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

Abstract

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.

Aspirin attenuates insulin resistance in muscle of diet-induced obese rats by inhibiting inducible nitric oxide synthase production and S-nitrosylation of IRβ/IRS-1 and Akt

“…Several reports have shown that salicylates or aspirin inhibit the production of iNOS induced by cytokines in different cell types and suggest that one of the therapeutic actions of these drugs could be mediated by a reduction in iNOS content and nitric oxide production [11,13]. This reduction in iNOS protein levels may be secondary to a decrease in NFκB activation [29]; however, a limitation of our study was that we did not directly measure NFκB activity in these experiments.…”

“…Furthermore, pharmacological or genetic blockage of iNOS production/activity ameliorates insulin resistance associated with obesity or lipopolysaccharide treatment by inhibiting S-nitrosylation of these proteins [9,10]. Interestingly, aspirin treatment can also decrease iNOS production and activity in several cell lines [11][12][13], but its effects on iNOS-induced insulin resistance in the muscle of obese rats has not yet been investigated.…”

Inhibition of p90 Ribosomal S6 Kinase-mediated CCAAT/Enhancer-binding Protein β Activation and Cyclooxygenase-2 Expression by Salicylate

“…Transfection and Promoter Activity Analysis-Cells at 60% confluence in a 35-mm dish were transfected with 2 g of COX-2 luciferase expression vector using Lipofectin according to a procedure previously described (28). 18 h after transfection, cells were incubated in serum-free medium for 24 h followed by stimulation with PMA for 4 h. Cells were harvested, and luciferase activity was measured in a Turner TD-20/20 luminometer.…”

“…Western Blot Analysis-Western blotting was performed by a procedure described previously (28). 20 g of proteins was loaded to each lane, separated by 4 -15% gradient SDS-polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane.…”

“…Nuclear Extract Preparation-Nuclear extracts were prepared by a procedure previously described (28). Cells were harvested in 1 ml of PBSI buffer (PBS buffer containing multiple protease inhibitors: 1 mM Na 3 VO 4 ; 10 mM NaF; 25 mM ␤-glycerophosphate; 0.1 mM PMSF; 0.06 mg/ml aprotinin; 1 g/ml leupeptin; and 0.5 mM dithiothreitol (DTT)) and spun down at 550 ϫ g for 5 min.…”