Endotoxin-mediated S-nitrosylation of p50 alters NF-kappa B-dependent gene transcription in ANA-1 murine macrophages.

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • delaTorre, A; Schroeder, RA; Punzalan, C; Kuo, PC

Published Date

  • April 1, 1999

Published In

Volume / Issue

  • 162 / 7

Start / End Page

  • 4101 - 4108

PubMed ID

  • 10201934

International Standard Serial Number (ISSN)

  • 0022-1767

Language

  • eng

Conference Location

  • United States