Abstract
Osteoclasts are the only somatic cells with bone-resorbing capacity and, as such, they have a critical role not only in normal bone homeostasis (called ‘bone remodelling’) but also in the pathogenesis of bone destructive disorders such as rheumatoid arthritis and osteoporosis1. A major focus of research in the field has been on gene regulation by osteoclastogenic cytokines such as receptor activator of NF-κB-ligand (RANKL, also known as TNFSF11) and TNF-α, both of which have been well documented to contribute to osteoclast terminal differentiation2,3. A crucial process that has been less well studied is the trafficking of osteoclast precursors to and from the bone surface, where they undergo cell fusion to form the fully differentiated multinucleated cells that mediate bone resorption. Here we report that sphingosine-1-phosphate (S1P), a lipid mediator enriched in blood4,5, induces chemotaxis and regulates the migration of osteoclast precursors not only in culture but also in vivo, contributing to the dynamic control of bone mineral homeostasis. Cells with the properties of osteoclast precursors express functional S1P1 receptors and exhibit positive chemotaxis along an S1P gradient in vitro. Intravital two-photon imaging of bone tissues showed that a potent S1P1 agonist, SEW2871, stimulated motility of osteoclast precursor-containing monocytoid populations in vivo. Osteoclast/monocyte (CD11b, also known as ITGAM) lineage-specific conditional S1P1 knockout mice showed osteoporotic changes due to increased osteoclast attachment to the bone surface. Furthermore, treatment with the S1P1 agonist FTY720 relieved ovariectomy-induced osteoporosis in mice by reducing the number of mature osteoclasts attached to the bone surface. Together, these data provide evidence that S1P controls the migratory behaviour of osteoclast precursors, dynamically regulating bone mineral homeostasis, and identifies a critical control point in osteoclastogenesis that may have potential as a therapeutic target.
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Acknowledgements
We thank U. H. von Andrian and I. B. Mazo for their help with the technique of intravital skull bone imaging. We also thank Y. Takuwa and N. Sugimoto for discussions, and P. M. Murphy and S.Venkatesan for their help in imaging in vitro chemotaxis using the EZ-Taxiscan. This work was supported in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, NIH, US Department of Health and Human Services, and by a fellowship grant to M.I. from the International Human Frontier Science Program.
Author Contributions M.I. performed most of experiments, with the assistance of J.G.E. and Y.S. for two-photon microscopy and for the in vitro osteoclast culture system, respectively. F.K. developed the unsupervised segmentation software and performed the computational analyses used to quantify the osteoclast-bone surface interface, with the assistance of M.M.-S. J.V. and R.L.P. generated CD11b-Cre transgenic and S1PR1loxP knock-in mice, respectively. R.N.G. helped M.I. in designing and interpreting experiments, as well as in writing the paper.
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Supplementary Information
This file contains Supplementary Figures 1-10 with Legends, Supplementary Tables 1-4, Supplementary Video Legends 1-7 and Supplementary References. (PDF 1213 kb)
Supplementary Video 1
This file shows in vitro chemotaxis of RAW264.7 cells in control conditions, detected using the EZ-Taxiscan device. Images were taken every minute for 2 hours. Scale bars represent 150 µm. (See legend in file s1). (MOV 1477 kb)
Supplementary Video 2
This file shows in vitro chemotaxis of RAW264.7 cells toward S1P gradient (10-8 M), detected using the EZ-Taxiscan device. Images were taken every minute for 2 hours. Scale bars represent 150 µm. (See legend in file s1). (MOV 1981 kb)
Supplementary Video 3
This file shows intravital two-photon imaging of mouse skull bone tissues of CX3CR1-EGFP knock-in (heterozygous) mice before application of SEW2871. Scale bars represent 50 µm. (See legend in file s1). (MOV 1073 kb)
Supplementary Video 4
This file shows intravital two-photon imaging of mouse skull bone tissues of CX3CR1-EGFP knock-in (heterozygous) mice 30 minutes after application of SEW2871. Scale bars represent 50 µm. (See legend in file s1). (MOV 1601 kb)
Supplementary Video 5
This file shows intravital two-photon imaging of mouse skull bone tissues of CSF1R-EGFP transgenic mice before application of SEW2871. Scale bars represent 50 µm. (See legend in file s1). (MOV 1299 kb)
Supplementary Video 6
This file shows intravital two-photon imaging of mouse skull bone tissues of CSF1R-EGFP transgenic mice 30 minutes after application of SEW2871. Scale bars represent 50 µm. (See legend in file s1). (MOV 1560 kb)
Supplementary Video 7
This file shows intravital two-photon imaging of mouse skull bone tissues of CX3CR1-EGFP knock-in (heterozygous) mice, pre-treated with FTY720 (3 mg/kg, i.p.) 4 hours before imaging. Scale bars represent 50 µm. (See legend in file s1). (MOV 988 kb)
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Ishii, M., Egen, J., Klauschen, F. et al. Sphingosine-1-phosphate mobilizes osteoclast precursors and regulates bone homeostasis. Nature 458, 524–528 (2009). https://doi.org/10.1038/nature07713
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DOI: https://doi.org/10.1038/nature07713
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