Down-regulation of glycosylphosphatidylinositol-specific phospholipase D induced by lipopolysaccharide and oxidative stress in the murine monocyte- macrophage cell line RAW 264.7

Abstract

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.

FIG. 1

Down-regulation of GPI-PLD mRNA by LPS stimulation. (A) Time- and dose-dependent response of GPI-PLD mRNA to LPS stimulation. RAW 264.7 cells were not stimulated (controls) or stimulated with different concentrations of LPS as indicated. Samples were taken at 1, 4, and 7 h for Northern blot analysis using 0.8% RNA denaturing gels. The results are representative of two independent Northern analyses that were hybridized sequentially with ³²P-labeled GPI-PLD and G3PDH cDNAs. Intensities of signals, here and in the following figures, were enhanced for presentation purposes; relative levels of GPI-PLD mRNA in stimulated cells in comparison to nonstimulated cells (100%) were calculated as described in Materials and Methods. (B) Reduction of GPI-PLD mRNA by LPS under different serum concentrations. The murine monocyte-macrophage cell lines J774 and RAW 264.7 were stimulated with LPS (5 ng/ml) in medium containing 2 or 10% FBS for 4 h. Total RNA was prepared and analyzed by Northern blotting using 0.8% RNA denaturing gels. Cells without LPS were analyzed in parallel. The results are representative of two independent Northern analyses.

FIG. 2

Down-regulation of GPI-PLD mRNA by oxidative stresses. (A) Time course response of GPI-PLD mRNA to H₂O₂ stimulation. RAW 264.7 cells were untreated (controls) or treated with 0.5 mM H₂O₂. Samples were taken at 1, 4, and 7 h, and total RNA was prepared and analyzed by Northern blotting. The results are representative of two independent Northern analyses. (B) MQ induces down-regulation of GPI-PLD mRNA. RAW 264.7 cells were stimulated with 50 μM MQ for 1 and 4 h. Cells without MQ were tested in parallel. Total RNA was prepared and analyzed by Northern blotting using 0.8% RNA denaturing gels. The results are representative of two independent Northern analyses.

FIG. 3

Effect of the antioxidant pretreatment on reduction of GPI-PLD mRNA in response to LPS and H₂O₂. (A) RAW 264.7 cells were untreated (none) or pretreated with 1 mM NAC or 1% DMSO for 1 h and then added with 5 ng of LPS per ml or 0.5 mM H₂O₂ for another 4 h. Cells without stimulation (control) were tested in parallel. Total RNA for each sample was isolated at the end of the stimulation and analyzed by Northern blotting. The results are representative of three independent experiments. (B) Relative levels of GPI-PLD mRNA in stimulated cells compared to nonstimulated cells under different pretreatments. The level of GPI-PLD mRNA in nonstimulated cells (control) was always set at 100% for either untreated (none) or antioxidant-treated cells. Results are depicted as means ± SE for three independent experiments. ∗, P < 0.05, comparing H₂O₂-stimulated cells with and without NAC pretreatment.

FIG. 4

Reduction of GPI-PLD mRNA by either LPS or H₂O₂ is completely inhibited by actinomycin D. (A) RAW 264.7 cells were untreated (control) or treated with 5 ng of LPS per ml or 0.5 mM H₂O₂ in the presence or absence of 1 μg of actinomycin D per ml for 4 h. Total RNA was extracted and analyzed by Northern blotting. The blots are representative of three independent experiments. (B) Percentages of relative levels of GPI-PLD mRNA in stimulated cells compared to nonstimulated cells with and without the presence of actinomycin D. Results are depicted as means ± SE for three independent experiments. ∗, P < 0.05; ∗∗, P < 0.01.

FIG. 5

Effect of LPS or H₂O₂ on the half-life of GPI-PLD mRNA. (A) RAW 264.7 cells were untreated (none) or pretreated with 5 ng of LPS per ml or 0.5 mM H₂O₂ for 2 h, and then actinomycin D was added to a final concentration of 1 μg/ml. At the times indicated, total RNA was extracted and analyzed by Northern blotting. The results are representative of three independent experiments. (B) Percentage of relative level of GPI-PLD mRNA as a function of time after the addition of actinomycin D. Details of calculation are described in Materials and Methods. Results are depicted as means ± SE for three independent experiments. ∗, P < 0.05, comparing half-lives of mRNA between H₂O₂-treated and untreated control cells at 4 h.

FIG. 6

Low GPI-PLD activity in RAW 264.7 sublines correlates with an increased stimulatory effect of LPS and increased mCD14 expression on the cell surface. RAW 264.7 sublines B, IA, IIA, and IIIA were obtained by stable transfection as described in Results. (A) RAW 264.6 cells (RAW) and the sublines were unstimulated or stimulated with 5 ng of LPS per ml for 4 h. Total RNA was extracted and analyzed by Northern blotting. Top, representative blot of three independent experiments hybridized with ³²P-labeled TNF-α cDNA; bottom, same blot hybridized with ³²P-G3PDH cDNA. (B) Comparison of mCD14 expression on the cell surface of RAW 264.7 cells (RAW) and the sublines. mCD14 was detected using phycoerythrin-conjugated rat anti-CD14 monoclonal antibody rmC5-3 as described in Materials and Methods. The histogram of fluorescence (FL2-H) intensity of each subline (black trace) was overlaid with that of RAW 264.7 cells (RAW). The fluorescence background shown by unstained RAW 264.7 cells was below 10, with a mean of 8.85. (C) Decreased GPI-PLD activity in RAW 264.7 sublines is associated with increased mCD14 presentation and induction of TNF-α mRNA by LPS. Three sets of data were aligned for comparison purposes. Top, cellular GPI-PLD specific activities in RAW 264.7 sublines B, IA, IIA, and IIIA (cultured in G418) relative to RAW 264.7 cells (RAW). Results are depicted as means ± SE for three independent experiments. Middle, comparison of statistical means of fluorescence intensities (mCD14) in RAW 264.7 sublines B, IA, IIA, and IIIA with that of RAW 264.7 cells (RAW). Results are depicted as means ± SE for three independent experiments. Bottom, induction of TNF-α mRNA by LPS in RAW 264.7 sublines B, IA, IIA, and IIIA relative to RAW 264.7 cells (RAW). The extent of induction was initially calculated by comparing relative level of TNF mRNA in LPS-stimulated cells to that of nonstimulated cells. For comparison purposes, the induction in RAW 264.7 cells was arbitrarily set at 1. Results are depicted as means ± SE for three independent experiments.

FIG. 7

TNF-α reduces GPI-PLD expression in human alveolar carcinoma cell line A549 but not in murine macrophage cell line RAW 264.7. (A) The GPI-PLD mRNA level in RAW 264.7 cells was not affected by TNF-α stimulation. RAW 264.7 cells were untreated or treated with TNF-α at the indicated concentrations for 4 h in either serum-free medium or 10% FBS-containing medium. Total RNA was prepared and analyzed by Northern blotting. Top, representative blots hybridized with ³²P-labeled GPI-PLD cDNA; bottom, same blot hybridized with ³²P-G3PDH. (B) Relative levels of GPI-PLD mRNA in stimulated cells compared to nonstimulated cells with and without the presence of serum. Results are depicted as means ± SE for three independent experiments. (C) Reduction of GPI-PLD mRNA upon TNF-α stimulation in A549 cells is independent of serum. A549 cells were untreated or treated with 500 U of TNF-α per ml in either serum-free medium or 10% FBS-containing medium. Samples were taken at 4 and 12 h, and total RNA was prepared and analyzed by Northern blotting. Shown are representative blots hybridized sequentially with ³²P-labeled GPI-PLD cDNA and ³²P-G3PDH along with quantification data. (D) TNF-α reduces the expression of SEAP driven by the human GPI-PLD promoter in A549 cells. The four promoter constructs are shown schematically on the left. The positions of five repeat sequences along the 5.5-kb 5′ untranslated region were predicted by using the Repeat Masker server at the University of Washington (http://ftp.genome.washington.edu/) and indicated by gray boxes. The TATA box of the strong promoter located within -502 to -1303 is indicated by a vertical bar. The distribution of 10 potential EBS (TTCC) detected within -1 to -1303 is also denoted by thin vertical bars. A549 cells were transiently transfected with promoter constructs containing a SEAP reporter gene. TNF-α (500 U/ml) treatment and measurement of SEAP activity are described in Materials and Methods. Results are depicted as means ± SE for four independent transfection experiments. ∗∗, P < 0.01; ∗∗∗, P < 0.001.