Isolation and characterization of a cDNA clone encoding a novel peptide (OSF) that enhances osteoclast formation and bone resorption

Using an expression cloning approach, we identified and cloned a novel intr acellular protein produced by osteoclasts that indirectly induces osteoclas t formation and bone resorption, termed OSF. Conditioned media from 293 cel ls transiently transfected with the 0.9 kb OSF cDNA clone stimulated osteoc last-like cell formation in both human and murine marrow cultures in the pr esence or absence 10(-9) M 1,25-dihydroxyvitamin D-3. In addition, conditio ned media from 293 cells transfected with the OSF cDNA clone enhanced the s timulatory effects of 1,25(OH)(2)D-3 on bone resorption in the fetal rat lo ng bone assay. In situ hybridization studies using antisense oligomers show ed expression of OSF mRNA in highly purified osteoclast-like cells from hum an giant cell tumors of the bone. Northern blot analysis demonstrated ubiqu itous expression of a 1.3 kb mRNA that encodes OSF in multiple human tissue s. Sequence analysis showed the OSF cDNA encoded a 28 kD peptide that conta ins a c-Src homology 3 domain (SH3) and ankyrin repeats, suggesting that it was not a secreted protein, but that it was potentially involved in cell s ignaling. Consistent with these data, immunoblot analysis using rabbit anti sera against recombinant OSF demonstrated OSF expression in cell lysates bu t not in the culture media. Furthermore, recombinant OSF had a high affinit y for c-Src, an important regulator of osteoclast activity. Taken together, these data suggest that OSF is a novel intracellular protein that indirect ly enhances osteoclast formation and osteoclastic bone resorption through t he cellular signal transduction cascade, possibly through its interactions with c-Src or other Src-related proteins. J Cell Physiol 177.636-645, 1998. (C) 1998 Wiley-Liss, Inc.