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Olivier Dormond, Lionel Ponsonnet, Meriem Hasmim, Alessandro Foletti, Curzio Regg
Manganese-induced integrin affinity maturation promotes recruitment of V3 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 1 and 3 integrins. In unstimulated HUVEC, 1 integrins were present in fibrillar adhesions, while V3 was detected in peripheral focal adhesions. Clustered 1 and 3 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 3 high affinity/LIBS epitopes at focal adhesions. MnCl2-induced V3 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 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced V3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl2 did not alter 1 integrin distribution or 1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl2-induced V3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity V3 to focal adhesions. Affinity-modulated V3 clustering requires PI3-K signaling and is negatively regulate by Src.
Thrombosis and Haemostasis, Schattauer
Print ISSN: 0340-6245
Volume: 92, 07/2004
Pages: 151 - 161