Specificity Protein 1-dependent p53-mediated Suppression of Human Manganese Superoxide Dismutase Gene Expression

Abstract

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-κB 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-κB-mediated MnSOD induction. Interaction between NPM and Sp1 or NF-κB 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. 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-κB 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-κB-mediated MnSOD induction. Interaction between NPM and Sp1 or NF-κB 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. Oxidative stress caused by reactive oxygen species is detoxified by intracellular antioxidant enzymes such as manganese superoxide dismutase (MnSOD), 2The abbreviations used are: MnSOD, manganese superoxide dismutase (SOD); hMnSOD, human MnSOD; NPM, nucleophosmin; NF-κB, nuclear factor κB; Sp1, specificity protein 1; TPA, 12-O-tetradecanoylphorbol-13-acetate; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; RT, reverse transcriptase; MOPS, 4-morpholinepropanesulfonic acid; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; I2E, intronic enhancer fragment. catalase, and glutathione. Among these primary antioxidant enzymes, MnSOD is the enzyme that must function in order for all aerobic organisms to survive. MnSOD is a highly regulated SOD encoded by the sod2 located in human chromosome 6q25.3 (1Dougall W.C. Nick H.S. Endocrinology. 1991; 129: 2379-2384Crossref Scopus (117) Google Scholar, 2Suzuki K. Tatsumi H. Satoh S. Sendra T. Naskata T. Fuji J. Takiguchi N. Am. J. Physiol. 1993; 265: H1173-H1178PubMed Google Scholar). The critical role of MnSOD as a cytoprotective enzyme is illustrated in both MnSOD knock-out and transgenic animal models. For instance, MnSOD knock-out mice develop cardiomyopathy and die within a few days after birth (3Li Y. Huang T.T. Carison E.J. Melov S. Ursell P.C. Olson J.L. Noble L.J. Yoshimura M.P. Berger C. Chan P.H. Nat. Genet. 1995; 11: 376-381Crossref PubMed Scopus (1462) Google Scholar). MnSOD knock-out mice treated with a SOD mimetic were protected from systemic toxicity and from neonatal death (4Melov S. Schneider J.A. Day B.J. Hinerfeld D. Coskun P. Mirra S.S. Crapo J.D. Wallace D.C. Nat. Genet. 1998; 18: 159-163Crossref PubMed Scopus (441) Google Scholar). Conversely, transgenic mice overexpressing human MnSOD experienced less injury resulting from inflammation (5Wispe J.R. Warner B.B. Clark J.C. Dey C.R. Neuman J. Glasser S.W. Crapo J.D. Chang L. Whitselt J.A. J. Biol. Chem. 1992; 267: 23937-23941Abstract Full Text PDF PubMed Google Scholar), cardio-toxic drugs (6Baker K. Marcus C.B. Huffman K. Kruk H. Malfroy B. Doctrow S.R. J. Pharmacol. Exp. Ther. 1998; 284: 215-221PubMed Google Scholar), and pathological and physiological conditions leading to brain injury (7Keller J.N. Kindy M.S. Holtsberg F.W. St. Clair D.K. Yen H.-C. Germeyer A.M. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar). The human MnSOD gene is a single-copy gene consisting of five exons interrupted by four introns with a typical splice junction (8Wan X.S. Devalaraja M.N. St. Clair D.K. DNA Cell Biol. 1994; 13: 1127-1136Crossref PubMed Scopus (195) Google Scholar). The sod2 from human, bovine, rat, and mouse share more than 90% homology in the coding sequence. The basal promoter of the sod2 has multiple transcription factor binding motifs containing Sp1 and Ap-2 binding sites. Functional studies in different cell lines with different levels of Sp1 and Ap-2 proteins suggest that cellular levels of these proteins differentially regulate the expression of the human MnSOD gene. Transcription factor Sp1 is essential and sufficient, whereas Ap-2 is unnecessary and antagonistic to the constitutive expression of the gene (9Xu Y. Porntadavity S. St. Clair D.K. Biochem. J. 2002; 362: 401-412Crossref PubMed Scopus (111) Google Scholar). Sp1 is a prototype member of a small family of transcription factors (Sp1, Sp2, Sp3, and Sp4) with homologous