title: Redox control of p53 in the transcriptional regulation of TGF-β1 target genes through SMAD cooperativity.
abstract: Transforming growth factor-β1 (TGF-β1) regulates the tissue response to injury and is the principal driver of excessive scarring leading to fibrosis and eventual organ failure. The TGF-β1 effectors SMAD3 and p53 are major contributors to disease progression. While SMAD3 is an established pro-fibrotic factor, the role of p53 in the TGF-β1-induced fibrotic program is not clear. p53 gene silencing, genetic ablation/subsequent rescue, and pharmacological inhibition confirmed that p53 was required for expression of plasminogen activator inhibitor-1 (PAI-1), a major TGF-β1 target gene and a key causative element in fibrotic disorders. TGF-β1 regulated p53 activity by stimulating p53(Ser15 and 9) phosphorylation and acetylation, promoting interactions with activated SMADs and subsequent binding of p53/SMAD3 to the PAI-1 promoter in HK-2 human renal tubular epithelial cells and HaCaT human keratinocytes. Immunohistochemistry revealed prominent co-induction of SMAD3, p53 and PAI-1 in the tubular epithelium of the obstructed kidney consistent with a potential in vivo role for p53 and SMADs in TGF-β1-driven renal fibrosis. TGF-β1-initiated phosphorylation of p53(Ser15) and up-regulation of expression of several pro-fibrotic genes, moreover, was dependent on the rapid generation of reactive oxygen species (ROS). shRNA silencing of the p22(Phox) subunit of NADP(H) oxidases in HK-2 cells partially attenuated (over 50%) p53(Ser15) phosphorylation and PAI-1 induction. These studies highlight the role of free radicals in p53 activation and subsequent pro-fibrotic reprogramming by TGF-β1 via the SMAD3-p53 transcriptional axis. Present findings provide a rationale for therapeutic targeting of SMAD3-p53 in aberrant TGF-β1 signaling associated with renal fibrosis.
authors.affiliation: Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, United States.
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Overstreet
authors.affiliation: Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, United States.
authors.forename: Rohan
authors.initials: R
authors.lastname: Samarakoon
authors.affiliation: Department of Urology, University of Florida School of Medicine, Gainesville, FL 32611, United States.
authors.forename: Kirstan K
authors.initials: KK
authors.lastname: Meldrum
authors.affiliation: Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, United States. Electronic address: higginp@mail.amc.edu.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Higgins
mesh_terms: D000107:Acetylation; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000378:metabolism; D005347:Fibroblasts; D005355:Fibrosis / Q000473:pathology*; D005786:Gene Expression Regulation; D006801:Humans; D015603:Keratinocytes; D007674:Kidney Diseases / Q000473:pathology; D007684:Kidney Tubules / Q000166:cytology; D051379:Mice; D018345:Mice, Knockout; D019255:NADPH Oxidases / Q000235:genetics; D010766:Phosphorylation; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D051900:Smad3 Protein / Q000378:metabolism*; D015533:Transcriptional Activation; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D017395:Plasminogen Activator Inhibitor 1; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C051321:SERPINE1 protein, human; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; D016159:Tumor Suppressor Protein p53; D019255:NADPH Oxidases; C086986:CYBA protein, human
keywords: Chromatin immunoprecipitation; Gene expression; PAI-1; Reactive oxygen species; SMADs; TGF-β1; Tissue fibrosis; Transcription; p53