Alpha-V-dependent outside-in signaling is required for the regulation of CD44 surface expression, MMP-2 secretion, and cell migration by osteopontin in human melanoma cells

The level of integrin αvβ3 and its ligand osteopontin (OPN) has been directly correlated to tumorigenicity of melanoma and other cancer cells. We have previously shown an increase in pp60c-Src kinase activity associated with integrin αvβ3 in melanoma cells (M21) treated with soluble OPN. pp60c-Src kinase activity was not observed in melanoma cells expressing αv that lacks the cytoplasmic domain (αv995). Results of the current study demonstrate that the amino acid sequence ‘995RPPQEEQERE1004’ in the β-turn of αv chain is required for the interaction of pp60c-Src. Our results suggest that the β-turn of αv chain may be indispensable for αv-associated signaling complex formation and outside-in signaling. To further analyze the αvβ3 signaling in melanoma cells, we over expressed OPN in M21 cells (M21/OPN). CD44 surface expression and MMP-2 activity in the conditioned medium were increased to a greater extent in M21/OPN cells as compared with M21 or αv995 cells. Also, M21/OPN cells exhibit increased motility, which is markedly reduced upon treatment with inhibitors to αv and MMP-2. Our findings suggest that the increase in MMP-2 activity is integrin-dependent as MMP-2 activity is reduced in cells treated with an inhibitor to αv or in αv995 cells expressing mutant αv.