R740S, a dominant V-ATPase a3 mutation, causes osteopetrosis in mice

Bone resorption involves osteoclast-mediated acidification via a vacuolar-type H+-ATPase (V-ATPase) localized to the ruffled border at the bone surface. V-ATPases are proton pumps composed of 13 subunits including the 'a' subunit. Of the 4 'a' isoforms in mammals, a3 is enriched in osteoclasts where it is essential for bone resorption. Humans with homozygous a3 mutations are severely osteopetrotic and do not live past 6 years without treatment (bone marrow transplant), while their heterozygous parents have no detectable phenotype. Homozygous a3^-/- mice have severe osteopetrosis and die by 6 weeks; a3^+/- mice have no reported abnormalities. We identified an a3 missense mutation (R740S) during a mouse genome-wide ethylnitrosourea (ENU) mutagenesis screen for dominant point mutations affecting bone mineral density (BMD). The mouse a3 R740 residue is perfectly conserved in mammalian and yeast 'a' isoforms, and in yeast is essential for proton translocation. Objective: To characterize the molecular and cellular mechanisms underlying osteopetrosis in Atp6i^R740S/+mice. Methods: DEXA, faxitron and micro-CT to quantify the BMD, visualize the skeleton and determine static histomorphometric parameters; culture of spleen cells with RANKL-MCSF and replating of osteoclasts generated onto dentine slices to quantify osteoclastogenesis and resorption respectively. Results: Atp6i^R740S/+ intercrosses revealed that mice homozygous for the R740S mutation died at birth or soon after, in contrast to both a3^-/- and a3^+/- mice. Atp6i^R740S/+mice have normal appearance, size and weight but exhibit higher BMD, greater trabecular bone mass and greater bone volume as percent of tissue volume compared with wildtype (WT) littermates, suggesting deficient bone resorption. Fewer and smaller (fewer nuclei/cell) osteoclasts formed in spleen cell cultures from Atp6i^R740S/+ versus WT mice, and Atp6i^R740S/+ osteoclasts had decreased resorption activity on dentine slices. Conclusion: R740S is the first dominant mutation described within a3 that results in osteopetrosis as a consequence of decreased osteoclastogenesis and osteoclast activity.