functional domains. Members of this family have an inhibitory domain in the N termini and four transcriptional activation domains (A, B, C, and D) (10Murata Y. Kim H.G. Rogers K.T. Udvadia A.J. Horowitz J.M. J. Biol. Chem. 1994; 269: 20674-20681Abstract Full Text PDF PubMed Google Scholar). Transcriptional domains A and B are required for general and full activation, whereas domain C possesses very low activation (11Coursey A.J. Tjian R. Cell. 1988; 55: 887-898Abstract Full Text PDF PubMed Scopus (1079) Google Scholar), and domain D is required for synergistic activation (12Pascal E. Tjian R. Genes Dev. 1991; 5: 1646-1656Crossref PubMed Scopus (356) Google Scholar). The Sp1 protein is capable of inducing homotypic, Sp1-Sp1 interaction (13Su W. Jackson S. Tjian R. Echols H. Genes Dev. 1991; 5: 820-826Crossref PubMed Scopus (259) Google Scholar) or forming heterotypic interactions with different classes of nuclear proteins such as TATA box-binding protein (TBP) (14Emili A. Greenblatt J. Ingeles C.J. Mol. Cell Biol. 1994; 14: 1582-1593Crossref PubMed Scopus (226) Google Scholar), C/EBP (15Lee Y.H. Williams S.C. Baer M. Sterneck E. Gonzalaz F.J. Johnson P.F. Mol. Cell Biol. 1997; 17: 2038-2047Crossref PubMed Google Scholar), and YY1 (16Seto E. Lewis B. Shenk T. Nature. 1993; 365: 462-464Crossref PubMed Scopus (249) Google Scholar). Because the MnSOD promoter does not contain a TATA or CAAT binding transcription of the MnSOD gene is on Sp1-Sp1 The sod2 of mice and enhancer elements in the of the gene D. J.M. Mol. Cell Biol. 1997; 17: PubMed Scopus Google Scholar, Y. Devalaraja M.N. H. E.J. St. Clair D.K. DNA Cell Biol. 18: PubMed Scopus Google Scholar). Functional of enhancer elements demonstrate that the intronic of NF-κB, and transcription factor binding sites. The activation of NF-κB is essential not for the induction of MnSOD by and tumor promoter. NF-κB and the of and B. in to a of NF-κB DNA binding sites with different within the gene M. 1993; PubMed Scopus Google Scholar, R. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). the role of Sp1 in the expression of MnSOD in is S. Y. St. Clair D.K. DNA Cell Biol. PubMed Scopus Google Scholar), the role of p53 in the expression of MnSOD is p53 is a transcriptional that an important role in tumor to p53-mediated function cellular are cell or death, both of the of containing is that the function is in by the of p53 that is a positive transcriptional of such as K. K. H. H. K. 2002; 17: Google Scholar). has also that p53 function as a negative of gene the expression of the of MnSOD D. B. B. M. PubMed Scopus Google Scholar). the mechanism by p53 gene is We have previously demonstrated that the tumor promoter MnSOD expression by activation of Sp1 and that NPM as a co-activator of NF-κB in the induction of MnSOD by cytokine and this we the by p53 MnSOD and demonstrate that p53 a negative role on MnSOD gene transcription by forming a complex with Sp1, leading to the of MnSOD gene transcription both constitutive and Cell human cell was from were in at in consisting of with and all were from antibody and were from and MnSOD and chromatin immunoprecipitation (ChIP) were from were from a p53 expression was by of The expression vector was from M. Horowitz were from of containing a was used to the to MnSOD basal promoter by this PCR with and sites were for at the of the gene. intronic enhancer containing the human MnSOD gene was used as the for PCR with a a The PCR was to the of the to basal promoter containing the of the gene. The were used to the reverse reverse of the human MnSOD basal promoter to the was the vector containing the gene. were of the human MnSOD basal promoter to a was expression Sp1 expression vector was in and were The presence of was by and of Sp1 binding sites in the MnSOD promoter were to our (9Xu Y. Porntadavity S. St. Clair D.K. Biochem. J. 2002; 362: 401-412Crossref PubMed Scopus (111) Google Scholar), in the resulting mutated sites were using to the and by The to a