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Osteoprotegerin exposure at different stages of osteoclastogenesis differentially affects osteoclast formation and function

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Abstract

This study aimed to investigate the effects of osteoprotegerin (OPG), a decoy receptor for receptor activator for nuclear factor κB ligand (RANKL), during the various stages of osteoclast differentiation, and additionally investigate its effects on osteoclast adhesion and activity. RAW264.7 murine monocytic cells were incubated with macrophage colony-stimulating factor and RANKL for 1, 3, 5, or 7 days, followed by an additional 24-h incubation in the presence or absence of OPG (80 ng/mL). We examined osteoclast differentiation and adhesion capacity using the tartrate-resistant acid phosphatase (TRAP) assay and immunofluorescence microscopy, and additionally examined cell growth in real time using the xCELLigence system. Furthermore, the expression levels of TRAP, RANK, integrin β3, matrix metalloproteinase 9, cathepsin K, carbonic anhydrase II, and vesicular-type H+-ATPase A1 were examined using western blotting. OPG exposure on day 1 enhanced the osteoclast growth curve as well as adhesion, and increased RANK and integrin β3 expression. In contrast, exposure to OPG at later time points (days 3–7) inhibited osteoclast differentiation, adhesion structure formation, and protease expression. In conclusion, the biological effects of OPG exposure at the various stages of osteoclast differentiation were varied, and included the enhanced adhesion and survival of preosteoclasts, the block of differentiation from the early to the terminal stages of osteoclastogenesis, and suppression of mature osteoclast activation following OPG exposure during the terminal differentiation stage, suggesting that the effects of OPG exposure differ based on the stage of differentiation.

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (Grant Numbers 31172373, 31302154, 31372495), the Specialized Research Fund for the Doctoral Program of Higher Education (Grant Number 20113250110003), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Graduate Innovation Project of Jiangsu Province (Grant Number CXZZ12_0917).

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    Authors and Affiliations

    1. College of Veterinary Medicine, Yangzhou University, 88 South University Ave., Yangzhou, Jiangsu, 225009, People’s Republic of China

      Hongyan Zhao, Jianhong Gu, Nannan Dai, Qian Gao, Dong Wang, Ruilong Song, Wei Liu, Yan Yuan, Jianchun Bian, Xuezhong Liu & Zongping Liu

    2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, People’s Republic of China

      Hongyan Zhao, Jianhong Gu, Nannan Dai, Qian Gao, Dong Wang, Ruilong Song, Wei Liu, Yan Yuan, Jianchun Bian, Xuezhong Liu & Zongping Liu

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    1. Hongyan Zhao
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    Correspondence to Zongping Liu.

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    Hongyan Zhao and Jianhong Gu have contributed equally to this work.

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    Zhao, H., Gu, J., Dai, N. et al. Osteoprotegerin exposure at different stages of osteoclastogenesis differentially affects osteoclast formation and function. Cytotechnology 68, 1325–1335 (2016). https://doi.org/10.1007/s10616-015-9892-7

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