Lipopolysaccharide-induced expression of cyclooxygenase-2 in mouse macrophages is inhibited by chloromethylketones and a direct inhibitor of NF-κB translocation

In macrophages, cyclooxygenase-2 (COX-2) is induced by cytokines, mitogens, or endotoxin. The present study investigates whether inhibitors of the nuclear transcription factor NF-kappa B affect lipopolysaccharide (LPS)-mediated expression of COX-2 mRNA, protein, and activity in the macrophage cell line T774.1A. The activation of COX-2 was assessed by measuring the accumulation of prostaglandin (PG) E-2 by radioimmunoassay. Expression of COX-2 mRNA and protein was detected by Northern and Western blot analysis, respectively. In the absence of LPS, mouse macrophages did not express COX-2 and generated low amounts of prostaglandin (PG) E-2. Treatment of T774.1A with LPS (0.1-30 mu g/ml) caused expression of COX-2 protein and activity. Induction of COX-2 activity along with the induction of COX-2 mRNA and protein by LPS was attenuated by the serine protease inhibitors N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK). A cell permeable peptide and a direct inhibitor of NF-kappa B translocation, SN50, attenuated the accumulation of PGE(2) in cell supernatant in a concentration-dependent manner. Our results show that induction of COX-2 by LPS in macrophages involves activation of NF-kappa B and point to a possible therapeutic use of protease inhibitors in inflammatory processes.