Osteoclast integrin α_Vβ₃ is present in the clear zone and contributes to cellular polarization

Abstract.

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 α_Vβ₃ integrin in primary rat osteoclasts and mouse osteoclast-like multinucleated cells (OCLs) formed in vitro. At the ultrastructural level, the specific immunoreactivity of both α_V and β₃ 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 α_V and β₃ 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.