Dual signaling by the αvβ3-integrin activates cytosolic PLA2in bovine pulmonary artery endothelial cells

Abstract

Vitronectin, which ligates the αvβ3-integrin, increases both lung capillary permeability and lung endothelial Ca2+. In stable monolayers of bovine pulmonary artery endothelial cells (BPAECs) viewed with confocal microscopy, multimeric vitronectin aggregated the apically located αvβ3-integrin. This caused arachidonate release that was inhibited by pretreating the monolayers with the anti-αvβ3monoclonal antibody (MAb) LM609. No inhibition occurred in the presence of the isotypic MAb PIF6, which recognizes the integrin αvβ5. Vitronectin also caused membrane translocation and phosphorylation of cytosolic phospholipase A2(cPLA2) as well as tyrosine phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 2. The cPLA2inhibitor arachidonyl trifluoromethylketone, the tyrosine kinase inhibitor genistein, and the MAPK kinase inhibitor PD-98059 all blocked the induced arachidonate release. PD-98059 did not inhibit the increase of cytosolic Ca2+or cPLA2translocation, although it blocked tyrosine phosphorylation of ERK2. Moreover, although the intracellular Ca2+chelator MAPTAM also inhibited arachidonate release, it did not inhibit tyrosine phosphorylation of ERK2. These findings indicate that ligation of apical αvβ3in BPAECs caused ERK2 activation and an increase of intracellular Ca2+, both conjointly required for cPLA2activation and arachidonate release. This is the first instance of a tyrosine phosphorylation-initiated “two-hit” signaling pathway that regulates an integrin-induced proinflammatory response.