Down-regulation of glycosylphosphatidylinositol-specific phospholipase D induced by lipopolysaccharide and oxidative stress in the murine monocyte- macrophage cell line RAW 264.7.
Serum glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) activity is reduced over 75% in systemic inflammatory response syndrome. To investigate the mechanism of this response, expression of the GPI-PLD gene was studied in the mouse monocyte-macrophage cell line RAW 264.7 stimulated with lipopolysaccharide (LPS; 0.5 to 50 ng/ml). GPI-PLD mRNA was reduced approximately 60% in a time- and dose-dependent manner. Oxidative stress induced by 0.5 mM H(2)O(2) or 50 microM menadione also caused a greater than 50% reduction in GPI-PLD mRNA. The antioxidant N-acetyl-L-cysteine attenuated the down-regulatory effect of H(2)O(2) but not of LPS. Cotreatment of the cells with actinomycin D inhibited down-regulation induced by either LPS or H(2)O(2). The half-life of GPI-PLD mRNA was not affected by LPS, or decreased slightly with H(2)O(2), indicating that the reduction in GPI-PLD mRNA is due primarily to transcriptional regulation. Stimulation with tumor necrosis factor alpha (TNF-alpha) resulted in approximately 40% reduction in GPI-PLD mRNA in human A549 alveolar carcinoma cells but not RAW 264.7 cells, suggesting that alternative pathways could exist in different cell types for down-regulating GPI-PLD expression during an inflammatory response and the TNF-alpha autocrine signaling mechanism alone is not sufficient to recapitulate the LPS-induced reduction of GPI-PLD in macrophages. Sublines of RAW 264.7 cells with reduced GPI-PLD expression exhibited increased cell sensitivity to LPS stimulation and membrane-anchored CD14 expression on the cell surface. Our data suggest that down-regulation of GPI-PLD could play an important role in the control of proinflammatory responses.
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Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
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D000818:Animals; D002460:Cell Line; D015536:Down-Regulation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D018384:Oxidative Stress; D010739:Phospholipase D / Q000235:genetics*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D010739:Phospholipase D; C065017:glycoprotein phospholipase D