BLOCKING LIGAND OCCUPANCY OF THE ALPHA-V-BETA-3 INTEGRIN INHIBITS INSULIN-LIKE-GROWTH-FACTOR-I SIGNALING IN VASCULAR SMOOTH-MUSCLE CELLS

Blocking alpha V beta 3 integrin occupancy results in attenuation of t he cellular migration response to insulin-like growth factor I (IGF-I) , To determine whether integrin antagonists alter other IGF-I-stimulat ed biologic actions, quiescent smooth muscle cells (SMCs) were exposed to echistatin and their ability to respond to IGF-I was determined. E chistatin (10(-7) M) inhibited IGF-I-stimulated DNA synthesis by 80%, and the protein synthesis response also was inhibited. Therefore block ing occupancy of alpha V beta 3 inhibited multiple target cell actions of IGF-I, To determine whether blocking alpha V beta 3 occupancy coul d alter IGF-I receptor-mediated signal transduction, the ability of IG F-I to stimulate phosphorylation of insulin receptor substrate-1 (IRS- 1) was analyzed. A 10-min exposure to 100 ng/ml of IGF-I resulted in a substantial increase in phosphorylated IRS-1, and echistatin (10(-7) M) blocked the IGF-I-induced IRS-1 phosphorylation response. Echistati n also attenuated downstream signaling because the capacity of the p85 subunit of phosphatidylinositol-3 kinase (PI-3 kinase) to bind to IRS -1 was blocked. In contrast, exposure of SMCs to vitronectin (1.0 mu g /cm(2)) or thrombospondin (0.25 mu g/cm(2)), two known ligands for alp ha V beta 3, resulted in enhancement of the IGF-I-stimulated IRS-1 res ponse. To determine whether these effects were caused by alterations i n receptor kinase activity, the IGF-I receptor was immunoprecipitated and then analyzed for phosphotyrosine. Echistatin (10-7 M) significant ly reduced IGF-I-stimulated tyrosine phosphorylation of the IGF-I rece ptor beta subunit, We conclude that occupancy of the alpha V beta 3 in tegrin is necessary for IGF-I to fully activate the kinase activity of the IGF-I receptor and phosphorylate IRS-1, Activation of the alpha V beta 3 receptor results in an interaction with the IGF-I signal trans duction pathway, which modulates SMCs responsiveness to IGF-I.