Peroxynitrite Is a Potent Inhibitor of NF-κB Activation Triggered by Inflammatory Stimuli in Cardiac and Endothelial Cell Lines

Sandra Levrand,Benoît Pesse,François Feihl,Bernard Waeber,Pal Pacher,Joëlle Rolli,Marie-Denise Schaller,Lucas Liaudet

doi:10.1074/jbc.m501977200

Abstract

Peroxynitrite is a potent oxidant and nitrating species proposed as a direct effector of myocardial damage in numerous cardiac pathologies. Whether peroxynitrite also acts indirectly, by modulating cell signal transduction in the myocardium, has not been investigated. Therefore, we examined a possible role for peroxynitrite on the activation of NF-kappaB, a crucial pro-inflammatory transcription factor, in cultured H9C2 cardiomyocytes. H9C2 cells were stimulated with tumor necrosis factor-alpha or lipopolysaccharide following a brief (20-min) exposure to peroxynitrite. NF-kappaB activation (phosphorylation and degradation of its inhibitor IkappaBalpha, nuclear translocation of NF-kappaB p65, and NF-kappaB DNA binding) triggered by lipopolysaccharide or tumor necrosis factor-alpha was abrogated by peroxynitrite. Peroxynitrite also inhibited NF-kappaB in two human endothelial cell lines activated with tumor necrosis factor-alpha or interleukin-1beta. These effects were related to oxidative but not nitrative chemistry and were still being observed while nitration was suppressed by epicatechin. The mechanism of NF-kappaB inhibition by peroxynitrite was a complete blockade of phosphorylation and activation of the upstream kinase IkappaB kinase (IKK) beta, required for canonical, pro-inflammatory NF-kappaB activation. At the same time, peroxynitrite activated phosphorylation of NF-kappaB-inducing kinase and IKKalpha, considered as part of an alternative, noncanonical NF-kappaB activation pathway. Suppression of IKKbeta-dependent NF-kappaB activation translated into a marked inhibition of the transcription of NF-kappaB-dependent genes by peroxynitrite. Thus, peroxynitrite has a dual effect on NF-kappaB, inhibiting canonical IKKbeta-dependent NF-kappaB activation while activating NF-kappaB-inducing kinase and IKKalpha phosphorylation, which suggests its involvement in an alternative pathway of NF-kappaB activation. These findings offer new perspectives for the understanding of the relationships between redox stress and inflammation.

Highlights

Peroxynitrite is a potent oxidant and nitrating species proposed as a direct effector of myocardial damage in numerous cardiac pathologies
Peroxynitrite Inhibits I␬B␣ Phosphorylation and Degradation and NF-␬B p65 Nuclear Translocation Elicited by LPS or TNF␣—Phosphorylation of I␬B␣, which targets I␬B␣ for degradation in the proteasome, allows inactive cytoplasmic NF-␬B dimers to translocate into the nucleus [1, 2]
PN has generally been considered as directly biotoxic toward cardiomyocytes, we provide the novel evidence that PN behaves as an important modulator of cell signal transduction at the level of NF-␬B

Summary

Introduction

Peroxynitrite is a potent oxidant and nitrating species proposed as a direct effector of myocardial damage in numerous cardiac pathologies. Peroxynitrite has a dual effect on NF-␬B, inhibiting canonical IKK␤-dependent NF-␬B activation while activating NF-␬B-inducing kinase and IKK␣ phosphorylation, which suggests its involvement in an alternative pathway of NF-␬B activation. These findings offer new perspectives for the understanding of the relationships between redox stress and inflammation. PN generation occurs during myocardial infarction [15], heart failure [16], and cardiomyopathy caused by anthracyclines [17] In such conditions, it is unknown whether PN only acts as a direct cytotoxic effector or may have further indirect effects related to the modulation of redox-sensitive cell signal transduction pathways such as NF-␬B. PN has a dual effect on NF-␬B, being an inhibitor of canonical IKK␤-dependent NF-␬B activation pathway while simultaneously activating the phosphorylation of NIK and IKK␣, suggesting its involvement in an alternative pathway of NF-␬B activation

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Results

Conclusion