Specificity protein 1-dependent p53-mediated suppression of human manganese superoxide dismutase gene expression.
Manganese superoxide dismutase (MnSOD) is a primary antioxidant enzyme necessary for the survival of aerobic life. Previously, we demonstrated that specificity protein 1 (Sp1) is essential for the basal transcription of the MnSOD gene. We also identified nucleophosmin (NPM), an RNA-binding protein, as an important co-activator of NF-kappaB in the induction of MnSOD by cytokine and tumor promoter. Here, using chromatin immunoprecipitation (ChIP) analysis, we demonstrate that Sp1 and NPM interact in vivo to enhance NF-kappaB-mediated MnSOD induction. Interaction between NPM and Sp1 or NF-kappaB at the promoter and enhancer of the MnSOD gene in vivo were verified by the presence of the PCR products from the promoter and enhancer elements in the ChIP assay. Unexpectedly, we also found p53, another transcription factor, to be a component of the complex detected by ChIP assay. The presence of p53 in this transcription complex was verified by immunoprecipitation of p53 proteins with antibody to Sp1 in nuclear extracts. Using a vector expressing full-length p53 cDNA, we demonstrated that p53 overexpression suppresses MnSOD mRNA and protein levels. Consistent with the negative role of p53 in the expression of the MnSOD gene, expression of small interfering RNA for p53 leads to an increase of MnSOD mRNA and protein levels. Using ChIP assays and immunoprecipitation, we further demonstrated that p53 interacts with Sp1 to suppress both the constitutive and 12-O-tetradecanoylphorbol-13-acetate-stimulated expression of the MnSOD gene. Inhibition of the MnSOD gene by p53 was abolished when Sp1 sites on the MnSOD promoter were mutated or when the Sp1 protein was reduced by siRNA approaches. Because expression of MnSOD protects against cell death, our findings reveal a previously unrecognized mechanism of p53-mediated cell death and demonstrate an intricate relationship between the positive and negative control of MnSOD expression.
Dhar
Sanjit Kumar
SK
Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536.
Xu
Yong
Y
Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536.
Chen
Yumin
Y
Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536.
St Clair
Daret K
DK
Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536. Electronic address: DSTL00@pop.uky.edu.
D016923:Cell Death; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D046912:Multiprotein Complexes; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D000090243:Nucleophosmin; D034741:RNA, Small Interfering / Q000494:pharmacology; D016329:Sp1 Transcription Factor / Q000378:metabolism / Q000502:physiology*; D013482:Superoxide Dismutase / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D046912:Multiprotein Complexes; D016328:NF-kappa B; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; D016159:Tumor Suppressor Protein p53; D000090243:Nucleophosmin; D013482:Superoxide Dismutase