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 the early events leading to T lymphocyte activation. We have examined the possibility that stimulation of phospholipase D (PLD) may regulate activation of transcription factor AP-1 in human T cells by transfecting human T lymphocyte Jurkat cells with a plasmid containing an AP-1 enhancer element and a chloramphenicol acetyltransferase reporter gene. We have detected activatable PLD in Jurkat cells, and we have found that 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 AP-1. Wortmannin, an inhibitor of receptor-coupled PLD activation, blocked the anti-CD3-induced increases in both PLD activity and AP-1 enhancer activity. We found a good correlation in the transfected cells between PLD activation and induction of AP-1 enhancer activity under different experimental conditions. Furthermore, ethanol, an inhibitor of the PLD pathway, blocked the anti-CD3-stimulated AP-1 enhancer activity. However, this anti-CD3-mediated response was not inhibited by neomycin, an inhibitor of phosphoinositide hydrolysis. The increases in AP-1 enhancer activity induced by PA or anti-CD3 mAb were efficiently abrogated by the presence of propranolol, an inhibitor of PA phosphohydrolase and protein kinase C (PKC). Furthermore, the PA- and the anti-CD3-induced increases in AP-1 enhancer activity were blocked by the presence of PKC inhibitors or by PKC down-regulation. These data indicate that PLD stimulation can activate the transcription factor AP-1 in T lymphocytes, and suggest that the induction of AP-1 enhancer factor activity by PA is mediated via PKC stimulation, either through a direct activating effect of PA or through PA-derived diacylglycerol formation. These data also provide evidence for a role of PLD-derived lipids in the induction of AP-1 enhancer activity resulting from stimulation of the TCR/CD3 complex, suggesting that increased PLD activity can play an important role in T lymphocyte activation.
Mollinedo
F
F
Center for Biological Research, Superior Council of Scientific Research, Madrid, Spain.
Gajate
C
C
Flores
I
I
D016718:Arachidonic Acid / Q000096:biosynthesis; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D018274:Electroporation; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D008213:Lymphocyte Activation / Q000502:physiology; D010712:Phosphatidic Acids / Q000032:analysis / Q000502:physiology*; D010739:Phospholipase D / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000502:physiology*; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000502:physiology; D015398:Signal Transduction / Q000276:immunology; D013601:T-Lymphocytes / Q000502:physiology*
D004075:Diglycerides; D007295:Inositol Phosphates; D010712:Phosphatidic Acids; D016755:Proto-Oncogene Proteins c-jun; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D016718:Arachidonic Acid; D015500:Chloramphenicol O-Acetyltransferase; D011493:Protein Kinase C; D010739:Phospholipase D