Salicylate Suppresses Macrophage Nitric-oxide Synthase-2 and Cyclo-oxygenase-2 Expression by Inhibiting CCAAT/Enhancer- binding Protein- 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(IFN). Cells were treated with sodium salicylate (10 -10 4 M) or vehicle for 30 min followed by LPS IFNfor up to 24 h. Salicylate suppressed NOS-2 and COX-2 protein levels and promoter activities stimulated by LPS IFNfor 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 (C/EBP ) 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/EBP binding at 4 h and did not alter its binding at 24 h. NOS-2 and COX-2 protein levels and C/EBP binding stimulated by LPS IFNfor 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/EBP binding, whereas its expression at a longer time point was contributed by IFN. Our findings implicate two distinct pathways for NOS-2 expression induced by LPS IFN. Salicylate at pharmacological concentrations is capable of suppressing the early phase of NOS-2 and COX-2 expression by blocking C/EBP binding.