Microarray and real-time RT-PCR were used to examine expression changes in primary bone marrow cells and RAW 264.7 cells in response to RANKL. In silico sequence analysis was performed on a novel gene which we designate OC-STAMP. Specific siRNA and antibodies were used to inhibit OC-STAMP RNA and protein, respectively, and TRAP+ multinucleated osteoclasts were counted. Antibodies were used to probe bone tissues and western blots of RAW cell extracts +/− RANKL. cDNA overexpression constructs were transfected into RAW cells and the effect on RANKL-induced differentiation was studied. OC-STAMP was very strongly up-regulated during osteoclast differentiation. Northern blots and sequence analysis revealed 2 transcripts of 2 kb and 3.7 kb differing only in 3'UTR length, consistent with predictions from genome sequence. The mRNA encodes a 498 amino acid, multi-pass transmembrane protein that is highly conserved in mammals. It has little overall homology to other proteins. The carboxy-terminal 193 amino acids, however, are significantly similar to the DC-STAMP family consensus sequence. DC-STAMP is 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 a previously unknown, RANKL-induced, multi-pass transmembrane protein that promotes the formation of multinucleated osteoclasts.

Osteoclasts differentiate from hematopoietic mononuclear precursor cells under the control of both colony stimulating factor-1 (CSF-1, or M-CSF) and receptor activator of NF-B ligand (RANKL, or TRANCE, TNFSF11) to carry out bone resorption. Using high density gene microarrays, we followed gene expression changes in long bone RNA when CSF-1 injections were used to restore osteoclast populations in the CSF-1-null toothless (csf1 tl /csf1 tl ) osteopetrotic rat. We found that ovarian cancer G-protein-coupled receptor 1 (OGR1, or GPR68) was strongly up-regulated, rising >6-fold in vivo after 2 days of CSF-1 treatments. OGR1 is a dual membrane receptor for both protons (extracellular pH) and lysolipids. Strong induction of OGR1 mRNA was also observed by microarray, real-time RT-PCR, and immunoblotting when mouse bone marrow mononuclear cells and RAW 264.7 pre-osteoclast-like cells were treated with RANKL to induce osteoclast differentiation. Anti-OGR1 immunofluorescence showed intense labeling of RANKL-treated RAW cells. The time course of OGR1 mRNA expression suggests that OGR1 induction is early but not immediate, peaking 2 days after inducing osteoclast differentiation both in vivo and in vitro. Specific inhibition of OGR1 by anti-OGR1 antibody and by small inhibitory RNA inhibited RANKL-induced differentiation of both mouse bone marrow mononuclear cells and RAW cells in vitro, as evidenced by a decrease in tartrate-resistant acid phosphatase-positive osteoclasts. Taken together, these data indicate that OGR1 is expressed early during osteoclastogenesis both in vivo and in vitro and plays a role in osteoclast differentiation.The catabolic removal of bone during skeletal formation and remodeling requires the specialized activity of multinucleated osteoclasts. Osteoclasts differentiate by fusion of hematopoietic mononuclear precursors in response to systemic and local signals, in particular colony-stimulating factor-1 (CSF-1, 2 or M-CSF) and the tumor necrosis factor family member receptor activator of NF-B ligand (RANKL, or TRANCE, TNFSF11) (1). Excessive osteoclast activity systemically leads to osteopenias such as osteoporosis, whereas local hyperactivity can lead to osteolysis as seen in tumor metastases to bone or in prosthesis loosening. Hypoactivity of osteoclasts can lead to sclerosing bone disorders, for example in genetic conditions such as osteopetrosis.Osteoclast differentiation is a complex process that requires the coordinated action of many gene products, including not only extrinsic factors such as CSF-1 and RANKL, which are supplied by osteoblasts locally in bone tissue, but also intrinsic factors required for osteoclast function. Mononuclear precursors must migrate to sites where resorption is needed, fuse to form multinucleated pre-osteoclasts, and attach firmly to bone. They develop highly specialized cellular structures, including an actin ring that forms a tight seal with the bone surface, and a highly convoluted plasma membrane domain called the ruffled border, which is the site of extremely a...

