BIOCHEMICAL-CHARACTERIZATION OF HUMAN OSTEOCLAST INTEGRINS - OSTEOCLASTS EXPRESS ALPHA-V-BETA-3, ALPHA-2-BETA-1, AND ALPHA-V-BETA-1 INTEGRINS

The study of osteoclast integrins has been previously hampered by the lack of a source of large numbers of purified osteoclasts. Osteoclasto ma, a human giant cell tumor of bone, supplied a rich source of osteoc lasts within a tissue containing many diverse cell types. Osteoclastom a integrin immunostaining confirmed the presence of the integrin alpha vbeta3 complex and the alpha2 and beta1 integrin subunits on osteoclas ts. However, weak integrin expression, for example with alphavbeta5, w as difficult to interpret. Purification with magnetic beads coated wit h vitronectin receptor monoclonal antibody (13C2) enabled osteoclast m embranes to be isolated with high purity and yield (57%) from osteocla stoma tissue. Positively (osteoclast-enriched) selected membranes were biochemically assessed for integrin expression by immunoprecipitation and visualization by non-radioactive enhanced chemiluminescence. Alph a1, alpha4, alpha6, alpha8, alphaM, alphaX, gpIIb, beta4, beta6, and b eta8 integrin chains were undetectable at a sensitivity of 1 ng. Alpha 3, alpha5, alphaL, beta2, and alphavbeta5 were found in the negatively selected osteoclastoma tissue but not in the positively purified oste oclast membranes. The presence of alphavbeta1 and alpha2beta1 dimers w as demonstrated biochemically on the immunoisolated osteoclast membran es. Osteoclast alphavbeta3 isolation by Arg-Gly-Asp (RGD) affinity chr omatography for NH2-terminal amino acid sequencing confirmed that the osteoclast vitronectin receptor was identical to that previously chara cterized on other cell types. In situ hybridization using human alphav riboprobes in osteoclasts from human and rodent bone further demonstr ated the high level and specificity of expression of alphav vitronecti n receptor in osteoclasts.