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 and U-937. Treatment of THP-1 or U-937 cells labelled with [32P]orthophosphate, [32P]acyl GPC or [3H]alkyl GPC with LPS, in the presence of 0.5% ethanol, resulted in the accumulation of labelled phosphatidylethanol (PEt) through PLD activation. LPS-mediated PLD activation of THP-1 or U-937 was inhibited by staurosporine (2 microM) and by protein kinase C (PKC) down-regulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) suggesting a role for PKC. In addition to LPS, TPA, ionomycin and cell-permeant analogs of diacylglycerol also stimulated [3H]PEt accumulation. The TPA-induced PEt accumulation was also completely abolished by staurosporine or down-regulation of PKC (> 95% inhibition). Furthermore, the LPS-mediated [32P]PEt formation was attenuated by either depletion of extracellular Ca2+ with EGTA (5 mM) or chelation of intracellular Ca2+ by BAPTA (30 microM). These results indicate that an increase in intracellular Ca2+ is necessary for LPS-mediated PLD activation. Further support for PKC activation by LPS was obtained by determining PKC activity in an in vitro assay of histone H1 phosphorylation using [gamma-32P]ATP. In untreated THP-1 cells, approximately 64% of the PKC activity was localized in the cytosol and 36% in the membrane fraction. Treatment of the cells with LPS (10 micrograms/ml, for 2 h) resulted in an increase of 10% of the membrane-associated PKC activity and a corresponding decrease in the cytosol fraction. These data provide evidence that one of the mechanisms of LPS-mediated signal transduction in human monocytic cell lines involves activation of PLD.
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
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Iwamoto
D000470:Alkaloids / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004075:Diglycerides / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D020404:Glycerophospholipids; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D010712:Phosphatidic Acids / Q000096:biosynthesis; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
C011439:1,2-diacylglycerol; D000470:Alkaloids; D004075:Diglycerides; D020404:Glycerophospholipids; D008070:Lipopolysaccharides; D010712:Phosphatidic Acids; C051521:phosphatidylethanol; D011493:Protein Kinase C; D010739:Phospholipase D; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium