PMID- 17317104 OWN - NLM STAT- MEDLINE DCOM- 20070807 LR - 20161019 IS - 0898-6568 (Print) IS - 0898-6568 (Linking) VI - 19 IP - 7 DP - 2007 Jul TI - TNF-alpha-induced NF-kappaB/RelA Ser(276) phosphorylation and enhanceosome formation is mediated by an ROS-dependent PKAc pathway. PG - 1419-33 AB - 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. FAU - Jamaluddin, Mohammad AU - Jamaluddin M AD - Department of Medicine, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1060, USA. mjamalud@utmb.edu FAU - Wang, Shaofei AU - Wang S FAU - Boldogh, Istvan AU - Boldogh I FAU - Tian, Bing AU - Tian B FAU - Brasier, Allan R AU - Brasier AR LA - eng GR - P01 AI062885/AI/NIAID NIH HHS/United States GR - P30 ES 06676/ES/NIEHS NIH HHS/United States GR - P01 AG021830/AG/NIA NIH HHS/United States GR - P30 ES006676/ES/NIEHS NIH HHS/United States GR - R01 AI 40218/AI/NIAID NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20070125 PL - England TA - Cell Signal JT - Cellular signalling JID - 8904683 RN - 0 (Antioxidants) RN - 0 (Interleukin-8) RN - 0 (Reactive Oxygen Species) RN - 0 (Transcription Factor RelA) RN - 0 (Tumor Necrosis Factor-alpha) RN - 17885-08-4 (Phosphoserine) RN - EC 2.3.1.48 (p300-CBP Transcription Factors) RN - EC 2.7.11.11 (Cyclic AMP-Dependent Protein Kinases) RN - EC 2.7.11.24 (Mitogen-Activated Protein Kinases) RN - YOW8V9698H (Dimethyl Sulfoxide) SB - IM MH - Animals MH - Antioxidants/metabolism MH - Cell Nucleus/drug effects/metabolism MH - Cyclic AMP-Dependent Protein Kinases/*metabolism MH - Dimethyl Sulfoxide/pharmacology MH - Enzyme Activation/drug effects MH - Humans MH - Interleukin-8/metabolism MH - Mice MH - Mitogen-Activated Protein Kinases/metabolism MH - Phosphoserine/*metabolism MH - Protein Binding/drug effects MH - Protein Transport/drug effects MH - Reactive Oxygen Species/*metabolism MH - Signal Transduction/drug effects MH - Transcription Factor RelA/*metabolism MH - Transcription, Genetic/drug effects MH - Tumor Necrosis Factor-alpha/*pharmacology MH - U937 Cells MH - p300-CBP Transcription Factors/metabolism EDAT- 2007/02/24 09:00 MHDA- 2007/08/08 09:00 CRDT- 2007/02/24 09:00 PHST- 2006/11/25 00:00 [received] PHST- 2007/01/12 00:00 [revised] PHST- 2007/01/15 00:00 [accepted] PHST- 2007/02/24 09:00 [pubmed] PHST- 2007/08/08 09:00 [medline] PHST- 2007/02/24 09:00 [entrez] AID - S0898-6568(07)00034-4 [pii] AID - 10.1016/j.cellsig.2007.01.020 [doi] PST - ppublish SO - Cell Signal. 2007 Jul;19(7):1419-33. doi: 10.1016/j.cellsig.2007.01.020. Epub 2007 Jan 25.