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 cells. Fusion-mediated giant cell formation is critical for osteoclast maturation; without it, bone resorption is inefficient. 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) V(0) domain (Atp6v0d2). 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.

Osteoclast stimulatory transmembrane protein and dendritic cell–specific transmembrane protein cooperatively modulate cell–cell fusion to form osteoclasts and foreign body giant cells

“…2D), indicating that OC-STAMP is required for osteoclast fusion independent of DC-STAMP. Other molecules reportedly involved in osteoclast cell-cell fusion, such as E-Cadherin and ATP6v0d2, (14,21) were equally expressed in OC-STAMP-deficient compared to WT osteoclasts (Fig. 2E), supporting the idea that OC-STAMP is required for osteoclast fusion rather than as a modulator of expression of fusion-related molecules.…”

“…(28) Likewise, Car2 regulates proton synthesis, and a subtype of proton pump reportedly functions in cell-cell fusion of osteoclasts and FBGCs. (21) OC-STAMP reportedly regulates osteoclast differentiation, (26) but the function of all three factors in cellcell fusion is not well known.…”

“…(3,21,25) Antibody-mediated block of OC-STAMP reportedly inhibits osteoclast formation and bone resorption. (26,30) Although, OC-STAMP-deficient mononuclear osteoclasts resorb bone, bone-resorbing area per well, as analyzed by pit formation assay, was significantly inhibited in OC-STAMPdeficient osteoclasts compared with WT osteoclasts (Fig.…”

“…(13) Therefore, although the functions of osteoclasts and FBGCs differ, they express common molecules, such as DC-STAMP, that function in cell-cell fusion. (3) To date, various molecules have been identified that regulate fusion of osteoclasts or macrophages, including DC-STAMP, ATP6v0d2, CD47, CD44, CD9, CD81, MFR, E-cadherin, and meltrin-a (3,(14)(15)(16)(17)(18)(19)(20)(21) ATP6v0d2-deficient mice show significant reductions in fusion of either cell type. (21) DC-STAMP-deficient osteoclasts or FBGCs show a complete lack of cell-cell fusion, a function specific for macrophage lineage fusion, since fertilization and myotube formation is normal in DC-STAMP-deficient mice.…”

“…(3) To date, various molecules have been identified that regulate fusion of osteoclasts or macrophages, including DC-STAMP, ATP6v0d2, CD47, CD44, CD9, CD81, MFR, E-cadherin, and meltrin-a (3,(14)(15)(16)(17)(18)(19)(20)(21) ATP6v0d2-deficient mice show significant reductions in fusion of either cell type. (21) DC-STAMP-deficient osteoclasts or FBGCs show a complete lack of cell-cell fusion, a function specific for macrophage lineage fusion, since fertilization and myotube formation is normal in DC-STAMP-deficient mice. (3) DC-STAMP was originally identified as a seventransmembrane protein expressed in dendritic cells (22) and then subsequently in interleukin 4 (IL-4)-induced macrophages and osteoclasts.…”