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 alpha(v)beta3 and its ligand osteopontin (OPN) has been directly correlated to tumorigenicity of melanoma and other cancer cells. We have previously shown an increase in pp(60c-Src) kinase activity associated with integrin alpha(v)beta3 in melanoma cells (M21) treated with soluble OPN. pp(60c-Src) kinase activity was not observed in melanoma cells expressing alpha(v) that lacks the cytoplasmic domain (alpha(v)995). Results of the current study demonstrate that the amino acid sequence '995RPPQEEQERE1004' in the beta-turn of alpha(v) chain is required for the interaction of pp(60c-Src). Our results suggest that the beta-turn of alpha(v) chain may be indispensable for alpha(v)-associated signaling complex formation and outside-in signaling. To further analyze the alpha(v)beta3 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 alpha(v)995 cells. Also, M21/OPN cells exhibit increased motility, which is markedly reduced upon treatment with inhibitors to alpha(v) 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 alpha(v) or in alpha(v)995 cells expressing mutant alpha(v).