AN INTERFERON-GAMMA-ACTIVATED SITE (GAS) IS NECESSARY FOR FULL EXPRESSION OF THE MOUSE INOS GENE IN RESPONSE TO INTERFERON-GAMMA AND LIPOPOLYSACCHARIDE

Mouse macrophages can be stimulated by interferon (IFN)-gamma and bact erial lipopolysaccharide (LPS) to pro duce nitric oxide (NO) as the re sult of expression of the inducible NO synthase (iNOS; EC 1.14.13.39) gene. The iNOS gene promoter contains a candidate gamma-interferon-act ivated site (GAS). In transfection studies reported here, it was demon strated that a luciferase reporter-gene construct, containing four syn thetic copies of the iNOS GAS, was inducible when transfected macropha ges were stimulated with either IFN-gamma, LPS, or a combination of th e two. Consistent with this finding were other transfection analyses, which showed that responsiveness of the intact iNOS promoter to these same agents was significantly reduced when two conserved nucleotide po sitions within the GAS were mutated. Oligonucleotide probes, which mim icked the iNOS GAS, formed a complex with proteins that appeared in th e nuclei of IFN-gamma or IFN-gamma + LPS treated macrophages within 30 min of stimulation, as shown by electrophoretic mobility shift assay. LPS alone also caused the the appearance of a nuclear protein capable of binding the iNOS GAS-containing oligonucleotide; however, in contr ast to binding induced by IFN-gamma, approximately 2 h of stimulation with LPS were required. The protein bound to the iNOS GAS containing o ligonucleotide reacted specifically with an antibody raised against St at1 alpha, regardless of the stimulus used. These data collectively su pport the conclusion that binding of Stat1 alpha to the iNOS promoter' s GAS is required for optimal induction of the iNOS gene by IFN-gamma and LPS.