Cytosolic phospholipase A(2)-mediated regulation of phospholipase D2 in leukocyte cell lines

Abstract

Phospholipase D (PLD) has been implicated in a variety of cellular processes, including inflammation, secretion, and respiratory burst, Two distinct PLD isoforms, designated PLD1 and PLD2, have been cloned; however, the regulatory mechanism for each PLD isoform is not clear, In our present study we investigated how PLD2 activity is regulated in mouse lymphocytic leukemia L1210 cells, which mainly contain PLD2 and in PLD2-transfected COS-7 cells, Intriguingly, A23187, a calcium ionophore that induces calcium influx, potently stimulates PLD activity in these two cell lints, suggesting that Ca2+ might be implicated in the regulation of the PLD2 activity. In addition to the A23187-induced PLD2 activation, A23187 also increases PLA(2)-mediated arachidonic acid release, and the A23187-stimulated PLD2 and PLA(2) activities could be blocked by pretreatment of the cells,vith cytosolic calcium-dependent PLA(2) (cPLA(2)) inhibitors, such as arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphonate in these two cell lines. Moreover, the A23187-induccd PLD2 and PLA(2) activities could be inhibited by cotransfection with antisense cPLA(2) oligonucleotide. These results suggest a role for cPLA(2) in the regulation of PLD2 activity in vivo. The inhibitory effect of arachidonyl trifluoromethyl ketone on the A23187-induced PLD2 activity could be recovered by addition of exogenous lysophosphatidylcholine. This study is the first to demonstrate that PLD2 activity is up-regulated by Ca2+ influx and that cPLA(2), may play a key role in the Ca2+-dependent regulation of PLD2 through generation of lysophosphatidylcholine.