v-ATPase V0 subunit d2–deficient mice exhibit impaired osteoclast fusion and increased bone formation

Abstract

Matrix-producing osteoblasts and bone-resorbing osteoclasts maintain bone homeostasis. Osteoclasts are multinucleated, giant cells of hematopoietic origin formed by the fusion of mononuclear pre-osteoclasts derived from myeloid cells1,2. Fusion-mediated giant cell formation is critical for osteoclast maturation; without it, bone resorption is inefficient2,3. To understand how osteoclasts differ from other myeloid lineage cells, we previously compared global mRNA expression patterns in these cells and identified genes of unknown function predominantly expressed in osteoclasts, one of which is the d2 isoform of vacuolar (H+) ATPase (v-ATPase) V0 domain (Atp6v0d2)4,5,6,7. Here we show that inactivation of Atp6v0d2 in mice results in markedly increased bone mass due to defective osteoclasts and enhanced bone formation. Atp6v0d2 deficiency did not affect differentiation or the v-ATPase activity of osteoclasts. Rather, Atp6v0d2 was required for efficient pre-osteoclast fusion. Increased bone formation was probably due to osteoblast-extrinsic factors, as Atp6v02 was not expressed in osteoblasts and their differentiation ex vivo was not altered in the absence of Atp6v02. Our results identify Atp6v0d2 as a regulator of osteoclast fusion and bone formation, and provide genetic data showing that it is possible to simultaneously inhibit osteoclast maturation and stimulate bone formation by therapeutically targeting the function of a single gene.

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Figure 1: Deletion of Atp6v0d2 leads to defective osteoclasts and increased bone formation.
Figure 2: Reduced mature osteoclast formation from Atp6v0d2−/− bone marrow cells.
Figure 3: Impaired fusion of Atp6v0d2−/− pre-osteoclasts.
Figure 4: Rescue of cell fusion in Atp6v0d2−/− osteoclasts by ADAM8 or ADAM12.

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Acknowledgements

We thank the Abramson Family Cancer Research Institute Transgenic Core for ES cell injection. We also thank members of the Choi lab for discussion and reading of the manuscript, T. Kitamura (University of Tokyo) for pMX vectors and PLAT-E cells, D. Fremont (Washington University) for recombinant M-CSF, M. Takami (Showa University) for dentine slices, and D. Adams (University of Connecticut Health Center Image Core) for μCT analysis. This work was supported in part by grants from the US National Institutes of Health (to Y.C., S.K.L., R.J.P. and J.A.L.).

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Correspondence to Yongwon Choi.

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Supplementary information

Supplementary Fig. 1

Supplementary Figure 1 (a) Mouse and human Atp6v0d2 amino acid sequences, identified in this study, are shown. (PDF 254 kb)

Supplementary Fig. 2

(a-b) Deficiency of Atp6v0d2 does not affect function of the kidney. (PDF 306 kb)

Supplementary Fig. 3

(a-b) Deficiency of Atp6v0d2 does not affect v-ATPase activity of osteoclasts. (PDF 235 kb)

Supplementary Fig. 4

Real-time PCR of RNA from wild-type (WT) and Atp6v0d2−/− osteoclasts. (PDF 121 kb)

Supplementary Fig. 5

Real-time PCR of ADAM family genes from wild-type (WT) and Atp6v0d2−/−osteoclasts. T (PDF 109 kb)

Supplementary Fig. 6

(a) DC-STAMP-deficient cells express Atp6v0d2 mRNA and protein at levels similar to wild-type cells. (PDF 183 kb)

Supplementary Fig. 7

(a) RANK expression or its signaling is not affected in the absence of Atp6v0d2. (PDF 198 kb)

Supplementary Table 1

(a) Structural Parameters measured by Microcomputed Tomography (b) Static histomorphometric parameters of bone structure in femur of wild-type (WT) and Atp6v0d2−/− (KO) mice. (PDF 55 kb)

Supplementary Methods (PDF 273 kb)

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Lee, S., Rho, J., Jeong, D. et al. v-ATPase V0 subunit d2–deficient mice exhibit impaired osteoclast fusion and increased bone formation. Nat Med 12, 1403–1409 (2006). https://doi.org/10.1038/nm1514

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