Salicylate suppresses macrophage nitric-oxide synthase-2 and cyclo-oxygenase-2 expression by inhibiting CCAAT/enhancer-binding protein-beta binding via a common signaling pathway.
We determined whether salicylate at pharmacological concentrations inhibits nitric-oxide synthase-2 (NOS-2) and cyclo-oxygenase-2 (COX-2) expressions in RAW 264.7 stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Cells were treated with sodium salicylate (10(-7)-10(-4) m) or vehicle for 30 min followed by LPS+IFN-gamma for up to 24 h. Salicylate suppressed NOS-2 and COX-2 protein levels and promoter activities stimulated by LPS+IFN-gamma for 4 h in a concentration-dependent manner but had no effect on NOS-2 expression stimulated by the combined agonists for 24 h. Results from promoter analysis indicate that the binding of CCAAT/enhancer-binding protein beta (C/EBPbeta) to its cognate site at -150/-142 on the NOS-2 promoter region was essential for NOS-2 expression at 4 h but not at 24 h. Salicylate reduced C/EBPbeta binding at 4 h and did not alter its binding at 24 h. NOS-2 and COX-2 protein levels and C/EBPbeta binding stimulated by LPS+IFN-gamma for 4 h were inhibited by a similar battery of signaling inhibitors, suggesting a common pathway for NOS-2 and COX-2 expression. Kinetic analysis indicates that NOS-2, similar to COX-2 expression, at 4 h was largely due to the action of LPS, which induced C/EBPbeta binding, whereas its expression at a longer time point was contributed by IFN-gamma. Our findings implicate two distinct pathways for NOS-2 expression induced by LPS+IFN-gamma. Salicylate at pharmacological concentrations is capable of suppressing the early phase of NOS-2 and COX-2 expression by blocking C/EBPbeta binding.
Cieslik
Katarzyna
K
Vascular Biology Research Center, Institute of Molecular Medicine and Division of Hematology, University of Texas-Houston Health Science Center, 6431 Fannin, Houston, TX 77030, USA.
Zhu
Ying
Y
Wu
Kenneth K
KK
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D051546:Cyclooxygenase 2; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D007093:Imidazoles / Q000494:pharmacology; D007371:Interferon-gamma / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D012459:Salicylates / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection
D002867:Chromones; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D007527:Isoenzymes; D008070:Lipopolysaccharides; D009025:Morpholines; D009687:Nuclear Proteins; D011725:Pyridines; D012459:Salicylates; D015534:Trans-Activators; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D007371:Interferon-gamma; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one