Received for publication April 28, 2006.
Revised September 6, 2006.
Accepted for publication September 7, 2006.

ESSENTIAL ROLE OF C-Rel IN NITRIC OXIDE SYNTHASE-2 TRANSCRIPTIONAL ACTIVATION: TIME-DEPENDENT CONTROL BY SALICYLATE

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- (LPS/IFN) on C-Rel DNA binding in RAW 264.7. LPS/IFN-stimulated C-Rel binding peaked at 4-8 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/IFN 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 previously reported that salicylate at 10^-5 M inhibited LPS/IFN-induced C/EBP binding at 4 h but not at 8 h or 24 h. A single dose of salicylate did not inhibit C-Rel binding at any time point. Addition of a second dose of salicylate 4 h prior to an indicated end point suppressed C-Rel but not C/EBP or IRF-1 binding at 8 h and 24 h. A single dose of salicylate added with LPS/IFN inhibited NOS-2 expression only at 4 h. However, salicylate supplement inhibited NOS-2 promoter activities, 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/IFN via evolving signaling pathways. We propose that salicylate inhibits C/EBP binding at 4 h and C-Rel binding at 8 h and 24 h by targeting related kinases.

Key words: Prostanoid, Nitric oxide, Nitric oxide synthases, P38 MAP Kinase, NFkappaB, Cyclooxygenases, RNA/siRNA

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