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Titre du document / Document title

TNF-α-induced NF-κB/Re1A Ser276 phosphorylation and enhanceosome formation is mediated by an ROS-dependent PKAc pathway

Auteur(s) / Author(s)

JAMALUDDIN Mohammad ; WANG Shaofei ; BOLDOGH Istvan ; BING TIAN ; BRASIER Allan R. ;

Résumé / Abstract

Tumor necrosis factor-a (TNF-a) is a potent mediator of inflammation, inducing expression of a gene network mediated by NF-KB. Previously we found that TNF-α-induced reactive oxygen species (ROS) production is required for NF-KB action because antioxidants inhibited TNF-α-inducible IL-8 expression without affecting its nuclear translocation. Here, we further investigated this ROS pathway controlling NF-KB/RelA dependent gene expression. We observed that TNF-a enhanced ROS production ∼ 2-fold 20 min after stimulation and significantly increased oxidative DNA damage (8-oxoguanine lesions) over controls. Treatment with chemically unrelated antioxidants specifically inhibited expression of TNF-inducible NF-KB-dependent genes without producing detectable cytotoxicity or affecting GAPDH expression. We found that TNF-α-induced NF-KB/RelA Ser276 phosphorylation, a modification critical for its transcriptional activity, was inhibited by abrogation of the ROS signaling pathway, whereas NF-KB/RelA Ser536 phosphorylation was not. Interestingly, antioxidant treatment selectively inhibited TNF-α-induced catalytic activity of cAMP dependent protein kinase A (PKAc) but not mitogen-stress related kinase-1 (MSK1), kinases known to phosphorylate RelA at Ser276. Using PKAc inhibitors and siRNA mediated PKAc knockdown, TNF-α-induced Ser276 phosphorylation and IL-8 expression were both significantly reduced, indicating PKAc is required for RelA Ser276 phosphorylation. Consistently, a site mutation of Rel A (Ser276 to Ala) in RelA-deficient embryonic fibroblasts failed to activate IL-8 Luciferase activity in response to TNF-a. Furthermore, TNF-α-inducible NF-KB/RelA interaction with the co-activator CBP/p300, essential for enhanceosome formation, was attenuated by antioxidant treatment. Using chromatin immunoprecipitation assay (ChIP), we observed that recruitment of p300 and RNA polymerase II (Pol II) to the IL-8 promoter was also abrogated by antioxidant. These results indicate that the ROS-mediated TNF-α-induced IL-8 transcription is regulated by NF-KB/RelA phosphorylation at the critical Ser276 residue by PKAc, resulting in stable enhanceosome formation on target genes. These studies provide insight into a novel antioxidant-sensitive pathway that can be targeted to inhibit NF-KB-mediated inflammation.

Revue / Journal Title

Cellular signalling    ISSN  0898-6568 

Source / Source

2007, vol. 19, no7, pp. 1419-1433 [15 page(s) (article)]

Langue / Language

Anglais

Editeur / Publisher

Elsevier, Kidlington, ROYAUME-UNI  (1988) (Revue)

Mots-clés d'auteur / Author Keywords

NF-κB

;

RelA

;

Phosphorylation

;

Reactive oxygen species

;

Enhanceosome

;

IL-8

;

Localisation / Location

INIST-CNRS, Cote INIST : 21777, 35400016281231.0060

Nº notice refdoc (ud4) : 18828163



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