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

Mohammad Jamaluddin, Shaofei Wang, Istvan Boldogh, Bing Tian, Allan R. Brasier

Research output: Contribution to journalArticle

144 Scopus citations

Abstract

Tumor necrosis factor-α (TNF-α) is a potent mediator of inflammation, inducing expression of a gene network mediated by NF-κB. Previously we found that TNF-α-induced reactive oxygen species (ROS) production is required for NF-κB action because antioxidants inhibited TNF-α-inducible IL-8 expression without affecting its nuclear translocation. Here, we further investigated this ROS pathway controlling NF-κB/RelA dependent gene expression. We observed that TNF-α 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-κB-dependent genes without producing detectable cytotoxicity or affecting GAPDH expression. We found that TNF-α-induced NF-κB/RelA Ser276 phosphorylation, a modification critical for its transcriptional activity, was inhibited by abrogation of the ROS signaling pathway, whereas NF-κB/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-α. Furthermore, TNF-α-inducible NF-κB/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-κB/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-κB-mediated inflammation.

Original languageEnglish (US)
Pages (from-to)1419-1433
Number of pages15
JournalCellular Signalling
Volume19
Issue number7
DOIs
StatePublished - Jul 1 2007

Keywords

  • Enhanceosome
  • IL-8
  • NF-κB
  • Phosphorylation
  • Reactive oxygen species
  • RelA

ASJC Scopus subject areas

  • Cell Biology

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