α(v)β3 integrin induces tyrosine phosphorylation-dependent Ca2+ influx in pulmonary endothelial cells

Abstract

The endothelial α(v)β3 integrin occurs luminally, where its ligation by soluble agents may induce inflammatory signaling. We tested this hypothesis in bovine pulmonary artery endothelial cell monolayers with the use of vitronectin and cross-linking antibodies to ligate and aggregate the integrin. We quantified the endothelial cytosclic Ca2+ concentration ([Ca2+](i)) according to the Fura 2 ratio imaging method in single cells of confluent monolayers. At baseline, endothelial [Ca2+](i) levels remained steady at 86 nmol/L for >20 minutes. Cross-linking of the α(v)β3 integrin through the sequential exposure of monolayers to anti-α(v)β3 monoclonal antibody LM609 and secondary IgG resulted in a [Ca2+](i) increase of 100% above baseline. This increase commenced in <0.5 minute, peaked in <2 minutes, and decayed to baseline in ≃5 minutes. Similar responses occurred after the addition of vitronectin (400 μg/mL). In contrast, external Ca2+ depletion blunted the cross-linking-induced [Ca2+](i) increase by 60%, a response that was completely inhibited when the monolayers were also pretreated with thapsigargin. Thus, the [Ca2+](i) increase was attributable in part to the release of Ca2+ from endosomal stores but mostly to Ca2+ influx across the plasma membrane. Induced aggregation of the α(v)β3 integrin enhanced tyrosine phosphorylation of phospholipase C-γ1 and increased the accumulation of inositol-14,4,5-trisphosphate. Genistein, a broad-spectrum tyrosine kinase inhibitor, abrogated both of these effects, as well as the α(v)β3-induced [Ca2+](i) increases. We conclude that aggregation of the endothelial α(v)/β3 integrin induces a rapid tyrosine phosphorylation- dependent increase in [Ca2+](i). This response may subserve the inflammatory role of α(v)β3 integrin in blood vessels.