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

Tumor necrosis factor-alpha, (TNF-alpha) is a potent mediator of inflammation, inducing expression of a gene network mediated by NF-kappa B. Previously we found that TNF-alpha-induced reactive oxygen species (ROS) production is required for NF-kappa B action because antioxidants inhibited TNF-alpha-inducible IL-8 expression without affecting its nuclear translocation. Here, we further investigated this ROS pathway controlling NF-kappa B/RelA dependent gene expression. We observed that TNF-alpha 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-kappa B-dependent genes without producing detectable cytotoxicity or affecting GAPDH expression. We found that TNF-alpha-induced NF-KB/RelA Ser 276 phosphorylation, a modification critical for its transcriptional activity, was inhibited by abrogation of the ROS signaling pathway, whereas NF-kappa B/RcIA Ser5(36) phosphorylation was not. Interestingly, antioxidant treatment selectively inhibited TNF-alpha-induced catalytic activity of cAMP dependent protein kinase A (PKAc) but not mitogen-stress related kinase-1 (MSK1), kinases known to phosphorylate RelA at Ser 276. Using PKAc inhibitors and siRNA mediated PKAc knockdown, TNF-alpha-induced Ser 276 phosphorylation and IL-8 expression were both significantly reduced, indicating PKAc is required for RelA Ser 276 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-alpha. Furthermore, TNF-alpha-inducible NF-kappa B/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-alpha-induced IL-8 transcription is regulated by NF-kappa B/RelA phosphorylation at the critical Ser 276 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-kappa B-mediated inflammation. (c) 2007 Elsevier Inc. All rights reserved.