Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 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 MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-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 beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological 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 alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.
Dormond
Olivier
O
Centre Pluridisciplinaire d'Oncologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
Ponsonnet
Lionel
L
Hasmim
Meriem
M
Foletti
Alessandro
A
Rüegg
Curzio
C
D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D019012:Integrin beta1 / Q000378:metabolism; D039661:Integrin beta3 / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D039302:Integrin alphaVbeta3; D019012:Integrin beta1; D039661:Integrin beta3; D009842:Oligopeptides; D008345:Manganese; C047981:arginyl-glycyl-aspartic acid; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases