15dPGJ₂ inhibition of LXR target genes mRNA expression is mediated by oxidative stress in human neutrophils.

<p>Neutrophils were preincubated at 37°C with or without 10 µM DEM, 100 µM TEMPO (A), 100 µM vitamin E analog (Vit E) or 1 mM GSH (B) for 1 h. Then the cells were treated or not with 10 µM 15dPGJ₂ for 1 h, and thereafter stimulated or not with 1 µM T0901317 for 18 h. Finally, the levels of LXRα target genes mRNAs were analyzed by real time RT-PCR. Statistical data (mean ± SEM, n = 3) were corrected for differences in β-actin mRNA levels and expressed as fold induction. * P<0.01 for T0901317–stimulated, 15dPGJ₂ (A and B) or DEM (A)-treated versus -untreated. ^◊ P<0.01 for 15dPGJ₂ and T0901317-stimulated, TEMPO (A), Vit E or GSH (B)-treated versus -untreated.</p

15dPGJ₂ inhibition of LXR target genes mRNA expression is mediated by ERK1/2 in human neutrophils.

<p>Neutrophils were pretreated at 37°C with or without 10 µM SB203580 or 1 µM SP600125, or with PD098059 at 40 µM (A) or at the indicated doses (B) for 1 h. Then the cells were incubated in the absence or presence of 10 µM 15dPGJ₂ for 1 h, and further stimulated or not with 1 µM T0901317 for 18 h. Finally, the levels of LXR target genes mRNA were analyzed by real time RT-PCR. Statistical data (mean ± SEM, n = 3) were corrected for differences in β-actin mRNA levels and are expressed as fold induction. * P<0.01 for T0901317-stimulated, 15dPGJ₂-treated versus -untreated. ** P<0.01 for T0901317-stimulated, PD098059-treated versus -untreated. ^◊ P<0.01 for 15dPGJ₂ plus T0901317-stimulated, PD098059-treated versus -untreated.</p

15dPGJ₂ promotes LXRα serine phosphorylation in human neutrophils.

<p>Neutrophils were preincubated at 37°C with or without 30 µM PD098059 for 30 min. Then the cells were treated or not with 10 µM 15dPGJ₂ for 30 min, and thereafter stimulated or not with 1 µM T0901317 for further 30 min. Finally, the cells were lysed, LXRα was immunoprecipitated and Western blotting analysis of phosphoserine (P-Ser) and LXRα levels was carried out as indicated in Experimental Procedures. P-Ser levels are given in arbitrary units. The blot is representative of a set of three experiments yielding similar results.</p

15dPGJ₂ prevents ligand-induced LXRα mRNA expression in a dose-dependent manner.

<p>Neutrophils or macrophages were left untreated (A), or else neutrophils were preincubated at 37°C for 1 h with or without 15dPGJ₂ at the indicated doses (B) or at a 10 µM concentration (C), and then they were treated or not with T0901317 for 18 h (B and C). Then LXRα and LXRβ mRNA levels were measured by real time RT-PCR (A and B), or LXRα protein levels were analyzed by Western blotting (C). Statistical data from real time RT-PCR experiments were normalized to LXRα or LXRβ levels found in human macrophages (A) and corrected for differences in β-actin mRNA levels (as endogenous gene) and expressed as fold induction (A and B). GAPDH bands in Western blots are shown for the sake of protein loading controls (C). In a different set of experiments, neutrophils were pretreated or not with 10 µM DEM for 30 min (D and E) and, after preincubation in the absence or presence of 1 µM T0901317 for 4 h (D and E), 10 µM 15dPGJ₂ was added and ROS levels were monitored by luminescence measurement for the next 6 h (D), or released IL-8 levels were quantitated by ELISA after an incubation period of 4 h (E). Neutrophil migration was quantitated in cells preincubated with or without 1 µM T0901317 for 4 h and then transferred to Transwell chambers and treated with 10 µM 15dPGJ₂ or 0.1 µM fMLP for 1 h. Results are expressed as the percentage of cells that migrated from the upper to the lower compartment (F). Each panel is representative of a set of three experiments yielding similar results. Values are plotted as the mean ± SEM (n = 3) (A, B, D, E and F). * P<0.01 for T0901317-stimulated, 15dPGJ₂ (B, F) or fMLP (F)-treated versus -untreated. ^◊ P<0.01 for 15dPGJ₂-treated versus untreated (E). ** P<0.01 for 15dPGJ₂ and T0901317-stimulated, DEM-treated versus -untreated (E).</p

15dPGJ₂ prevents T0901317-induced ABCA1, ABCG1 and SREBP1c mRNA expression in human neutrophils and macrophages.

<p>Neutrophils (A-C) or macrophages (D) were preincubated at 37°C with or without 10 µM 15dPGJ₂ for 1 h, and thereafter they were stimulated or not with 1 µM T0901317 for 18 h. The levels of mRNA from the indicated genes were analyzed by real time RT-PCR. Statistical data (mean ± SEM, n = 3) were corrected for differences in β-actin mRNA levels and expressed as fold induction. * P<0.01 for T0901317-stimulated, 15dPGJ₂-treated versus -untreated.</p