Abstract
To determine the role of C-Rel in nitric-oxide synthase-2 (NOS-2) transcriptional activation, we evaluated the effect of lipopolysaccharide and interferon-γ (LPS/IFNγ) on C-Rel DNA binding in RAW 264.7. LPS/IFNγ-stimulated C-Rel binding peaked at 4 to 8 h and declined at 24 h. Transfection of cells with a C-Rel small interfering RNA abrogated C-Rel binding at all time points. LPS/IFNγ produced superoxide at 4 h, which subsided at 8 h. C-Rel binding and NOS-2 expression were abrogated by superoxide dismutase or apocynin at 4 h, suggesting a key role that superoxide plays in mediating C-Rel binding and NOS-2 transactivation only at 4 h. We have reported previously that salicylate at 10-5 M inhibited LPS/IFNγ-induced CCAAT/enhancer binding protein β (C/EBPβ) binding at 4 h but not at 8 or 24 h. A single dose of salicylate did not inhibit C-Rel binding at any time point. The addition of a second dose of salicylate 4 h before an indicated endpoint suppressed C-Rel but not C/EBPβ or interferon-γ-regulated factor-1 binding at 8 and 24 h. A single dose of salicylate added with LPS/IFNγ inhibited NOS-2 expression only at 4 h. However, salicylate supplement inhibited NOS-2 promoter activities and mRNA and protein levels throughout 24 h. Signal profiling with a panel of inhibitors revealed time-dependent switch of signaling pathways. These results demonstrate temporal regulation of transactivator binding by LPS/IFNγ via evolving signaling pathways. We propose that salicylate inhibits C/EBPβ binding at 4 h and C-Rel binding at 8 and 24 h by targeting related kinases.
Footnotes
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This work is supported by grants from the National Institutes of Health (National Heart, Lung and Blood Institute R01-HL50675 and National Institute of Neurological Diseases and Stroke P50-NS23327).
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Current affiliation: Department of Pediatrics-Neonatology, Medical School, University of Texas Health Science Center at Houston, Houston, Texas.
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ABBREVIATIONS: NOS-2, nitric-oxide synthase-2; COX, cyclooxygenase; C/EBPβ, CCAAT/enhancer binding protein β; LPS, lipopolysaccharide; IFNγ, interferon γ; IRF-1, interferon-γ regulated factor-1; siRNA, small interfering RNA; MAPK, mitogen-activated protein kinase; RSK, p90 ribosomal S6 kinase; mTOR, mammalian target of rapamycin; Jak, Janus-activated kinase; PI-3K, phosphoinositide 3-kinase; ROS, reactive oxygen species; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; GFP, green fluorescent protein; NF-κB, nuclear factor κB; BZT, 1,2,3-benzenetriol; SOD, superoxide dismutase; qPCR, quantitative polymerase chain reaction; ChIP, chromatin immunoprecipitation; PBSI, phosphate-buffered saline buffer containing Na3VO4, NaF, β-glycerophosphate, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, and dithiothreitol; PD98059, 2′-amino-3′-methoxyflavone; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; AG490, α-cyano-(3,4-dihydroxy)-N-benzylcinnamide; GF109203X, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride.
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↵1 K.A.C. and W.-G.D. contributed equally to this manuscript.
- Received April 27, 2006.
- Accepted September 7, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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