Essential role of C-Rel in nitric-oxide synthase-2 transcriptional activation: time-dependent control by salicylate.
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.
Cieslik
Katarzyna A
KA
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.
Deng
Wu-Guo
WG
Wu
Kenneth K
KK
D000818:Animals; D001483:Base Sequence; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers; D051379:Mice; D019001:Nitric Oxide Synthase / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D020569:Proto-Oncogene Proteins c-rel / Q000378:metabolism / Q000502:physiology*; D020156:Salicylic Acid / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism; D015533:Transcriptional Activation / Q000502:physiology*
D022782:CCAAT-Enhancer-Binding Protein-beta; D017931:DNA Primers; D020569:Proto-Oncogene Proteins c-rel; D013481:Superoxides; D019001:Nitric Oxide Synthase; D020156:Salicylic Acid