TNF-alpha-induced NF-kappaB/RelA Ser(276) 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-kappaB. Previously we found that TNF-alpha-induced reactive oxygen species (ROS) production is required for NF-kappaB action because antioxidants inhibited TNF-alpha-inducible IL-8 expression without affecting its nuclear translocation. Here, we further investigated this ROS pathway controlling NF-kappaB/RelA dependent gene expression. We observed that TNF-alpha enhanced ROS production approximately 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-kappaB-dependent genes without producing detectable cytotoxicity or affecting GAPDH expression. We found that TNF-alpha-induced NF-kappaB/RelA Ser(276) phosphorylation, a modification critical for its transcriptional activity, was inhibited by abrogation of the ROS signaling pathway, whereas NF-kappaB/RelA Ser(536) 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 (Ser(276) to Ala) in RelA-deficient embryonic fibroblasts failed to activate IL-8 Luciferase activity in response to TNF-alpha. Furthermore, TNF-alpha-inducible NF-kappaB/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-kappaB/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-kappaB-mediated inflammation.
Jamaluddin
Mohammad
M
Department of Medicine, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1060, USA. mjamalud@utmb.edu
Wang
Shaofei
S
Boldogh
Istvan
I
Tian
Bing
B
Brasier
Allan R
AR
D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010768:Phosphoserine / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D051996:Transcription Factor RelA / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D020298:U937 Cells; D050880:p300-CBP Transcription Factors / Q000378:metabolism
D000975:Antioxidants; D016209:Interleukin-8; D017382:Reactive Oxygen Species; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D010768:Phosphoserine; D050880:p300-CBP Transcription Factors; D017868:Cyclic AMP-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D004121:Dimethyl Sulfoxide