An Interferon-γ-activated Site (GAS) Is Necessary for Full Expression of the Mouse iNOS Gene in Response to Interferon-γ and Lipopolysaccharide

Mouse macrophages can be stimulated by interferon (IFN)-γ and bacterial lipopolysaccharide (LPS) to produce nitric oxide (NO) as the result of expression of the inducible NO synthase (iNOS; EC 1.14.13.39) gene. The iNOS gene promoter contains a candidate γ-interferon- activated site (GAS). In transfection studies reported here, it was demonstrated that a luciferase reporter-gene construct, containing four synthetic copies of the iNOS GAS, was inducible when transfected macrophages were stimulated with either IFN-γ, LPS, or a combination of the 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 positions within the GAS were mutated. Oligonucleotide probes, which mimicked the iNOS GAS, formed a complex with proteins that appeared in the nuclei of IFN-γ or IFN-γ+ 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 contrast to binding induced by IFN-γ, approximately 2 h of stimulation with LPS were required. The protein bound to the iNOS GAS-containing oligonucleotide reacted specifically with an antibody raised against Stat1α, regardless of the stimulus used. These data collectively support the conclusion that binding of Stat1α to the iNOS promoter's GAS is required for optimal induction of the iNOS gene by IFN-γ and LPS. Mouse macrophages can be stimulated by interferon (IFN)-γ and bacterial lipopolysaccharide (LPS) to produce nitric oxide (NO) as the result of expression of the inducible NO synthase (iNOS; EC 1.14.13.39) gene. The iNOS gene promoter contains a candidate γ-interferon- activated site (GAS). In transfection studies reported here, it was demonstrated that a luciferase reporter-gene construct, containing four synthetic copies of the iNOS GAS, was inducible when transfected macrophages were stimulated with either IFN-γ, LPS, or a combination of the 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 positions within the GAS were mutated. Oligonucleotide probes, which mimicked the iNOS GAS, formed a complex with proteins that appeared in the nuclei of IFN-γ or IFN-γ+ 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 contrast to binding induced by IFN-γ, approximately 2 h of stimulation with LPS were required. The protein bound to the iNOS GAS-containing oligonucleotide reacted specifically with an antibody raised against Stat1α, regardless of the stimulus used. These data collectively support the conclusion that binding of Stat1α to the iNOS promoter's GAS is required for optimal induction of the iNOS gene by IFN-γ and LPS.