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

Rebeca Pérez,Roberto Melero,María A Balboa,Jesús Balsinde

doi:10.1074/jbc.m402562200

Abstract

Group VIA calcium-independent phospholipase A2 (iPLA2) 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 iPLA2 roles have rested heavily on the use of bromoenol lactone as an iPLA2-specific inhibitor, but this compound actually inhibits other enzymes and lipid pathways unrelated to PLA2, which makes it difficult to define the contribution of iPLA2 to specific functions. In previous work, we pioneered the use of antisense technology to decrease cellular iPLA2 activity as an alternative approach to study iPLA2 functions. In the present study, we followed the opposite strategy and prepared U937 cells that exhibited enhanced iPLA activity by stably expressing a plasmid containing iPLA2 cDNA. Compared with control cells, the iPLA2 -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 iPLA2 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 iPLA2-overexpressing cells. Increased phospholipid hydrolysis and fatty acid release also occurred in these cells. Unexpectedly, however, abrogation of U937 cell iPLA2 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 iPLA2-mediated phospholipid hydrolysis occurs during apoptosis, iPLA2 may actually be dispensable for the apoptotic process to occur. Thus, beyond a mere destructive role, iPLA2 may play other roles during apoptosis.

Highlights

Phospholipases A2 constitute a diverse group of enzymes whose common feature is to hydrolyze the fatty acid at the sn-2 position of phospholipids
According to their biochemical characteristics, the phospholipase A2 (PLA2) enzymes are generally grouped into three major subfamilies, viz. secreted PLA2 enzymes, cytosolic Ca2ϩ-dependent PLA2, and cytosolic Ca2ϩ-independent PLA2 [1,2,3,4,5,6]. sPLA2 enzymes are extracellular low molecular mass enzymes that require millimolar Ca2ϩ concentrations for activity. cPLA2, the ␣ isoform, is an intracellular enzyme that plays a pivotal role in receptor-coupled arachidonic acid (AA) release and prostaglandin production
We consistently failed to obtain expression of an independent phospholipase A2 (iPLA2) dominant-negative mutant in which the catalytic Ser465 had been replaced by Ala. These data appear to suggest that large increases or decreases in the intracellular iPLA2 activity content are injurious to U937 cells, highlighting the importance that this class of enzymes may have in the regulation of homeostatic phospholipid metabolism

Summary

Introduction

Phospholipases A2 constitute a diverse group of enzymes whose common feature is to hydrolyze the fatty acid at the sn-2 position of phospholipids. Role of iPLA2 in AA Incorporation into U937 Cell Phospholipids Exposed to H2O2—Rather than reflecting enzyme activation per se, iPLA2-mediated fatty acid release in response to H2O2 is thought to occur because of a facilitated interaction of the enzyme with its substrate, secondary to H2O2-induced membrane oxidation [20].

Results

Conclusion