of was The mutated to with the mutated in were as in the were between and sites. of were and in the binding sites were by DNA The sites were used to for the sites. and were in with and with were with after a as by the were with DNA containing the or basal promoter of the human MnSOD gene in the gene. containing was also with vector as an control at a of after the were with and in were and in a at a of an were treated with the were with and in p53 expression vector was with MnSOD promoter containing vector in Sp1 expression vector and p53 expression vector or control were with MnSOD promoter containing vector in p53 and Sp1 siRNA were in to were treated with a for in the as the were in cell were and the were with the in with the in a were from A of was and at for at to cell were in A containing and with the of at a of 1 the and were The cell was on for of was and the was for The and nuclear were by at at for the nuclear were in B containing and at a of 1 and on for proteins in the were by at at for studies were with nuclear from or in 1 The used for immunoprecipitation were and mouse of nuclear was at with of with the of protein was to the of the antibody and nuclear and at were by at for by with the all the with the protein were were in the to and a the proteins were detected by ChIP were on a for and treated with and for to DNA was by to to a of were using containing 1 and 1 were by at for at and in containing and chromatin was to DNA on to between to chromatin was at for to the The protein of the was of chromatin from was in ChIP and The chromatin was by of chromatin was with the NPM and Sp1 control was by and chromatin were to and were at and with the of for at with to the were by at with 1 of in the order of low complex complex by with 1 The were by or For analysis, to the were for with of and to proteins were detected by For PCR were for at with of of was by the at for in the presence of of were with by for 1 at in the presence of DNA was by and by in for PCR DNA was by PCR For of enhancer elements of the MnSOD gene, the and to and to and For the was and reverse were We also the transcription factor binding of the of MnSOD gene using and reverse from to of NF-κB and Sp1 were with and The were on and in of containing and 1 The of was and at were used within after of nuclear protein and of binding containing and of were were for at the of the complex to the after were by 1 of the primary antibody to the binding and the to 1 at were from on from control or treated were used to the levels of transcription factors or For cellular were by were with and to and to a The was by with The was with the The were by in and for at 1 at a with the were in the primary antibody for at at or at The primary were in containing in a of The were for with The were with the antibody at a of for at The were with for and with for were 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levels of Sp1, NPM, and NF-κB family in when both control and treated nuclear were to and using to protein, the of Sp1, and NPM not or in the after with the increase in NF-κB and Sp1 levels is by an increase in DNA binding we NF-κB and Sp1 DNA binding using nuclear from DNA binding of both NF-κB and Sp1 a and the of the interactions between Sp1 and we the cellular protein with cellular DNA in vivo by using The were to chromatin immunoprecipitation using antibody to a member of the NF-κB family of transcription factors in The of chromatin immunoprecipitation was by PCR and For analysis, chromatin were to and with an antibody for member of the NF-κB in an antibody to was to and not and B, and the interaction with or after chromatin immunoprecipitation using Sp1 antibody was to and The levels of or NPM after Unexpectedly, we also found p53 protein with Sp1 antibody that p53 a role in MnSOD For PCR of the MnSOD these proteins to we DNA protein and DNA for PCR Using the DNA as the both the promoter and intronic enhancer were detected DNA that was from chromatin immunoprecipitation