Negative regulation by phosphatidylinositol 3-kinase of inducible nitric oxide synthase expression in macrophages

Triggering of the macrophage cell line RAW 264.7 with LPS promotes a transi ent activation of phosphatidylinositol 3-kinase (PI3-kinase). Incubation of activated macrophages with wortmannin and LY294002, two inhibitors of PI3- kinase, increased the amount of inducible nitric oxide synthase (iNOS) and the synthesis of nitric oxide. Treatment with wortmannin promoted a prolong ed activation of NF-kappa B in LPS-treated cells as well as an increase in the promoter activity of the iNOS gene as deduced from transfection experim ents using a 1.7-kb fragment of the 5' flanking region of the iNOS gene. Co transfection of cells with a catalytically active p110 subunit of PI3-kinas e impaired the responsiveness of the iNOS promoter to LPS stimulation, wher eas transfection with a kinase-deficient mutant of p110 maintained the up-r egulation in response to wortmannin, These results indicate that PI3-kinase plays a negative role in the process of macrophage activation and suggest that this enzyme might participate in the mechanism of action of antiinflam matory cytokines.