Role of intracellular osteopontin in the osteoclast fusion and tumor cell invasion

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Osteoclasts (OC) are the multinucleated cells which is responsible for the bone resorption in both physiological and pathological bone metabolism. Precursors of OC need to migrate and fuse together to form the multinucleated mature cells, which is an important step in OC differentiation. That osteopontin (OPN) has a prominent role in bone resorption was first indicated by the demonstration that it colocalizes with the avβ3 integrin and mediates the attachment of OC to the bone. However, it is still unclear at which stages of OC differentiation and activation that OPN functions. To clarify this, we examined the OC generated from bone marrow mononuclear precursors by laser scanning confocal microscopy. In prefusion OC and in motile multinucleated OC, staining for OPN colocalized with CD44 and β3 integrin, which are the OPN receptors, in cell processes. OPN was also found to colocalize with Cdc42 and Racl in filopodia and ruffled membranes, respectively. Immunocytochemical analysis of OC derived from OPN-null and CD44-null showed diminished perimembranous staining for CD44 and OPN, respectively, indicating a mutual interdependency of these proteins for their localization at cell surfaces. Moreover, OC generated from these mutant mice showed reduced cell spreading, limited protrusion of pseudopodia and impaired cell fusion compared to wild-type controls, consistent with the putative role of the OPN and CD44 in mediating cytoskeletal rearrangements. These studies indicate that OPN colocalizes with CD44 within osteoclast cell processes, and that OPN may be important in regulating CD44-mediated cytoskeletal changes associated with OC migration, fusion and resorption. Furthermore, it was confirmed that OPN played a critical role in wound healing process in squamous carcimona cells, since the front-line cells which protrude pseudopods and migrate toward the space between cell layers expressed OPN and CD44 strongly.