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Nramp1 functionality increases inducible nitric oxide synthase transcription via stimulation of IFN regulatory factor 1 expression

Nramp1 functionality increases inducible nitric oxide synthase transcription via stimulation of IFN regulatory factor 1 expression
Nramp1 functionality increases inducible nitric oxide synthase transcription via stimulation of IFN regulatory factor 1 expression
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 macrophagecell 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- 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 Nramp1express 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.
0022-1767
1994-1998
Fritsche, G.
019a735f-5448-4d9b-b585-3faa7d7d45cc
Dlaska, M.
f11c5d6d-434f-4122-b677-fca2912c5949
Barton, C.H.
5dfb4e1a-d559-4b58-9036-31de1e6c9ad8
Theurl, I.
459712ab-3401-49e3-a8dd-229f989032a9
Garimorth, K.
0017eb64-612a-4a09-8847-7a79050f6cc4
Weiss, G.
9461d2ef-d058-4e79-96e4-5920c1420fb0
Fritsche, G.
019a735f-5448-4d9b-b585-3faa7d7d45cc
Dlaska, M.
f11c5d6d-434f-4122-b677-fca2912c5949
Barton, C.H.
5dfb4e1a-d559-4b58-9036-31de1e6c9ad8
Theurl, I.
459712ab-3401-49e3-a8dd-229f989032a9
Garimorth, K.
0017eb64-612a-4a09-8847-7a79050f6cc4
Weiss, G.
9461d2ef-d058-4e79-96e4-5920c1420fb0

Fritsche, G., Dlaska, M., Barton, C.H., Theurl, I., Garimorth, K. and Weiss, G. (2003) Nramp1 functionality increases inducible nitric oxide synthase transcription via stimulation of IFN regulatory factor 1 expression. Journal of Immunology, 171 (4), 1994-1998.

Record type: Article

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 macrophagecell 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- 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 Nramp1express 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.

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Published date: 15 August 2003

Identifiers

Local EPrints ID: 24076
URI: http://eprints.soton.ac.uk/id/eprint/24076
ISSN: 0022-1767
PURE UUID: 2be67cc7-adc1-4573-8062-d03bb9a8e988

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Date deposited: 21 Mar 2006
Last modified: 15 Mar 2024 06:52

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Contributors

Author: G. Fritsche
Author: M. Dlaska
Author: C.H. Barton
Author: I. Theurl
Author: K. Garimorth
Author: G. Weiss

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