Calcium-independent phospholipase A 2 mediates CREB phosphorylation in double-stranded RNA-stimulated endothelial cells

One of the products of a calcium-independent phospholipase A 2 (iPLA 2 ) attack of plasmenylcholine, lysoplasmenylcholine, has previously been shown to activate cAMP-dependent protein kinase (PKA). Because endothelial cells respond to some agonists in part by the activation of iPLA 2 , the present study was designed to determine whether double-stranded RNA (dsRNA), the primary activator of the antiviral response in endothelial cells, elicits cAMP response element binding protein (CREB) phosphorylation through a mechanism mediated by iPLA 2 . dsRNA stimulated CREB phosphorylation in bovine pulmonary artery endothelial cells that was inhibited by the iPLA 2 inhibitor, bromoenol lactone, and the PKA inhibitor, H-89. Additionally, the product of iPLA 2 hydrolysis of plasmenylcholine and lysoplasmenylcholine elicited CREB phosphorylation in bovine pulmonary endothelial cells. Taken together, the present studies suggest that dsRNA as well as other agonists of endothelial cells elicit signaling mechanisms that include in part CREB phosphorylation mediated by iPLA 2 . —Martinson, B. D., C. J. Albert, J. A. Corbett, R. B. Wysolmerski, and D. A. Ford. Calcium-independent phospholipase A 2 mediates CREB phosphorylation in doublestranded RNA-stimulated endothelial cells. J. Lipid Res. 2003. 44: 1686–1691. Supplementary key words endothelium • cAMP response element binding protein • cAMP-dependent Lysophospholipid production in most mammalian cells is regulated by the coordinated activities of the phospholipase A 2 family and lysolipid acyl CoA transferases. Calcium-independent phospholipase A 2 (iPLA 2 ) has previously been shown to be the predominant phospholipase A 2 in mammalian myocardium and is activated during brief episodes of ischemia (1–3). Because purified cAMPdependent protein kinase (PKA) is activated by both lysophosphatidylcholine and lysoplasmenylcholine through a cAMP-independent mechanism, a putative role of iPLA 2 in the ischemic heart was predicted as a mediator resulting in PKA activation (4). Indeed, recent studies have demonstrated that iPLA 2 likely mediates cAMP response element binding protein (CREB) phosphorylation and subsequent cfos expression in response to brief ischemia through the activation of PKA by lysoplasmenylcholine or lysophosphatidylcholine (5). Double-stranded RNA (dsRNA) accumulates at various stages of viral replication and plays a role in the activation of the antiviral response in virally infected cells (6). We have recently shown that dsRNA elicits iPLA 2 -mediated CREB phosphorylation in RAW 264.7 cells (7). Because one of the primary host target cells of viral infection are the vascular endothelial cells, the role of iPLA 2 as a mediator of dsRNA stimulation of endothelial cell signaling was determined. The present studies demonstrate that dsRNA elicits CREB phosphorylation in endothelial cells that is sensitive to inhibition by the iPLA 2 inhibitor, bromoenol lactone (BEL). Additionally, the mechanism of this signaling appears to be mediated by lysoplasmenylcholine or lysophosphatidylcholine because they independently elicit CREB phosphorylation. Furthermore, dsRNA-stimulated CREB phosphorylation likely is mediated through iPLA 2 -derived lyso choline glycerophospholipids stimulating PKA because this signaling pathway was susceptible to inhibition by the PKA inhibi-