Osteoclasts are bone-resorbing, multinucleated giant cells that are essential for bone remodeling and are formed through cell fusion of mononuclear precursor cells. Although receptor activator of nuclear factor–κB ligand (RANKL) has been demonstrated to be an important osteoclastogenic cytokine, the cell surface molecules involved in osteoclastogenesis are mostly unknown. Here, we report that the seven-transmembrane receptor-like molecule, dendritic cell–specific transmembrane protein (DC-STAMP) is involved in osteoclastogenesis. Expression of DC-STAMP is rapidly induced in osteoclast precursor cells by RANKL and other osteoclastogenic stimulations. Targeted inhibition of DC-STAMP by small interfering RNAs and specific antibody markedly suppressed the formation of multinucleated osteoclast-like cells. Overexpression of DC-STAMP enhanced osteoclastogenesis in the presence of RANKL. Furthermore, DC-STAMP directly induced the expression of the osteoclast marker tartrate-resistant acid phosphatase. These data demonstrate for the first time that DC-STAMP has an essential role in osteoclastogenesis.

Histone deacetylase (HDAC) inhibitors are emerging as a new class of anticancer therapeutic agents and have been demonstrated to induce differentiation in some myeloid leukemia cell lines. In this study, we show that HDAC inhibitors have a novel action on osteoclast differentiation. The effect of 2 HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), on osteoclastogenesis was investigated using rat and mouse bone marrow cultures and a murine macrophage cell line RAW264. Both TSA and NaB inhibited the formation of preosteoclast-like cells (POCs) and multinucleated osteoclastlike cells (MNCs) in rat bone marrow culture. By reverse transcription-polymerase chain reaction analysis, TSA reduced osteoclastspecific mRNA expression of cathepsin K and calcitonin receptor (CTR). In contrast, TSA and NaB did not affect the formation of bone marrow macrophages (BMMs) induced by macrophage colony-stimulating factor as examined by nonspecific esterase staining. Fluorescence-activated cell sorting analysis showed that TSA did not affect the surface expression of macrophage markers for CD11b and F4/80 of BMMs. TSA and NaB also inhibited osteoclast formation and osteoclast-specific mRNA expression in RAW264 cells stimulated with receptor activator of nuclear factor-B (NF- B IntroductionHistone deacetylase (HDAC) inhibitors are known as agents that modulate the expression of genes by increasing histone acetylation, thereby regulating chromatin structure and transcription. 1,2 However, these inhibitors were not discovered based on their ability to inhibit HDAC activity. HDAC inhibitors include several structurally diverse natural products. Currently, there are several classes of HDAC inhibitors, including butyrate, hydroxamic acid, benzamide, and cyclic peptides. The simplest compound, butyrate, is a short-chain fatty acid derived from bacterial metabolism of dietary fiber in the colon. Butyrate was thought to be important for proper epithelial cell regulation, but was also found to have an antiproliferative and differentiation-inducing activity on various human colon carcinoma cells, normal cells, and neoplastic cells. [3][4][5] On the other hand, a hydroxamic acid, trichostatin A (TSA), is a more potent HDAC inhibitor that was identified as having potential therapeutic value against cancer in screens for agents that induce differentiation of murine erythroleukemia cells. 6,7 These HDAC inhibitors induce differentiation, inhibit cell proliferation, and induce apoptosis of tumor cells in cultures and animal models [3][4][5][6][7]8 and are emerging as a new class of potential therapeutic agents for the treatment of solid and hematologic malignancies.The effect of both sodium butyrate (NaB) and TSA on myeloid cell differentiation was well investigated using human promyelocytic leukemia cell lines, HL-60, U937, and a novel myeloid cell line, SN-1. 3,9,10 NaB treatment enhanced the promoter activity of a myeloid marker, the integrin CD11c/CD18 gene, in U937 cells and triggered differentiation of these 3 cell lines toward m...

Pagetic osteoclasts are greatly increased in number and size and have increased numbers of nuclei per cell compared to normal osteoclasts. The mechanisms responsible for enhanced osteoclast formation in Paget's disease are unknown. We have used our recently described model system for pagetic osteoclast formation to evaluate culture media conditioned by these atypical multinucleated cells (MNC) to determine if pagetic osteo-

Macrophage inflammatory protein-1 (MIP-1 ) is a member of the CC chemokines. We have previously reported the use of a whole bone marrow culture system to show that MIP-1 stimulates the formation of osteoclastlike multinucleated cells. Here we use rat bone marrow cells deprived of stromal cells, and clones obtained from murine macrophage-like cell line RAW264 to show that MIP-1 acts directly on cells in osteoclast lineage. We obtained several types of RAW264 cell clones, one of these clones, designated as RAW264 cell D clone (D clone), showed an extremely high response to receptor activator of NF B ligand (RANKL) and tumor necrosis factor-(TNF-), while the other clone, RAW264 cell N clone (N clone), demonstrated no response to RANKL or TNF-. Although both clones expressed receptor activator NF B (RANK) before being stimulated for differentiation, only the D clone expressed cathepsin K when cells were stimulated to differentiate to osteoclasts. MIP-1 stimulated the formation of mononuclear preosteoclastlike cells from rat bone marrow cells deprived of stromal cells. MIP-1 also stimulated formation of osteoclast-like multinucleated cells from the D clone, when these cells were stimulated with RANKL and TNF-. These findings provide strong evidence to show that MIP-1 acts directly on cells in the osteoclast lineage to stimulate osteoclastogenesis. Furthermore, pretreatment of RAW264 cell D clone with MIP-1 significantly induced adhesion properties of these cells to primary osteoblasts, suggesting a crucial role for MIP-1 in the regulation of the interaction between osteoclast precursors and osteoblasts in osteoclastogenesis.