using not PCR Because we previously identified NPM as a co-activator of NF-κB in the induction of MnSOD by cytokine and TPA, we also ChIP with NPM to the promoter and enhancer elements of the MnSOD gene. ChIP that NPM is with both the promoter and enhancer elements of the MnSOD gene. further the specificity of transcription factors binding to the promoter and enhancer we the DNA from the ChIP DNA using the between to of the MnSOD gene. in we were to MnSOD using ChIP DNA were to of the gene using the of DNA that was used as p53 with p53 was identified in complex with Sp1, we the that p53 be a component of the transcription complex the expression of the Using against p53, we found that p53 antibody was to Sp1 and NPM not that p53 not interact with NF-κB family Consistent with this and Sp1 not p53 and was not to or p53 The interaction between and and Sp1 after a and that Sp1 with p53 in and the interaction proteins within the promoter and enhancer sites. p53 Transcription of the MnSOD p53 a role in MnSOD gene we p53 in using the vector expressing were with a MnSOD promoter to and p53 expression of p53 to an increase of p53 protein with the vector of p53 to a in whereas with the vector on gene The transcription of the MnSOD gene was also after of for with the the role of p53 on the gene, we p53 in and the mRNA levels were by in p53 overexpression p53 mRNA levels and mRNA levels. Consistent with the on MnSOD p53 overexpression to protein levels with in the vector control mRNA and protein levels of after the induction of MnSOD by was reduced in p53 with vector treated B and p53 siRNA further the role of p53 in the expression of MnSOD, we used the siRNA were with the control siRNA or p53 siRNA, and the and mRNA levels were of p53 by expression of p53 siRNA a on MnSOD gene transcription as by gene and MnSOD mRNA levels A and of p53 siRNA the p53 mRNA not the gene p53 siRNA expression also the mRNA levels of a p53 gene, whereas mRNA further the p53 on we p53 expression vector in and the p53 gene expression. p53 overexpression mRNA expression further that p53 are regulated by p53 in p53 with Sp1 and NPM at we not the interaction of p53 with NF-κB Sp1 and NPM both interact with NF-κB to enhance MnSOD expression. p53 with MnSOD expression at the promoter or enhancer we p53 in and a ChIP to the of NPM and Sp1 to the of the MnSOD gene. ChIP with Sp1 that Sp1 and p53 in in both and and after with control in after with control in p53 more Sp1 and NPM in the of whereas of NPM with Sp1 or p53 suggest that in p53 with Sp1 when NPM is to an protein such as NF-κB family p53 interaction is at the promoter or enhancer we a ChIP with p53 antibody in vector or immunoprecipitation with p53 antibody and PCR of promoter to were as in we not the intronic enhancer using the DNA as we positive from that p53 interaction with Sp1 in the promoter not at the enhancer p53 interaction in the MnSOD promoter is Sp1 we Sp1 siRNA with p53 expression vector and a ChIP using a p53 The in that Sp1 siRNA p53 the promoter whereas Sp1 siRNA does not have on the p53 gene such as promoter that the of p53 on MnSOD transcription is of MnSOD by p53 in a we previously demonstrated that Sp1 is essential and for the basal promoter of MnSOD (9Xu Y. Porntadavity S. St. Clair D.K. Biochem. J. 2002; 362: 401-412Crossref PubMed Scopus (111) Google Scholar). further the role of p53 on MnSOD promoter activation, we used MnSOD promoter and with p53 expression vector Consistent with our of five sites in the to promoter caused a of basal and levels of gene of Sp1 sites in MnSOD promoter a low of transcriptional this of MnSOD transcriptional is reduced with MnSOD promoter that the of p53 on MnSOD transcription is on Sp1 sites in the promoter. p53 to the Sp1 and for the binding with Sp1, we with nuclear from of p53 not the Sp1 binding basal or with p53 proteins using Sp1 that p53 was to to the Sp1 of MnSOD by p53 further that p53 leading to the of MnSOD gene transcription is on the of Sp1, we