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Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption

Abstract

Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequently become activated to be competent for bone resorption through programs primarily governed by receptor activator of nuclear factor-κB ligand in cooperation with macrophage colony–stimulating factor1,2,3. Proteins prominently expressed at late phases of osteoclastogenesis and with a supportive role in osteoclast function are potential therapeutic targets for bone-remodeling disorders. In this study, we used a proteomics approach to show that abundance of the brain-type cytoplasmic creatine kinase (Ckb) is greatly increased during osteoclastogenesis. Decreasing Ckb abundance by RNA interference or blocking its enzymatic activity with a pharmacological inhibitor, cyclocreatine, suppressed the bone-resorbing activity of osteoclasts grown in vitro via combined effects on actin ring formation, RhoA GTPase activity and vacuolar ATPase function. Activities of osteoclasts derived from Ckb−/− mice were similarly affected. In vivo studies showed that Ckb−/− mice were better protected against bone loss induced by ovariectomy, lipopolysaccharide challenge or interleukin-1 treatment than wild-type controls. Furthermore, administration of cyclocreatine or adenoviruses harboring Ckb small hairpin RNA attenuated bone loss in rat and mouse models. Our findings establish an important role for Ckb in the bone-resorbing function of osteoclasts and underscore its potential as a new molecular target for antiresorptive drug development.

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Figure 1: Ckb expression increases during osteoclastogenesis in response to RANKL.
Figure 2: Suppression of osteoclast function by Ckb-sh–mediated knockdown and pharmacological inhibition.
Figure 3: Effects of Ckb deficiency on in vitro and in vivo bone resorption.
Figure 4: Effects of Ckb inhibition on bone destruction in rats and mice.

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Acknowledgements

We thank S.I. Kim, H.B. Kwak, J.Y. Yang, T.K. Yu and J.S. Ko for technical help and discussion. We also thank T. Kitamura (University of Tokyo) for Plat-E cells. This work was supported by the 21C Frontier Functional Proteomics Project grants FPR08B1-170 (to H.-H.K.) and FPR08A1-070 (to Y.K.P.), the Research Program for New Drug Target Discovery grant M10748000257-07N4800-25710 (to H.-H.K.) from the Ministry of Education, Science & Technology, Korea and NKB-KWF grant 2002-2763 (to B.W.).

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E.-J.C. designed and performed most of the experiments and wrote the manuscript. J.H. performed the experiments by assisting with tissue collection; DNA, RNA and protein isolation; and analyses. F.O. performed the experiments by assisting with breeding and analyses of Ckb-knockout mice. Y.J.L. carried out histological analysis. J.R. and H.J.K. performed the V-ATPase assay. Y.L., H.-M.K. and J.-Y.C. participated in histomorphometrical analysis. S.W.L and J.Y.K. performed proteomic experiments. Y.K.P. generated adenoviruses. C.S.S., Z.H.L. and S.T. participated in generation and analyses of various in vivo bone resorption models. B.W. was responsible for establishing Ckb-knockout mice and participated in data interpretation and discussion. Z.H.L. and H.-H.K. conceived the study. H.-H.K. supervised the study and wrote the manuscript.

Corresponding authors

Correspondence to Bé Wieringa or Hong-Hee Kim.

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Supplementary Figs. 1–8 and Supplementary Methods (PDF 4931 kb)

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Chang, EJ., Ha, J., Oerlemans, F. et al. Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption. Nat Med 14, 966–972 (2008). https://doi.org/10.1038/nm.1860

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