Osteoclasts differentiate from hematopoietic precursors under systemic and local controls. Chemokines and receptors direct leukocyte traffic throughout the body and may help regulate site-specific bone resorption. We investigated bone gene expression in vivo during rapid osteoclast differentiation induced by colonystimulating factor 1 (CSF-1) in Csf1-null toothless (tl/tl) rats. Long-bone RNA from CSF-1-treated tl/tl rats was analyzed by high-density microarray over a time course. TRAP (tartrate-resistant acid phosphatase)-positive osteoclasts appeared on day 2, peaked on day 4, and decreased slightly on day 6, as marrow space was expanding. TRAP and cathepsin K mRNA paralleled the cell counts. We examined all chemokine and receptor mRNAs on the arrays. CCL9 was strongly induced and peaked on day 2, as did its receptor, CCR1, and regulatory receptors c-Fms (CSF-1 receptor) and RANK (receptor activator of nuclear factor B).

Bone resorption is regulated by a complex system of hormones and cytokines that cause osteoblasts/stromal cells and lymphocytes to produce factors including RANKL, that ultimately result in the differentiation and activation of osteoclasts, the bone resorbing cells.We used a microarray approach to identify genes upregulated in RANKL-stimulated osteoclastprecursor cells. Osteoclast expression was confirmed by multiple tissue Northern and in situ hybridization analysis. Gene function studies were carried out by siRNA analysis.We identified a novel gene, which we termed nha-oc/NHA2, which is strongly upregulated during RANKL-induced osteoclast differentiation in vitro and in vivo. nha-oc/NHA2 encodes a novel cation/proton antiporter (CPA), and is the mouse orthologue of a human gene identified in a database search: HsNHA2. nha-oc/NHA2 is selectively expressed in osteoclasts. NHA-oc/NHA2 protein localizes to the mitochondria, where it mediates Na + -dependent changes in mitochondrial pH and Na + Acetate induced-mitochondrial passive swelling. RNA silencing of nha-oc/nha2 reduces osteoclast differentiation and resorption, suggesting a role for NHA-oc/NHA2 in these processes.nha-oc/NHA2 therefore is a novel member of the CPA family, and is the first mitochondrial NHA characterized to date. nha-oc/NHA2 is also unique in that it is the first eukaryotic and tissue specific CPA2 characterized to date. NHA-oc/NHA2 displays the expected activities of a bona fide CPA and plays a key role(s) in normal osteoclast differentiation and function.

Bone morphogenetic proteins (BMPs) play pivotal roles in bone and cartilage growth and repair. Through phenotypes of short ear mice, which have BMP-5 mutations, a role for BMP-5 in some specific aspects of skeletogenesis and cartilage growth is known. This report examines BMP-5 expression in the growth plate and in differentiating cultures of primary chondrocytes, and the effects of addition of BMP-5 or its inhibition by anti-BMP-5 antibody in chondrocyte cultures. By laser capture microdissection and immunohistochemistry, we found that BMP-5 is expressed in proliferating zone chondrocytes and that the expression increases sharply with hypertrophic differentiation. A similar pattern was observed in differentiating cultures of primary chondrocytes, with BMP-5 expression increasing as cells differentiated, in contrast to other BMPs. BMP-5 added to cultures increased cell proliferation early in the culture period and also stimulated cartilage matrix synthesis. Also, BMP-5 addition to the cultures activated phosphorylation of Smad 1/5/8 and p38 MAP kinase and caused increased nuclear accumulation of phospho-Smads. Anti-BMP-5 antibody inhibited the endogenous BMP-5, reducing cell proliferation and phospho-Smad nuclear accumulation. Together, the results demonstrate that BMP-5 is normally an important regulator of chondrocyte proliferation and differentiation. Whether other BMPs may compensate in BMP-5 lossof-function mutations is discussed.