Ameloblastin Regulates Osteogenic Differentiation by Inhibiting Src Kinase via Cross Talk between Integrin β1 and CD63

Ameloblastin, the most abundant non-amelogenin enamel matrix protein, plays a role in ameloblast differentiation. Here we found that ameloblastin was expressed in osteosarcoma cells; to explore the potential functions of ameloblastin in osteoblasts, we investigated whether this protein is involved in osteogenic differentiation and bone formation on the premise that CD63, a member of the transmembrane-4 glycoprotein superfamily, interacts with integrins in the presence of ameloblastin. Ameloblastin bound to CD63 and promoted CD63 binding to integrin β1. The interaction between CD63 and integrin β1 induced Src kinase inactivation via the binding of CD63 to Src. The reduction of Src activity and osteogenic differentiation mediated by ameloblastin was abrogated by treatment with anti-CD63 antibody and overexpression of constitutive active Src, respectively. Moreover, amelobastin upregulated the formation of stress-fibre and focal adhesions and downregulated cell migration in association with RhoA regulation via Src activity. Therefore, our results suggest that ameloblastin is expressed in osteoblasts and functions as a promoting factor for osteogenic differentiation via a novel pathway through the interaction between CD63 and integrin β1.