Dual signaling by the alpha(v)beta(3)-integrin activates cytosolic PLA(2) in bovine pulmonary artery endothelial cells

Vitronectin, which ligates the alpha (v)beta (3)-integrin, increases both l ung 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 alpha (v )beta (3)-integrin. This caused arachidonate release that was inhibited by pretreating the monolayers with the anti-alpha (v)beta (3) monoclonal antib ody (MAb) LM609. No inhibition occurred in the presence of the isotypic MAb PIF6, which recognizes the integrin alpha (v)beta (5). Vitronectin also ca used membrane translocation and phosphorylation of cytosolic phospholipase A(2) (cPLA(2)) as well as tyrosine phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 2. The c PLA(2) inhibitor arachidonyl trifluoromethylketone, the tyrosine kinase inh ibitor genistein, and the MAPK kinase inhibitor PD-98059 all blocked the in duced arachidonate release. PD-98059 did not inhibit the increase of cytoso lic Ca2+ or cPLA(2) translocation, although it blocked tyrosine phosphoryla tion of ERK2. Moreover, although the intracellular Ca2+ chelator MAPTAM als o inhibited arachidonate release, it did not inhibit tyrosine phosphorylati on of ERK2. These findings indicate that ligation of apical alpha (v)beta ( 3) in BPAECs caused ERK2 activation and an increase of intracellular Ca2+, both conjointly required for cPLA2 activation and arachidonate release. Thi s is the first instance of a tyrosine phosphorylation-initiated "two-hit" s ignaling pathway that regulates an integrin-induced proinflammatory respons e.