INVOLVEMENT OF PHOSPHOLIPASE-D IN THE ACTIVATION OF TRANSCRIPTION FACTOR AP-1 IN HUMAN T-LYMPHOID JURKAT CELLS

The induction of the AP-1 transcription factor has been ascribed to th e early events leading to T lymphocyte activation. We have examined th e possibility that stimulation of phospholipase D (PLD) may regulate a ctivation of transcription factor AP-1 in human T cells by transfectin g human T lymphocyte Jurkat cells with a plasmid containing an AP-1 en hancer element and a chloramphenicol acetyltransferase reporter gene. We have detected activatable PLD in Jurkat cells, and we have found th at addition of phosphatidic acid (PA), the physiologic product of PLD action on phospholipids, is rapidly incorporated into Jurkat cells and leads to activation of transcription factor AP-1. Treatment of Jurkat cells with anti-CD3 mAb activated both PLD and transcription factor A P-1. Wortmannin, an inhibitor of receptor-coupled PLD activation, bloc ked the anti-CD3-induced increases in both PLD activity and AP-1 enhan cer activity. We found a good correlation in the transfected cells bet ween PLD activation and induction of AP-1 enhancer activity under diff erent experimental conditions. Furthermore, ethanol, an inhibitor oi t he PLD path?way, blocked the anti-CD3-stimulated AP-1 enhancer activit y. However, this anti-CD3-mediated response was not inhibited by neomy cin, an inhibitor of phosphoinositide hydrolysis. The increases in AP- 1 enhancer activity induced by PA or anti-CD3 mAb were efficiently abr ogated by the presence of propranolol, an inhibitor oi PA phosphohydro lase and protein kinase C (PKC). Furthermore, the PA- and the anti-CD3 -induced increases in AP-1 enhancer activity were blocked by the prese nce of PKC inhibitors or by PKC down-regulation. These data indicate t hat PLD stimulation can activate the transcription factor AP-1 in T ly mphocytes, and suggest that the induction of AP-1 enhancer factor acti vity by PA is mediated via PKC stimulation, either through a direct ac tivating effect of PA or through PA-derived diacylglycerol formation. These data also provide evidence for a role of PLD-derived lipids in t he induction of AP-1 enhancer activity resulting from stimulation of t he TCR/CD3 complex, suggesting that increased PLD activity can play an important role in T lymphocyte activation.