Undercarboxylated osteocalcin inhibits the early differentiation of osteoclast mediated by Gprc6a.

Hailong Wang,Jian Chen,Hongyu Zhang,Zhifeng Guo,Yinghui Li,Zihan Xu,Jinqiao Li,Feng Wu,Xiukun Sui,Zhongquan Dai,Chao Yang,Bai Ding

doi:10.7717/peerj.10898

Abstract

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.

Highlights

Osteocalcin (OCN) is one of the most abundant noncollagen proteins and initially considered as an osteoblast- secreted biomarker of bone turnover for the clinical diagnosis (Kruse & Kracht, 1986)
Previous studies have demonstrated that OCN could recruit and promote osteoclast differentiation, but the mechanism was still not well understood, especially undercarboxylated OCN (ucOCN)
We tried to evaluate the effects of ucOCN on the migration, proliferation, and differentiation of osteoclast recruitment process using RAW264.7 cells

Summary

Introduction

Osteocalcin (OCN) is one of the most abundant noncollagen proteins and initially considered as an osteoblast- secreted biomarker of bone turnover for the clinical diagnosis (Kruse & Kracht, 1986). The carboxylated osteocalcin (cOCN) has a stronger affinity with hydroxyapatite (HA) in the presence of calcium ions and deposits into the bone matrix (Hauschka & Carr, 1982; Li et al, 2016). OCN is thought to play a vital role in skeletal development. Previous studies have showed that the percentage of undercarboxylated OCN (ucOCN) was increased early and remained high during spaceflight, which means OCN maybe play an important role during bone alternation during microgravity exposure (Caillot-Augusseau et al, 2000). The exact function of OCN in bone and the whole body is still unknown (Diegel et al, 2020)

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Discussion

Conclusion