p47phox participates in activation of RelA in endothelial cells

Ying Gu, You Cheng Xu, Ru Feng Wu, Fiemu E. Nwariaku, Rhonda F. Souza, Sonia C. Flores, Lance S. Terada

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Activation of endothelial cell NF-κB by interleukin (IL)-1 constitutes an event critical to the progression of the innate immune response. In this context, oxidants have been associated with NF-κB activation, although the molecular source and mechanism of targeting have remained obscure. We found that RelA, essential for NF-κB activation by IL-1, was associated with the NADPH oxidase adapter protein p47phox in yeast two-hybrid, coprecipitation, and in vitro binding studies. RelA and p47-GFP also colocalized in endothelial cells in focal submembranous dorsoventral protrusions. Overexpression of p47phox synergized with IL-1β in the activation of an artificial κB-luciferase reporter and specifically augmented IL-1β-induced RelA transactivation activity. p47phox overexpression also greatly increased IL-1β-stimulated RelA phosphorylation, whereas it had no effect on I-κB degradation or on RelA nuclear translocation of κB binding. The tandem SH3 domains of p47phox were found to associate with a proline-rich mid-region of RelA (RelA-PR) located between the Rel homology and transactivation domains. The RelA-PR peptide blocked interaction of p47phox and RelA, and ectopic expression of RelA-PR abrogated IL-1β-induced transactivation of the NF-κB-dependent E-selectin promoter. Further, suppression of NADPH oxidase function through the inhibitor diphenylene iodonium, the superoxide dismutase mimetic Mn(III) tetrakis(4-benzoic acid)porphyrin (MnTBAP), or expression of a dominant interfering mutant of a separate NADPH oxidase subunit (p67(V204A)) decreased IL-1β-induced E-selectin promoter activation, suggesting that p47phox facilitates NF-κB activation through linkage with the NADPH oxidase. IL-1β rapidly increased tyrosine phosphorylation of IL-1 type I receptor-associated proteins, suggesting that oxidants may operate through inactivation of local protein-tyrosine phosphatases in the proximal IL-1β signaling pathway leading to RelA activation.

Original languageEnglish (US)
Pages (from-to)17210-17217
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number19
DOIs
StatePublished - May 9 2003

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Endothelial cells
Interleukin-1
Endothelial Cells
Chemical activation
NADPH Oxidase
Transcriptional Activation
Phosphorylation
E-Selectin
Oxidants
Interleukin-1 Type I Receptors
Protein Tyrosine Phosphatases
src Homology Domains
Coprecipitation
Luciferases
Innate Immunity
Proline
Yeast
Superoxide Dismutase
Tyrosine
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

p47phox participates in activation of RelA in endothelial cells. / Gu, Ying; Xu, You Cheng; Wu, Ru Feng; Nwariaku, Fiemu E.; Souza, Rhonda F.; Flores, Sonia C.; Terada, Lance S.

In: Journal of Biological Chemistry, Vol. 278, No. 19, 09.05.2003, p. 17210-17217.

Research output: Contribution to journalArticle

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abstract = "Activation of endothelial cell NF-κB by interleukin (IL)-1 constitutes an event critical to the progression of the innate immune response. In this context, oxidants have been associated with NF-κB activation, although the molecular source and mechanism of targeting have remained obscure. We found that RelA, essential for NF-κB activation by IL-1, was associated with the NADPH oxidase adapter protein p47phox in yeast two-hybrid, coprecipitation, and in vitro binding studies. RelA and p47-GFP also colocalized in endothelial cells in focal submembranous dorsoventral protrusions. Overexpression of p47phox synergized with IL-1β in the activation of an artificial κB-luciferase reporter and specifically augmented IL-1β-induced RelA transactivation activity. p47phox overexpression also greatly increased IL-1β-stimulated RelA phosphorylation, whereas it had no effect on I-κB degradation or on RelA nuclear translocation of κB binding. The tandem SH3 domains of p47phox were found to associate with a proline-rich mid-region of RelA (RelA-PR) located between the Rel homology and transactivation domains. The RelA-PR peptide blocked interaction of p47phox and RelA, and ectopic expression of RelA-PR abrogated IL-1β-induced transactivation of the NF-κB-dependent E-selectin promoter. Further, suppression of NADPH oxidase function through the inhibitor diphenylene iodonium, the superoxide dismutase mimetic Mn(III) tetrakis(4-benzoic acid)porphyrin (MnTBAP), or expression of a dominant interfering mutant of a separate NADPH oxidase subunit (p67(V204A)) decreased IL-1β-induced E-selectin promoter activation, suggesting that p47phox facilitates NF-κB activation through linkage with the NADPH oxidase. IL-1β rapidly increased tyrosine phosphorylation of IL-1 type I receptor-associated proteins, suggesting that oxidants may operate through inactivation of local protein-tyrosine phosphatases in the proximal IL-1β signaling pathway leading to RelA activation.",
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T1 - p47phox participates in activation of RelA in endothelial cells

AU - Gu, Ying

AU - Xu, You Cheng

AU - Wu, Ru Feng

AU - Nwariaku, Fiemu E.

AU - Souza, Rhonda F.

AU - Flores, Sonia C.

AU - Terada, Lance S.

PY - 2003/5/9

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