Manganese-induced integrin affinity maturation promotes recruitment of αVβ3 integrin to focal adhesions in endothelial cells:: evidence for a role of phosphatidylinositol 3-kinase and Src

Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl2 to increase integrin affinity and monitored clustering of beta1 and beta3 integrins. In unstimulated HUVEC, beta1 integrins were present in fibrillar adhesions, while alphaVbeta3 was detected in peripheral focal adhesions. Clustered beta1 and beta3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl2-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta3 high affinity/LIBS epitopes at focal adhesions. MnCl2-induced alphaVbeta3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two parmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 34, mutant protein. Conversely, over-expression of active PI 34, and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alphaVbeta3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl2 did not alter P I integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl2-induced alphaVbeta3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alphaVbeta3 to focal adhesions. Affinity-modulated alphaVbeta3 clustering requires P13-K signaling and is negatively regulate by Src.