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pp. family consensus sequence. DC-STAMP is usually a transmembrane protein required for osteoclast precursor fusion. Knockdown of OC-STAMP mRNA by siRNA and protein inhibition by antibodies significantly suppressed the formation of tartrate-resistant acid phosphatase (TRAP) +, multinucleated cells in differentiating osteoclast cultures, with many TRAP+ mononuclear cells present. Conversely, overexpression of OC-STAMP increased osteoclastic differentiation of RAW 264.7 cells. We conclude that OC-STAMP is usually a previously unknown, RANKL-induced, multi-pass transmembrane protein that promotes the formation of multinucleated osteoclasts. INTRODUCTION Skeletal homeostasis requires coordinated action by bone-forming osteoblasts and bone-resorbing osteoclasts (Marks and Odgren, 2002). Deficient bone resorption prospects to sclerotic bone, as seen in osteopetrosis, whereas excessive resorption is usually central to the pathogenesis of osteoporosis, tumor metastasis to bone, arthritis, periodontal disease, and prosthetic joint implant loosening. Understanding the mechanisms that control the differentiation and activity of osteoclasts is usually therefore of central importance to many widespread clinical conditions. The differentiation of active, multinucleated osteoclasts from mononuclear hematopoietic precursors is usually a Rabbit Polyclonal to FOXD3 complex process requiring endocrine signals, local signals from growth factors in the bone environment, and the successful expression of the many gene products needed for the precursors to fuse to form multinucleated cells; for the osteoclast to attach to the bone surface; to secrete protons, ions, and proteases; and to ingest the solubilized bone matrix and transport it through the cell for export (Balemans et al., 2005; Boyle et al., 2003). Multinucleated osteoclasts are created by fusion of mononuclear, hematopoietic cells of the monocyte/macrophage lineage (Marks and Walker, 1981; Walker, 1975). Osteoclast precursors respond to signals from colony-stimulating factor-1 (CSF-1; M-CSF) and express RANK, the receptor for the TNF superfamily member RANKL (TRANCE, OPGL, ODF)(Boyle et al., 2003). CSF-1 and RANKL are supplied in the bone environment by osteoblasts. Normally, both CSF-1 and RANKL are required for osteoclast differentiation, although it is possible to circumvent this pathway by means of TNF- and TGF- (Kim et al., 2005). To understand better how osteoclasts differentiate and carry out resorptive activity, we have used high-density microarrays to investigate global gene expression changes that accompany osteoclast differentiation (Yang et al., 2006a; Yang et al., 2006b). In the course of these experiments, we recognized a previously uncharacterized gene that is strongly up-regulated during Cefonicid sodium osteoclast differentiation. The gene product is usually unrelated to other known proteins with the Cefonicid sodium notable exception of a long stretch of its carboxy-terminal region that bears significant similarity to the DC-STAMP protein family consensus. DC-STAMP is usually a multi-pass transmembrane protein that was originally recognized in gene expression screens of dendritic Cefonicid sodium cells (Hartgers et al., 2000). Further work showed that DC-STAMP expression responded strongly to RANKL and played an important role in osteoclast differentiation. Blocking or knocking down DC-STAMP inhibited osteoclast differentiation and overexpressing it Cefonicid sodium increased osteoclast differentiation Cefonicid sodium in response to RANKL (Kukita et al., 2004). In addition, DC-STAMP knockout mice have a unique osteoclast phenotype in that they have large numbers of mononuclear, TRAP-positive osteoclasts that are able to resorb bone, albeit inefficiently, leading to moderate osteopetrosis (Yagi et al., 2005). DC-STAMP-negative cells are unable to initiate cell-cell fusion, but they are able to fuse with DC-STAMP-positive cells, providing the first mechanistic insights into the process of fusion (Vignery, 2005; Yagi et al., 2005). The ligand for DC-STAMP remains unknown. Proteins made up of the DC-STAMP family consensus sequence are not limited to vertebrates with mineralized skeletons. The Conserved Domain name Database (Marchler-Bauer et al., 2005) lists several dozen DC-STAMP consensus-containing proteins, including mouse, human, and rat DC-STAMP as well as the protein we describe here and its vertebrate orthologues. In addition, there are several other.