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Nramp1 functionality increases inducible nitric oxide synthase transcription via stimulation of IFN regulatory factor
Gernot Fritsche1, Margit Dlaska, Howard Barton
1Department of Internal Medicine, University Hospital of Innsbruck, Innsbruck, Austria.
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.
Insights
Functional Natural-resistance associated macrophage protein 1 (Nramp1) enhances nitric oxide (NO) production in macrophages. This increased NO expression, driven by sustained iNOS transcription, may explain Nramp1
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Natural-resistance associated macrophage protein 1 (Nramp1) is crucial for resistance against intracellular pathogens.
- The precise mechanism by which Nramp1 confers resistance remains incompletely understood.
- Nitric oxide (NO) is a key antimicrobial effector molecule produced by macrophages.
Purpose of the Study:
- To investigate the role of functional Nramp1 in regulating nitric oxide (NO) production in macrophages.
- To elucidate the molecular mechanisms underlying Nramp1-mediated regulation of inducible NO synthase (iNOS) expression.
- To determine how Nramp1 influences macrophage antimicrobial activity.
Main Methods:
- Utilized RAW264.7 macrophage cell lines stably transfected with functional (RAW-37) or nonfunctional (RAW-21) Nramp1.
- Stimulated cells with interferon-gamma (IFN-γ) and lipopolysaccharide (LPS).
- Assessed iNOS enzymatic activity, iNOS mRNA levels, iNOS promoter activity via reporter gene assays, and expression of IFN regulatory factor 1 (IRF-1) and STAT-1.
Main Results:
- Nramp1-expressing cells (RAW-37) exhibited higher iNOS activity and prolonged iNOS mRNA levels compared to Nramp1-deficient cells (RAW-21).
- Reporter gene assays revealed increased and sustained iNOS promoter activity in RAW-37 cells, linked to elevated IRF-1 and STAT-1 binding.
- A mutation in the IRF-1 binding site of the iNOS promoter abolished Nramp1-dependent differences in iNOS transcription.
Conclusions:
- Functional Nramp1 enhances NO production by promoting sustained iNOS transcription.
- STAT-1-mediated stimulation of IRF-1 expression appears to be a key mechanism for prolonged iNOS activation in Nramp1-expressing cells.
- Enhanced NO production contributes to the protective role of Nramp1 against intracellular pathogens.