p53 in with reduced levels of Sp1 using Sp1 Sp1 siRNA Sp1 protein, and p53 overexpression p53 protein levels of p53 with control siRNA MnSOD and of p53 with Sp1 siRNA both the basal and on MnSOD promoter. further that of MnSOD transcription by p53 is we p53 in with levels of Sp1 using Sp1 expression The expression of Sp1 and p53 proteins was by of nuclear of Sp1 MnSOD transcription in a of p53 reduced both the basal and MnSOD transcription p53 overexpression to a of MnSOD transcription of MnSOD in to conditions is the mechanism by transcription of the MnSOD gene is is not gene transcriptional is highly on transcription factors such as NF-κB and We and have demonstrated that NF-κB is essential for the induction of sod2 in to TPA, tumor and D. 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St. Clair PubMed Scopus Google Scholar). the using ChIP and assays we that Sp1, and NPM are of the transcription factor complex MnSOD expression at the chromatin We also identified p53 as a component of this complex that the positive of Sp1 on MnSOD gene The presence of p53 in the MnSOD promoter complex was verified by the in the of PCR products that p53 interacts with these transcription factors at the We have also previously identified NPM as a co-activator of NF-κB in the induction of MnSOD by C. St. Clair D.K. J. Biol. Chem. 1994; Full Text Full Text PDF Scopus Google Scholar). has that NPM to p53 and p53 from S. A. 1998; PubMed Scopus Google Scholar). this we that NPM with p53 at the as detected by and ChIP assays and and that the of p53 after not is to that p53 with NPM p53 and p53 interaction with the p53 binding sites in the MnSOD gene are not a of the MnSOD gene that a p53 binding is located at of MnSOD promoter with a The is located at to Sp1 binding sites. that p53 to the and the of Sp1 to with DNA by the Sp1 this was we not p53 binding to the promoter containing this not when we p53 protein in the of using Sp1 we found that p53 not to the Sp1 Sp1 binding to P. P. A. S. M. S. L. S. L.J. M. M. PubMed Scopus Google Scholar) that the p53 transcription factor binding is at of the transcription in the human MnSOD gene. the promoter used in our of the the of p53 binding to the MnSOD is also these that is not that the of p53 on the MnSOD gene is to p53 binding to MnSOD promoter to p53 interaction with Sp1 The mechanism by p53 MnSOD transcription is not have for p53-mediated transcriptional with transcription P. J. J.L. C. PubMed Scopus Google Scholar), binding of p53 A.J. Mol. Cell Biol. PubMed Scopus Google Scholar), and complex with proteins M. J. A.J. Genes Dev. 13: PubMed Scopus Google Scholar). mechanism by the p53 with at the promoter and interfering with the function of the by of a complex leading to the of gene transcription J. S. Cell PubMed Scopus Google Scholar). For instance, the of complex was to be the mechanism of promoter Y. A. M. J. M. Y. Mol. Cell Biol. 1998; 18: PubMed Scopus Google Scholar). have that p53 interfering with basal transcription and the binding K. M. W. J. K. PubMed Scopus Google Scholar). has also that the of chromatin and of proteins to the promoter A.J. Mol. Cell Biol. PubMed Scopus Google Scholar, A. M. H. S. L. P. C.B. S. 2002; PubMed Scopus Google Scholar). that Sp1 binding sites are necessary for both constitutive and expression of MnSOD, suggest that p53 both basal and levels of MnSOD gene transcription by interfering with the basal transcriptional complex by p53 with Sp1 at the promoter is to have a on MnSOD induction in to is by the Sp1 is essential for both and expression of the MnSOD p53 has on MnSOD expression when all Sp1 sites in the basal promoter were overexpression of Sp1 reduced expression of Sp1 the of p53 on MnSOD expression. findings are very p53 as a factor in cellular to whereas MnSOD is a protein are in from is that the tumor function of p53 be in by transcriptional to suppress the expression of cytoprotective such as We of for the of p53 expression vector and M. for Sp1 expression