Journal of Biological Chemistry
Volume 277, Issue 51, 20 December 2002, Pages 49304-49310
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GENES: STRUCTURE AND REGULATION
Salicylate Suppresses Macrophage Nitric-oxide Synthase-2 and Cyclo-oxygenase-2 Expression by Inhibiting CCAAT/Enhancer-binding Protein-β Binding via a Common Signaling Pathway*

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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-γ (IFN-γ). Cells were treated with sodium salicylate (10−7-10−4m) or vehicle for 30 min followed by LPS+IFN-γ for up to 24 h. Salicylate suppressed NOS-2 and COX-2 protein levels and promoter activities stimulated by LPS+IFN-γ 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 β (C/EBPβ) 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/EBPβ binding at 4 h and did not alter its binding at 24 h. NOS-2 and COX-2 protein levels and C/EBPβ binding stimulated by LPS+IFN-γ 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/EBPβ binding, whereas its expression at a longer time point was contributed by IFN-γ. Our findings implicate two distinct pathways for NOS-2 expression induced by LPS+IFN-γ. Salicylate at pharmacological concentrations is capable of suppressing the early phase of NOS-2 and COX-2 expression by blocking C/EBPβ binding.

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Published, JBC Papers in Press, October 11, 2002, DOI 10.1074/jbc.M205030200

*

This work was supported by Grants P50 NS-23327 and R01 HL-50675 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.