title: Nramp1 functionality increases inducible nitric oxide synthase transcription via stimulation of IFN regulatory factor 1 expression.
abstract: Natural-resistance associated macrophage protein 1 (Nramp1) encodes a transmembrane phagolysosomal protein exerting resistance toward infections with intracellular pathogens by a mechanism not fully elucidated so far. We used the murine macrophage cell line RAW264.7, stably transfected with functional (RAW-37) or nonfunctional (RAW-21) Nramp1, to study for differences in the expression of NO, a central antimicrobial effector molecule of macrophages. Following stimulation with IFN-gamma and LPS, Nramp1-expressing cells exhibit higher enzymatic activity of inducible NO synthase (iNOS) and increased cytoplasmic iNOS mRNA levels than RAW-21 cells. Time-course experiments showed that iNOS-mRNA levels remain increased in RAW-37 cells after prolonged cytokine stimulation while they decrease in RAW-21 cells. Reporter gene assays with iNOS-promoter luciferase constructs demonstrated an increased and prolonged promoter activity in Nramp1-resistant vs susceptible cells. This was paralleled by increased IFN regulatory factor 1 (IRF-1) expression and binding affinity to the iNOS promoter in RAW-37 cells, which may be related to enhanced STAT-1 binding affinity in these cells. A point mutation within the IRF-1 binding site of the iNOS promoter abolished the differences in iNOS transcription between RAW-21 and RAW-37 cells. Cells carrying functional Nramp1 express increased amounts of NO, which may be related to STAT-1-mediated stimulation of IRF-1 expression with subsequent prolonged activation of iNOS transcription. Enhanced NO expression may partly underlie the protection against infection with intracellular pathogens by Nramp1 functionality.
authors.affiliation: Department of Internal Medicine, University Hospital of Innsbruck, Innsbruck, Austria.
authors.forename: Gernot
authors.initials: G
authors.lastname: Fritsche
authors.forename: Margit
authors.initials: M
authors.lastname: Dlaska
authors.forename: Howard
authors.initials: H
authors.lastname: Barton
authors.forename: Igor
authors.initials: I
authors.lastname: Theurl
authors.forename: Katja
authors.initials: K
authors.lastname: Garimorth
authors.forename: Günter
authors.initials: G
authors.lastname: Weiss
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D027682:Cation Transport Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D015536:Down-Regulation / Q000187:drug effects / Q000276:immunology; D004789:Enzyme Activation / Q000235:genetics / Q000276:immunology; D005786:Gene Expression Regulation / Q000276:immunology; D007113:Immunity, Innate / Q000235:genetics; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000494:pharmacology; D007501:Iron / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000201:enzymology* / Q000276:immunology / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D012486:Salmonella typhimurium / Q000276:immunology; D014158:Transcription, Genetic / Q000276:immunology*; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics* / Q000276:immunology*; D000099173:Natural Resistance-associated Macrophage Protein 1
chemical_list: D027682:Cation Transport Proteins; D004268:DNA-Binding Proteins; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma; D007501:Iron; D008070:Lipopolysaccharides; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites; D010750:Phosphoproteins; D012333:RNA, Messenger; D000099173:Natural Resistance-associated Macrophage Protein 1; C494258:Irf1 protein, mouse; C496317:Nos2 protein, mouse