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.

[1]  C. Goldring,et al.  In vivo footprinting of the mouse inducible nitric oxide synthase gene: inducible protein occupation of numerous sites including Oct and NF-IL6. , 1996, Nucleic acids research.

[2]  R. Schreiber,et al.  Targeted Disruption of the Stat1 Gene in Mice Reveals Unexpected Physiologic Specificity in the JAK–STAT Signaling Pathway , 1996, Cell.

[3]  E. Petricoin,et al.  Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[4]  A. Larner,et al.  Induction of early response genes by interferons, interleukins, and growth factors by the tyrosine phosphorylation of latent transcription factors. Implications for chronic inflammatory diseases. , 1995, Arthritis and rheumatism.

[5]  J. Hotchkiss,et al.  Potential genotoxicity of chronically elevated nitric oxide: a review. , 1995, Mutation research.

[6]  C. Nathan,et al.  Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase , 1995, Cell.

[7]  T. Billiar.  Nitric Oxide Novel Biology with Clinical Relevance , 1995, Annals of surgery.

[8]  S. Russell,et al.  Contribution of clonally distinct subpopulations to heterogeneous production of inducible nitric oxide synthase by LPS-stimulated mouse macrophages , 1994 .

[9]  C. Lowenstein,et al.  Population dynamics of inducible nitric oxide synthase production by LPSand LPS/IFNγ-stimulated mouse macrophages , 1994 .

[10]  S. McKnight,et al.  An interleukin-4-induced transcription factor: IL-4 Stat. , 1994, Science.

[11]  C. Nathan,et al.  Role of interferon regulatory factor 1 in induction of nitric oxide synthase , 1994, The Journal of experimental medicine.

[12]  T. Rein,et al.  In vivo footprinting of the IRF-1 promoter: inducible occupation of a GAS element next to a persistent structural alteration of the DNA. , 1994, Nucleic acids research.

[13]  N. Copeland,et al.  Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation , 1994, Molecular and cellular biology.

[14]  J. Darnell,et al.  Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. , 1994, Science.

[15]  B. Groner,et al.  Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. , 1994, The EMBO journal.

[16]  O. Silvennoinen,et al.  Signaling by the cytokine receptor superfamily: JAKs and STATs. , 1994, Trends in biochemical sciences.

[17]  T. Suzuki,et al.  Role of endogenous interferon-beta in lipopolysaccharide-triggered activation of the inducible nitric-oxide synthase gene in a mouse macrophage cell line, J774. , 1994, The Journal of biological chemistry.

[18]  J. Darnell,et al.  Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. , 1994, Science.

[19]  D. Levy,et al.  A novel interferon-alpha-regulated, DNA-binding protein participates in the regulation of the IFP53/tryptophanyl-tRNA synthetase gene. , 1994, The Journal of biological chemistry.

[20]  T. Taniguchi,et al.  Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. , 1994, Science.

[21]  C. Nathan,et al.  Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. , 1994, The Journal of biological chemistry.

[22]  D. Morrison,et al.  Necessity and sufficiency of beta interferon for nitric oxide production in mouse peritoneal macrophages , 1994, Infection and immunity.

[23]  S. Snyder,et al.  Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[24]  T. Suzuki,et al.  Influence of 3' half-site sequence of NF-kappa B motifs on the binding of lipopolysaccharide-activatable macrophage NF-kappa B proteins. , 1993, The Journal of biological chemistry.

[25]  M. Marletta,et al.  Nitric oxide synthase structure and mechanism. , 1993, The Journal of biological chemistry.

[26]  C. Nathan,et al.  Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide , 1993, The Journal of experimental medicine.

[27]  H. Sadowski,et al.  Cell-free activation of a DNA-binding protein by epidermal growth factor , 1993, Nature.

[28]  S. Snyder,et al.  Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing. Molecular basis for the synergy between interferon-gamma and lipopolysaccharide. , 1993, The Journal of biological chemistry.

[29]  C. Nathan,et al.  Nitric oxide as a secretory product of mammalian cells , 1992, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[30]  C. Nathan,et al.  Calmodulin is a subunit of nitric oxide synthase from macrophages , 1992, The Journal of experimental medicine.

[31]  D. von der Ahe,et al.  Constitutive expression of the urokinase plasminogen activator gene in murine RAW264 macrophages involves distal and 5' non-coding sequences that are conserved between mouse and pig. , 1991, Nucleic acids research.

[32]  J. Garthwaite.  Glutamate, nitric oxide and cell-cell signalling in the nervous system , 1991, Trends in Neurosciences.

[33]  R. Busse,et al.  Calcium‐dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin , 1990, FEBS letters.

[34]  S. Moncada,et al.  The anti‐aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide , 1987, British journal of pharmacology.

[35]  S. Moncada,et al.  Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor , 1987, Nature.

[36]  S. Snyder,et al.  Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[37]  P. Sharp,et al.  A high-efficiency HeLa cell nuclear transcription extract. , 1988, DNA.