Phospholipase D is required in the signaling pathway leading to p38 MAPK activation in neutrophil-like HL-60 cells, stimulated by N-formyl-methionyl-leucyl-phenylalanine

Human acute myelogenous leukemia cells (HL-60 Cells) can be induced to diff erentiate to neutrophils by exposure to dibutyryl-cyclic AMP. The different iation of HL-60 cells allowed the mitogen-activated protein kinases p38 and p44/p42 to be rapidly and transiently activated upon stimulation with N-fo rmyl-methionyl-leucyl-phenylalanine (fMLP). Western blot analysis using pho sphospecific p38 and p44/p42 mitogen-activated protein kinase antibodies sh owed that increasing concentrations of ethanol or 1-butanol but not 2-butan ol (0.05-0.5%) inhibited fMLP-induced p38 activation but did not inhibit p4 4/p42 activation. These data indicated that activation of phospholipase D ( PLD) was required for activation of p38 but not p44/p42. We compared the ef fect of fMLP with those of tumor necrosis factor a (TNF alpha) and granuloc yte-macrophage colony-stimulating factor (GAT-CSF). We found that ethanol d id not inhibit p38 phosphorylation upon stimulation with either GM-CSF or T NF alpha. These results suggested that in cells stimulated with RULP, PLD w as upstream of p38. To further test the involvement of PLD, we used antisen se inhibition of human PLD1 expression. Treatment with antisense oligonuele otides inhibited p38 but not p44/p42 phosphorylation. These data supported a role for human PLD1 in fMLP-induced p38-activation in neutrophil-like HL- 60 cells. In addition, the results obtained with TNF alpha and GM-CSF demon strated that p38 activation occurred independently of PLD activation.