Osteoclast integrin alphaVbeta3 is present in the clear zone and contributes to cellular polarization

Osteoclasts are primary bone-resorbing cells with highly polarized cytoplasmic structures, such as ruffled borders and clear zones. In the present study, we have examined the subcellular localization and function of the alphaVbeta3 integrin in primary rat osteoclasts and mouse osteoclast-like multinucleated cells (OCLs) formed in vitro. At the ultrastructural level, the specific immunoreactivity of both alphaV and beta3 subunits is localized not only along the plasma membranes of osteoclast ruffled borders and basolateral membranes but also in clear zones. Addition of GRGDS (Gly-Arg-Gly-Asp-Ser) peptide to the culture medium during a pit formation assay reduces resorbed areas on dentine slices in a dose-dependent manner. Treatment with the GRGDS peptide also dose-dependently inhibits the formation of a ringed structure of F-actin (an "actin ring") in OCLs on dentine slices. Electron-microscopic analysis has revealed that OCLs treated with GRGDS peptide at 1 mM can adhere to dentine slices with unusually broad or poorly defined clear zones but do not form the ruffled burder structure. These results suggest that integrin alphaV and beta3 subunits localized in the ruffled border/clear zone complex of osteoclasts are essential for the functional osteoclast-to-bone matrix interaction and the structural polarization of osteoclasts.