ECHISTATIN INHIBITS THE MIGRATION OF MURINE PREFUSION OSTEOCLASTS ANDTHE FORMATION OF MULTINUCLEATED OSTEOCLAST-LIKE CELLS

The vitronectin receptor alpha(v)beta(3) is highly expressed in osteoc lasts and was shown to play a critical role in osteoclast function in vivo. The objective of this study was to examine the role of alpha(v)b eta(3) integrin in osteoclast formation in vitro using the inhibitory disintegrin echistatin, an RGD-containing snake venom. We documented b y immunocytochemistry and Northern blot analysis that during murine os teoclast-like cell (OCL) formation in a coculture of mouse osteoblasti c MB1.8 cells and bone marrow cells there is increased expression of t he alpha(v) and beta(3) integrin subunits. Echistatin binds preferenti ally to the membrane fraction of isolated enriched OCLs (IC50 = 0.6 nM ), and this binding is inhibited by vitronectin receptor-blocking poly clonal antibodies. Additionally, cross-linking of radiolabeled echista tin to OCLs, followed by immunoprecipitation with antibodies to vitron ectin or fibronectin receptors, shows that alpha(v)beta(3) integrin is the predominant receptor for echistatin in this system. In this cocul ture, echistatin completely inhibits the formation of multinucleated O CLs, but not that of mononuclear prefusion OCLs (pOCs). This inhibitio n is RGD and dose dependent (IC50 = 0.7 nM). We tested the hypothesis that inhibition of OCL formation may be due to interference with pOC m igration and found that echistatin inhibited macrophage colony-stimula ting factor-induced migration and fusion of pOCs (IC50 = 1 and 0.6 nM, respectively). Echistatin inhibition of pOCs migration and fusion is also RGD dependent. These results suggest that the integrin alpha(v)be ta(3) plays a role in pOC migration, which can explain the inhibitory effect of echistatin on multinucleated osteoclast; formation in vitro.