Investigation on the role of osteopontin and CD44 in the osteoclast development

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Osteopontin (OPN) is a highly phosphorylated glycoprotein that is expressed in bone by cells of both the osteoblast and osteoclast lineages. Through an RGD motif, OPN can mediate cell attachment with a preference for the av β3 integrin. Thus, OPN has been shown to have an important role in osteoclastic resorption and to provide a substratum for the attachment of bone lining cells. OPN is also a ligand for CD44, through which it promotes directed cell migration. Since OPN and CD44 are highly expressed in migrating cells, we examined the relationship between these proteins in osteoclasts derived from normal mice and mice with targeted disruption of either the OPN or CD44 genes. Analysis of multinucleated osteoclasts isolated from rat femurs revealed that OPN was primarily localized around the nucleus but was also present in lamellar structures and cell processes which also stained for CD44 and β3 integrin. In non-permeabilized cells, strong surface staining for CD44 and β3, whereas little staining for OPN was observed, reflecting the intracellular location of the OPN and the extracellular location of epitopes for the CD44 and β3. In cells treated with cycloheximide, perinuclear staining for OPN was lost, but iOPN staining was retained within cell processes. Osteoclasts generated from the OPN- or CD44-null mice showed reduced cell spreading, limited protrusion of pseudopodia and impaired cell fusion, resorptive activity compared to wild-type controls. Exogenous OPN could partially restore resorption depth in OPN-null osteoclasts, but not cell mobility or cell fusion indicating that iOPN, co-localizing with CD44 within cell processes and filopodia, is important for the formation and normal function of osteoclasts. These studies indicate that an iOPN co-localizes with CD44 within osteoclast cell processes and filopodia, and that this form of OPN may be important in regulating CD44-mediated cytoskeletal changes associated with osteoclast migration, fusion and resorption.