Induction and Stabilization of IB by Nitric Oxide Mediates Inhibition of NF-B

doi:10.1074/jbc.270.23.14214

Induction and Stabilization of IB by Nitric Oxide Mediates Inhibition of NF-B (*)

From the (1) Cardiovascular Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Masssachusetts 02115

^§ To whom correspondence should be addressed:
Cardiovascular Division, Dept. of Medicine, 221 Longwood Ave., LMRC-307, Boston, MA 02115.
Tel.: 617-732-6538; Fax: 617-732-6961.

Abstract

To determine the mechanism(s) by which the endogenous mediator nitric oxide (NO) inhibits the activation of transcription factor NF-κB, we stimulated human vascular endothelial cells with tumor necrosis factor-α in the presence of two NO donors, sodium nitroprusside and S-nitrosoglutathione. Electrophoretic mobility shift assays demonstrated that both NO donors inhibited NF-κB activation by tumor necrosis factor-α. This effect was not mediated by guanylyl cyclase activation since the cGMP analogue 8-bromo-cGMP had no similar effect. Inhibition of endogenous constitutive NO production by L-N-monomethylarginine, however, activated NF-κB, suggesting tonic inhibition of NF-κB under basal conditions. NO had little or no effects on other nuclear binding proteins such as AP-1 and GATA. Immunoprecipitation studies showed that NO stabilized the NF-κB inhibitor, IκBα, by preventing its degradation from NF-κB. NO also increased the mRNA expression of IκBα, but not NF-κB subunits, p65 or p50, and transfection experiments with a chloramphenicol acetyltransferase reporter gene linked to the IκBα promoter suggested transcriptional induction of IκBα by NO. We propose that the induction and stabilization of IκBα by NO are important mechanisms by which NO inhibits NF-κB and attenuate atherogenesis.

Footnotes

↵* This work was supported by National Institutes of Health Grants HL02508 (to J. K. L.) and HL34636 (to P. L.) and an American Heart Association grant-in-aid (to J. K. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PDTC

pyrrolidine dithiocarbamate

EMSA

electrophoretic mobility shift assay

GSNO

S-nitrosoglutathione

CAT

chloramphenicol acetyltransferase

TNFα

tumor necrosis factor

kb

kilobase pair(s)

PBS

phosphate-buffered saline

L-NMA

L-N-monomethylarginine.