Undercarboxylated osteocalcin (ucOCN) on osteoclast and bone resorption are not well understood. In the present study, preosteoclast RAW264.7 cells and bone marrow mononuclear cells (BMMs) were treated with ucOCN purified from prokaryotic bacteria. Our results showed that ucOCN attenuated the proliferation of RAW264.7 cells with a concentration dependant manner by MTS assay. Scrape wounding assay revealed the decreased motility of RAW264.7 cells after ucOCN treatment. RT-qPCR results manifested the inhibitory effects of ucOCN on the expression of osteoclastic marker genes in RAW264.7 cells during inducing differentiation of RANKL. It was also observed that ucOCN inhibited the formation of multinucleated cells from RAW264.7 cells and BMMs detected by TRAP staining. The number and area of bone resorb pits were also decreased after treatment with ucOCN during their osteoclast induction by toluidine blue staining. The formation and integrity of the osteoclast actin ring were impaired by ucOCN by immunofluorescent staining. Time dependant treatment of ucOCN during osteoclastic induction demonstrated the inhibitory effects mainly occurred at the early stage of osteoclastogenesis. Signaling analysis of luciferase activity of the CRE or SRE reporter and ERK1/2 phosphorylation showed the selective inhibitor or siRNA of Gprc6a (a presumptive ucOCN receptor) could attenuate the promotion of ucOCN on CRE-luciferase activity. Taken together, we provided the first evidence that ucOCN had negative effects on the early differentiation and bone resorption of osteoclasts via Gprc6a.
骨钙素（OCN）是最丰富的非胶原蛋白，被认为是一种内分泌因子。然而，未羧化骨钙素（ucOCN）对破骨细胞和骨吸收的作用尚未完全明确。在本研究中，用从原核细菌中纯化得到的ucOCN处理破骨前体细胞RAW264.7和骨髓单个核细胞（BMMs）。我们的结果表明，通过MTS法检测，ucOCN以浓度依赖性方式减弱RAW264.7细胞的增殖。划痕损伤试验显示，ucOCN处理后RAW264.7细胞的运动性降低。RT-qPCR结果表明，在RANKL诱导分化过程中，ucOCN对RAW264.7细胞中破骨细胞标志物基因的表达具有抑制作用。通过抗酒石酸酸性磷酸酶（TRAP）染色还观察到，ucOCN抑制RAW264.7细胞和BMMs形成多核细胞。在用甲苯胺蓝染色诱导破骨细胞的过程中，ucOCN处理后骨吸收凹坑的数量和面积也减少。通过免疫荧光染色发现，ucOCN破坏了破骨细胞肌动蛋白环的形成和完整性。在破骨细胞诱导过程中对ucOCN进行时间依赖性处理表明，抑制作用主要发生在破骨细胞生成的早期。对CRE或SRE报告基因的荧光素酶活性以及ERK1/2磷酸化的信号分析表明，Gprc6a（一种推测的ucOCN受体）的选择性抑制剂或小干扰RNA（siRNA）可减弱ucOCN对CRE荧光素酶活性的促进作用。综上所述，我们首次提供证据表明，ucOCN通过Gprc6a对破骨细胞的早期分化和骨吸收具有负面影响。
