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

Transcription of liver X receptor is down-regulated by 15-deoxy-Δ12,14-prostaglandin J2 through oxidative stress in human neutrophils

Liver X receptors (LXRs) are ligand-activated transcription factors of the nuclear receptor superfamily. They play important roles in controlling cholesterol homeostasis and as regulators of inflammatory gene expression and innate immunity, by blunting the induction of classical pro-inflammatory genes. However, opposite data have also been reported on the consequences of LXR activation by oxysterols, resulting in the specific production of potent pro-inflammatory cytokines and reactive oxygen species (ROS). The effect of the inflammatory state on the expression of LXRs has not been studied in human cells, and constitutes the main aim of the present work. Our data show that when human neutrophils are triggered with synthetic ligands, the synthesis of LXRα mRNA became activated together with transcription of the LXR target genes ABCA1, ABCG1 and SREBP1c. An inflammatory mediator, 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), hindered T0901317-promoted induction of LXRα mRNA expression together with transcription of its target genes in both neutrophils and human macrophages. This down-regulatory effect was dependent on the release of reactive oxygen species elicited by 15dPGJ2, since it was enhanced by pro-oxidant treatment and reversed by antioxidants, and was also mediated by ERK1/2 activation. Present data also support that the 15dPGJ2-induced serine phosphorylation of the LXRα molecule is mediated by ERK1/2. These results allow to postulate that down-regulation of LXR cellular levels by pro-inflammatory stimuli might be involved in the development of different vascular diseases, such as atherosclerosis.Funding provided by the Ministerio de Educación y Ciencia (BFU2006-13802) and the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P08-CVI-03550) (P06-CTS-01936) Consejería de Salud, Junta de Andalucía (CS 0116/2007)

Heme oxygenase-1 expression is down-regulated by angiotensin II and under hypertension in human neutrophils

Angiotensin II (Ang II) is a peptide hormone able to elicit a strong production of reactive oxygen species by human neutrophils. In this work, we have addressed whether expression of heme oxygenase-1 (HO-1), an antioxidant enzyme, becomes altered in these cells upon Ang II treatment or under hypertension conditions. In neutrophils from healthy and hypertensive subjects, induction of HO-1 mRNA and protein expression with a parallel increase in enzyme activity took place upon treatment with 15-deoxy-Δ12,14-PGJ2 (15dPGJ2). However, Ang II prevented HO-1 synthesis by normal neutrophils in vitro, and HO-1 expression was depressed in neutrophils from hypertensive patients in comparison with cells from healthy subjects. In addition, Ang II treatment led to a reduced HO-1 enzyme activity to levels similar to those found in neutrophils from hypertensive patients. NO donors reversed the inhibition of 15dPGJ2-dependent HO-1 expression in neutrophils from hypertensive patients, and conversely, inhibition of inducible NO synthase (NOS2) activity counteracted the stimulatory effect of 15dPGJ2 on HO-1 expression in normal human neutrophils. Moreover, Ang II canceled 15dPGJ2-dependent induction of NOS2 mRNA synthesis. Present findings indicate that down-regulation of HO-1 expression in neutrophils from hypertensive subjects is likely exerted through the inhibition of NOS2 expression. Additionally, they underscore the potential usefulness of NO donors as new, therapeutic agents against hypertension