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.

Murine BM was fractionated using a series of hematopoietic markers to characterize its osteoclast progenitor populations. We found that the early osteoclastogenic activity in total BM was recapitulated by a population of cells contained within the CD11b −/low CD45R − CD3 − CD115 high fraction.Introduction: Osteoclasts are of hematopoietic origin and they have been shown to share the same lineage as macrophages. We further characterized the phenotype of osteoclast progenitor populations in murine bone marrow (BM) by analyzing their cell surface markers. Materials and Methods:We used fluorescence-activated cell sorting (FACS) to identify the subsets of BM cells that contained osteoclast progenitors. We fractionated BM according to several markers and cultured the sorted populations for a period of 2-6 days with macrophage-colony stimulating factor (M-CSF) and RANKL. The numbers of multinucleated osteoclast-like cells (OCLs) that formed in the cultures were counted. Results: We found that the CD45R

Notch receptors are determinants of cell fate and function and play a central role in skeletal development and bone remodeling. Hajdu Cheney syndrome, a disease characterized by osteoporosis and fractures, is associated with NOTCH2 mutations resulting in a truncated stable protein and gain-of-function. We created a mouse model reproducing the Hajdu Cheney syndrome by introducing a 6955C3 T mutation in the Notch2 locus leading to a Q2319X change at the amino acid level. Notch2Q2319X heterozygous mutants were smaller and had shorter femurs than controls; and at 1 month of age they exhibited cancellous and cortical bone osteopenia. As the mice matured, cancellous bone volume was restored partially in male but not female mice, whereas cortical osteopenia persisted in both sexes. Cancellous bone histomorphometry revealed an increased number of osteoclasts and bone resorption, without a decrease in osteoblast number or bone formation. Osteoblast differentiation and function were not affected in Notch2 Q2319X cells. The pre-osteoclast cell pool, osteoclast differentiation, and bone resorption in response to receptor activator of nuclear factor B ligand in vitro were increased in Notch2 Q2319X mutants. These effects were suppressed by the ␥-secretase inhibitor LY450139. In conclusion, Notch2 Q2319X mice exhibit cancellous and cortical bone osteopenia, enhanced osteoclastogenesis, and increased bone resorption.Notch proteins are four single-pass transmembrane receptors that play a critical role in cell fate decisions ( Fig. 1) (1-4). Notch regulates cell renewal and plays a role in skeletal development and homeostasis and in osteoblast and osteoclast differentiation (4 -8). Jagged1 and -2 and DeltaLike1, -3, and -4 are the five classic Notch ligands (4). Notch-ligand interactions result in the proteolytic cleavage and release of the Notch intracellular domain (NICD), 2 which translocates to the nucleus to form a complex with recombination signal-binding protein for immunoglobulin J region (Rbpj) and Mastermind-like to regulate transcription (9 -12). This canonical signaling pathway leads to the transcription of Hairy Enhancer of Split (Hes)1, -5, and -7 and Hes related with YRPW motif (Hey)1, -2, and -L. Skeletal cells express Notch1, Notch2, and low levels of Notch3 transcripts (13-15). Activation of Notch in undifferentiated and differentiated osteoblasts inhibits cell differentiation and function and causes osteopenia (16,17). In contrast, activation of Notch1 in osteocytes causes a pronounced increase in bone mass due to a suppression of bone resorption (18). Results from the conditional inactivation of Notch1 and Notch2 in the developing skeleton confirmed the inhibitory role of Notch in osteoblastogenesis (6,19). Whereas substantial work has characterized the consequences of Notch1 gain-of-function in the skeleton, there is limited knowledge on the function of Notch2 in the postnatal skeleton. This knowledge is particularly important because Notch1 and Notch2 do not have redundant functions, and Notch1 inhibits, wh...

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In bone, early events in inflammation involve the production and release of primary proinflammatory cytokines, such as interleukin-1 beta. By activation of target cells, these cytokines are thought to induce a second wave of cytokines, including monocyte chemoattractant protein-1 (MCP-1). MCP-1 is a cytokine that stimulates chemotaxis of monocytes. Experiments were undertaken to examine the expression of MCP-1 in bone-associated cells in vivo. To observe in vivo expression of MCP-1, an inflammatory lesion was created in the murine mandible. Immunohistochemistry experiments using specific antibodies to MCP-1 were conducted to identify MCP-1-expressing cells in inflamed and noninflamed bone. We found that osteoblasts were the principal cells expressing MCP-1 in inflamed bone. There was little or no MCP-1 expression in noninflamed bone. Immunohistochemistry experiments were carried out to assess monocyte recruitment during osseous inflammation. The number of MCP-1-positive cells was significantly correlated to the number of monocytes/macrophages present (n = 15; r = 0.69; P < = 0.01). These in vivo results strongly suggest that MCP-1 is an important mediator involved in the recruitment of monocytes/macrophages in inflamed bone.