CCAAT/enhancer-binding protein mediates carbon monoxide-induced suppression of cyclooxygenase-2

Abstract

Cyclooxygenase-2 (COX-2) is a key enzyme involved in the inflammatory process that is rapidly induced in macrophages in response to LPS. Carbon monoxide (CO), a byproduct of heme oxygnease-1, can suppress proinflammatory response in various in vitro and in vivo models of inflammation. This study was undertaken to examine whether CO can regulate (and if so, to delineate the mechanism by which CO regulates) LPS-induced COX-2 expression in macrophages. RAW 264.7 murine macrophages were stimulated with LPS (0-10 ng/ml) with or without CO (500 ppm). Northern and Western blot analysis was done. Progstaglandin E(2) and nitrite concentration was measured from cell culture supernatant. Electrophoretic mobility shift assay was performed to assess nuclear factor binding. CO downregulated LPS-induced COX-2 mRNA and protein expression. CO also inhibited LPS-induced prostaglandin E(2) secretion (P < 0.05). CO also decreased LPS-induced CCAAT/enhancer-binding protein (C/EBP) beta and delta protein expression in LPS-treated RAW 264.7 cells. Gel shift analysis revealed that CO treatment decreased LPS-induced activation of protein binding to C/EBP consensus oligonucleotides of murine cyclooxygenase-2 promoter. CO also decreased LPS-induced nitric oxide synthase-2 protein expression and nitrite production, and decreased LPS-induced activation of protein binding to C/EBP consensus oligonucleotides of murine nitric oxide synthase-2 promoter. CO may act as an important regulator of inflammation by virtue of its ability to regulate C/EBPs.

Figure 1.

CO suppresses COX-2 expression in LPS-stimulated RAW 264.7 cells. (A) Total RNA was extracted from cells stimulated with LPS (2.5 ng/ml) for indicated times in the absence or presence of 500 ppm of CO, and Northern blot analysis was performed. Ethidium bromide staining was done to show equal loading. Bar graph represents mean ± SD of densitometry value of three independent experiments. (B) Total cellular proteins were extracted 4 h after stimulation with various doses of LPS in the presence and absence of 500 ppm of CO and Western blot analysis was performed for COX-2 expression. Data representative of three independent experiments. RA, room air; CO, 500 ppm CO. *P < 0.05. (C) Alveolar macrophages were harvested with or without LPS treatment for 4 h in the absence or presence of CO (500 ppm) and COX-2 production was determined by Western blot analysis. Findings were representative of three independent experiments.

Figure 2.

CO dose-dependently suppresses LPS-induced COX-2 protein expression. Whole cell lysates were harvested at 4 h after LPS (2.5 ng/ml) treatment with or without two different doses of CO treatment (250 and 500 ppm) for Western blot analysis. Data are representative of two independent experiments.

Figure 3.

CO decreases LPS-induced prostaglandin(PG)E₂ production in RAW 264.7 macrophages. Cells were treated for 4 h with LPS (2.5 ng/ml) in the presence or absence of CO (500 ppm). PGE₂ level was measured by immunoassay. Data represent means ± SD of samples from three independent experiments. RA, room air; CO, 500 ppm CO. *P < 0.05.

Figure 4.

CO decreases C/EBP expression in the nucleus of LPS-stimulated RAW 264.7 cells. Nuclear protein extracts were harvested at 0, 1, 2, and 4 h after stimulation with LPS (2.5 ng/ml) in the presence or absence of 500 ppm of CO. Data are representative of three independent experiments. RA, room air; CO, 500 ppm CO.

Figure 5.

CO attenuates nuclear protein binding to C/EBP element of murine cox-2 promoter. EMSA was performed using nuclear extracts from RAW264.7 macrophages treated with LPS (2.5 ng/ml) with or without CO (500 ppm). Cells were harvested 0, 2, and 4 h after treatment. Specificity of binding was examined by carrying out supershift assay by adding specific antibodies to C/EBP β and C/EBP δ. Data are representative of three independent experiments. RA, room air.

Figure 6.

CO attenuates LPS-induced cox-2 promoter via C/EBP. RAW264.7 cells were transiently transfected with either WT or C/EBP mutants (C/EBP-1 or C/EBP-2) overnight before stimulation with LPS (10 ng/ml) for 24 h in the absence or presence of CO (500 ppm). Luciferase assays were performed using Promega Dual Luciferase kit according to manufacturer's protocol. Experiments performed in triplicate in 96-well plates. Open bars, RA + LPS; shaded bars, CO + LPS.

Figure 7.

CO suppresses nitric oxide synthase-2 (NOS-2) expression and nitrite production in LPS-stimulated RAW 264.7 cells. (A) Total cellular proteins were extracted 0, 2, 4, 6, and 10 h after stimulation with LPS (2.5 ng/ml) in the presence and absence of 500 ppm of CO. Data are representative of three independent experiments. (B) Whole cell lysates were harvested at 4 h after LPS (2.5 ng/ml) treatment with or without two different doses of CO treatment (250 and 500 ppm) for Western blot analysis. Data are representative of two independent experiments. (C) Cells were treated for 4 h with LPS (2.5 ng/ml) in the presence or absence of CO (500 ppm). Nitrite production in culture supernatant was measured by Griess reaction. Data represent means ± SD of samples from three independent experiments. RA, room air; CO, 500 ppm CO.

Figure 8.

CO attenuates nuclear protein binding to C/EBP element of murine nos-2 promoter. EMSA was performed using nuclear extracts from RAW264.7 macrophages treated with LPS (2.5 ng/ml) with or without CO (500 ppm). Cells were harvested 0 and 4 h after treatment. Specificity of binding was examined by carrying out supershift assay by adding specific antibodies to C/EBPα, C/EBP β, C/EBP δ, and C/EBPε. Data are representative of three independent experiments.