LIPOPOLYSACCHARIDE-MEDIATED SIGNAL-TRANSDUCTION THROUGH PHOSPHOLIPASE-D ACTIVATION IN MONOCYTIC CELL-LINES

Lipopolysaccharide (LPS)-induced phospholipase D (PLD) activation was investigated in undifferentiated monocytic leukemic cell lines THP-1 a nd U-937. Treatment of THP-1 or U-937 cells labelled with [P-32]orthop hosphate, [P-32]acyl GPC or [H-3]alkyl GPC with LPS, in the presence o f 0.5% ethanol, resulted in the accumulation of labelled phosphatidyle thanol (PEt) through PLD activation. LPS-mediated PLD activation of TH P-1 or U-937 was inhibited by staurosporine (2 mu M) and by protein ki nase C (PKC) down-regulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) suggesting a role for PKC. In addition to LPS, TPA, ionomycin a nd cell-permeant analogs of diacylglycerol also stimulated [H-3]PEt ac cumulation. The TPA-induced PEt accumulation was also completely aboli shed by staurosporine or down-regulation of PKC (> 95% inhibition). Fu rthermore, the LPS-mediated [P-32]PEt formation was attenuated by eith er depletion of extracellular Ca2+ with EGTA (5 mM) or chelation of in tracellular Ca2+ by BAPTA (30 mu M). These results indicate that an in crease in intracellular Ca2+ is necessary for LPS-mediated PLD activat ion. Further support for PKC activation by LPS was obtained by determi ning PKC activity in an in vitro assay of histone H1 phosphorylation u sing [gamma-P-32]ATP. In untreated THP-1 cells, approximately 64% of t he PKC activity was localized in the cytosol and 36% in the membrane f raction. Treatment of the cells with LPS (10 mu g/ml, for 2 h) resulte d in an increase of 10% of the membrane-associated PKC activity and a corresponding decrease in the cytosol fraction. These data provide evi dence that one of the mechanisms of LPS-mediated signal transduction i n human monocytic cell lines involves activation of PLD.