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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

    Walsh, M.C. et al. Osteoimmunology: interplay between the immune system and bone metabolism. Annu. Rev. Immunol. 24, 33–63 (2006).

    CAS  Article  Google Scholar 

  2. 2

    Teitelbaum, S.L. Bone resorption by osteoclasts. Science 289, 1504–1508 (2000).

    CAS  Article  Google Scholar 

  3. 3

    Yagi, M. et al. DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells. J. Exp. Med. 202, 345–351 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Nishi, T., Kawasaki-Nishi, S. & Forgac, M. Expression and function of the mouse V-ATPase d subunit isoforms. J. Biol. Chem. 278, 46396–46402 (2003).

    CAS  Article  Google Scholar 

  5. 5

    Sun-Wada, G.H., Yoshimizu, T., Imai-Senga, Y., Wada, Y. & Futai, M. Diversity of mouse proton-translocating ATPase: presence of multiple isoforms of the C, d and G subunits. Gene 302, 147–153 (2003).

    CAS  Article  Google Scholar 

  6. 6

    Smith, A.N., Borthwick, K.J. & Karet, F.E. Molecular cloning and characterization of novel tissue-specific isoforms of the human vacuolar H(+)-ATPase C, G and d subunits, and their evaluation in autosomal recessive distal renal tubular acidosis. Gene 297, 169–177 (2002).

    CAS  Article  Google Scholar 

  7. 7

    Rho, J. et al. Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis. DNA Cell Biol. 21, 541–549 (2002).

    CAS  Article  Google Scholar 

  8. 8

    Nishi, T. & Forgac, M. The vacuolar (H+)-ATPases—nature's most versatile proton pumps. Nat. Rev. Mol. Cell Biol. 3, 94–103 (2002).

    CAS  Article  Google Scholar 

  9. 9

    Stevens, T.H. & Forgac, M. Structure, function and regulation of the vacuolar (H+)-ATPase. Annu. Rev. Cell Dev. Biol. 13, 779–808 (1997).

    CAS  Article  Google Scholar 

  10. 10

    Suda, T., Jimi, E., Nakamura, I. & Takahashi, N. Role of 1α,25-dihydroxyvitamin D3 in osteoclast differentiation and function. Methods Enzymol. 282, 223–235 (1997).

    CAS  Article  Google Scholar 

  11. 11

    Kadono, Y. et al. Strength of TRAF6 signalling determines osteoclastogenesis. EMBO Rep. 6, 171–176 (2005).

    CAS  Article  Google Scholar 

  12. 12

    Li, Y.P., Chen, W., Liang, Y., Li, E. & Stashenko, P. Atp6i-deficient mice exhibit severe osteopetrosis due to loss of osteoclast-mediated extracellular acidification. Nat. Genet. 23, 447–451 (1999).

    CAS  Article  Google Scholar 

  13. 13

    Takami, M., Woo, J.T. & Nagai, K. Osteoblastic cells induce fusion and activation of osteoclasts through a mechanism independent of macrophage-colony-stimulating factor production. Cell Tissue Res. 298, 327–334 (1999).

    CAS  Article  Google Scholar 

  14. 14

    Chen, E.H. & Olson, E.N. Unveiling the mechanisms of cell-cell fusion. Science 308, 369–373 (2005).

    CAS  Article  Google Scholar 

  15. 15

    Verrier, S., Hogan, A., McKie, N. & Horton, M. ADAM gene expression and regulation during human osteoclast formation. Bone 35, 34–46 (2004).

    CAS  Article  Google Scholar 

  16. 16

    Abe, E., Mocharla, H., Yamate, T., Taguchi, Y. & Manolagas, S.C. Meltrin-alpha, a fusion protein involved in multinucleated giant cell and osteoclast formation. Calcif. Tissue Int. 64, 508–515 (1999).

    CAS  Article  Google Scholar 

  17. 17

    Choi, S.J., Han, J.H. & Roodman, G.D. ADAM8: a novel osteoclast stimulating factor. J. Bone Miner. Res. 16, 814–822 (2001).

    CAS  Article  Google Scholar 

  18. 18

    Martin, T.J. & Sims, N.A. Osteoclast-derived activity in the coupling of bone formation to resoption. Trends Mol. Med. 11, 76–81 (2005).

    CAS  Article  Google Scholar 

  19. 19

    Rho, J., Gong, S., Kim, N. & Choi, Y. TDAG51 is not essential for Fas/CD95 regulation and apoptosis in vivo. Mol. Cell. Biol. 21, 8365–8370 (2001).

    CAS  Article  Google Scholar 

  20. 20

    Lee, S.K. et al. Interleukin-7 influences osteoclast function in vivo but is not a critical factor in ovariectomy-induced bone loss. J. Bone Miner. Res. 21, 695–702 (2006).

    Article  Google Scholar 

  21. 21

    Montero, A. et al. Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation. J. Clin. Invest. 105, 1085–1093 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Jacquin, C., Gran, D.E., Lee, S.K., Lorenzo, J.A. & Aguila, H.L. Identification of multiple osteoclast precursor populations in murine bone marrow. J. Bone Miner. Res. 21, 67–77 (2006).

    Article  Google Scholar 

Download references


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.).

Author information



Corresponding author

Correspondence to Yongwon Choi.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading