Endotoxin-mediated S-nitrosylation of p50 alters NF-kappa B-dependent gene transcription in ANA-1 murine macrophages.
Nitric oxide (NO) regulates cellular function, in part, by S-nitrosylating active site thiol groups of proteins. Ex vivo S-nitrosylation of NF-kappa B p50 significantly decreases its capacity for DNA binding. To determine the cellular relevance of this observation, we utilized the ANA-l murine macrophage model of endotoxin (LPS)-mediated NO synthesis. In selected instances, the NO synthase inhibitor, L-arginine methyl ester (L-NAME; 100 microM), or the NO donor, S-nitroso-N-acetylcysteine (SNAC; 100 microM), was added. In contrast to that of LPS cells, nuclear extracts from LPS + L-NAME cells demonstrated increased NF-kappa B DNA binding on gel shift analysis. Addition of SNAC to LPS + L-NAME cells restored binding to a level equivalent to that of LPS cells. Spectrophotometric analysis of NF-kappa B p50 immunoprecipitates demonstrated S-NO bonds exclusively in LPS cells; these p50 protein isolates retained the same DNA binding characteristics as that of the nuclear extracts. Transfection assays utilizing NF-kappa B-dependent promoter-reporter constructs demonstrated increased activity in LPS + L-NAME cells compared with LPS cells; nuclear run-on assays confirmed increased transcription of the corresponding genes. These results suggest that LPS-mediated NO synthesis is associated with S-nitrosylation of NF-kappa B p50 and inhibition of NF-kappa B-dependent DNA binding, promoter activity, and gene transcription. We conclude that NO can regulate gene transcription by S-nitrosylation of NF-kappa B.
Duke Scholars
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Related Subject Headings
- Protein Binding
- Promoter Regions, Genetic
- Nitric Oxide Synthase Type II
- Nitric Oxide Synthase
- Nitric Oxide
- NF-kappa B p50 Subunit
- NF-kappa B
- Mice
- Macrophages
- Macrophage Colony-Stimulating Factor
Citation
Published In
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Protein Binding
- Promoter Regions, Genetic
- Nitric Oxide Synthase Type II
- Nitric Oxide Synthase
- Nitric Oxide
- NF-kappa B p50 Subunit
- NF-kappa B
- Mice
- Macrophages
- Macrophage Colony-Stimulating Factor