Role of group VIA calcium-independent phospholipase A2 in arachidonic acid release, phospholipid fatty acid incorporation, and apoptosis in U937 cells responding to hydrogen peroxide

Group VIA calcium-independent phospholipase A(2) (iPLA(2)) has been shown to play a major role in regulating basal phospholipid deacylation reactions in certain cell types. More recently, roles for this enzyme have also been suggested in the destruction of membrane phospholipid during apoptosis and after oxidant injury. Proposed iPLA(2) roles have rested heavily on the use of bromoenol lactone as an iPLA(2)-specific inhibitor, but this compound actually inhibits other enzymes and lipid pathways unrelated to PLA(2), which makes it difficult to define the contribution of iPLA(2) to specific functions. In previous work, we pioneered the use of antisense technology to decrease cellular iPLA(2) activity as an alternative approach to study iPLA(2) functions. In the present study, we followed the opposite strategy and prepared U937 cells that exhibited enhanced iPLA(2) activity by stably expressing a plasmid containing iPLA(2) cDNA. Compared with control cells, the iPLA(2)-overexpressing U937 cells showed elevated responses to hydrogen peroxide with regard to both arachidonic acid mobilization and incorporation of the fatty acid into phospholipids, thus providing additional evidence for the key role that iPLA(2) plays in these events. Long-term exposure of the cells to hydrogen peroxide resulted in cell death by apoptosis, and this process was accelerated in the iPLA(2)-overexpressing cells. Increased phospholipid hydrolysis and fatty acid release also occurred in these cells. Unexpectedly, however, abrogation of U937 cell iPLA(2) activity by either methyl arachidonyl fluorophosphonate or an antisense oligonucleotide did not delay or decrease the extent of apoptosis induced by hydrogen peroxide. These results indicate that, although iPLA(2)-mediated phospholipid hydrolysis occurs during apoptosis, iPLA(2) may actually be dispensable for the apoptotic process to occur. Thus, beyond a mere destructive role, iPLA(2) may play other roles during apoptosis.