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Mol Pharmacol. 2006 Dec;70(6):2004-14. Epub 2006 Sep 8.

Essential role of C-Rel in nitric-oxide synthase-2 transcriptional activation: time-dependent control by salicylate.

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Vascular Biology Research Center at the Brown Foundation Institute of Molecular Medicine and Division of Hematology, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.


To determine the role of C-Rel in nitric-oxide synthase-2 (NOS-2) transcriptional activation, we evaluated the effect of lipopolysaccharide and interferon-gamma (LPS/IFNgamma) on C-Rel DNA binding in RAW 264.7. LPS/IFNgamma-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/IFNgamma 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/IFNgamma-induced CCAAT/enhancer binding protein beta (C/EBPbeta) 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/EBPbeta or interferon-gamma-regulated factor-1 binding at 8 and 24 h. A single dose of salicylate added with LPS/IFNgamma 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/IFNgamma via evolving signaling pathways. We propose that salicylate inhibits C/EBPbeta binding at 4 h and C-Rel binding at 8 and 24 h by targeting related kinases.

[Indexed for MEDLINE]

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