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

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.