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Originally published In Press as doi:10.1074/jbc.M205030200 on October 11, 2002

J. Biol. Chem., Vol. 277, Issue 51, 49304-49310, December 20, 2002
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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*

Katarzyna Cieslik, Ying Zhu, and Kenneth K. WuDagger

From the Vascular Biology Research Center, Institute of Molecular Medicine and Division of Hematology, University of Texas-Houston Health Science Center, Houston, Texas 77030

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.

* 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.

Dagger To whom correspondence should be addressed: Vascular Biology Research Center, Institute of Molecular Medicine and Division of Hematology, University of Texas-Houston Health Science Center, 6431 Fannin, Houston, TX 77030. Tel.: 713-500-6801; Fax: 713-500-6812; E-mail: Kenneth.K.Wu@uth.tmc.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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