p53 Suppresses the Nrf2-dependent Transcription of Antioxidant Response Genes

Cells respond to the shift of intracellular environment toward pro-oxidant conditions by activating the transcription of numerous “antioxidant” genes. This response is based on the activation of the Nrf2 transcription factor, which transactivates the genes containing in their promoters the antioxidant response cis-elements (AREs). If the oxidative stress provokes DNA damage, a second response of the cell takes place, based on the activation of p53, which induces cell cycle arrest and/or apoptosis. Here we have explored the cross-talk between these two regulatory mechanisms. The results show that p53 counteracts the Nrf2-induced transcription of three ARE-containing promoters of the x-CT, NQO1, and GST-α1 genes. Endogenous transcripts of these antioxidant genes accumulate as a consequence of Nrf2 overexpression or exposure to electrophile diethylmaleate, but these effects are again blocked by p53 overexpression or endogenous p53 activation. Chromatin immunoprecipitation experiments support the hypothesis that this p53-dependent trans-repression is due to the direct interaction of p53 with the ARE-containing promoters. Considering that p53-induced apoptosis requires an accumulation of reactive oxygen species, this negative control on the Nrf2 transactivation appears to be aimed to prevent the generation of a strong anti-oxidant intracellular environment that could hinder the induction of apoptosis. Cells respond to the shift of intracellular environment toward pro-oxidant conditions by activating the transcription of numerous “antioxidant” genes. This response is based on the activation of the Nrf2 transcription factor, which transactivates the genes containing in their promoters the antioxidant response cis-elements (AREs). If the oxidative stress provokes DNA damage, a second response of the cell takes place, based on the activation of p53, which induces cell cycle arrest and/or apoptosis. Here we have explored the cross-talk between these two regulatory mechanisms. The results show that p53 counteracts the Nrf2-induced transcription of three ARE-containing promoters of the x-CT, NQO1, and GST-α1 genes. Endogenous transcripts of these antioxidant genes accumulate as a consequence of Nrf2 overexpression or exposure to electrophile diethylmaleate, but these effects are again blocked by p53 overexpression or endogenous p53 activation. Chromatin immunoprecipitation experiments support the hypothesis that this p53-dependent trans-repression is due to the direct interaction of p53 with the ARE-containing promoters. Considering that p53-induced apoptosis requires an accumulation of reactive oxygen species, this negative control on the Nrf2 transactivation appears to be aimed to prevent the generation of a strong anti-oxidant intracellular environment that could hinder the induction of apoptosis. The shift of the intracellular environment toward pro-oxidant conditions, due to the accumulation of reactive oxygen species (ROS) 2The abbreviations used are: ROS, reactive oxygen species; ARE, antioxidant response element; GST, glutathione S-transferase; DEM, diethylmaleate; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; WT, wild type; FACS, fluorescence-activated cell sorter. or to other electrophilic insults, induces a prompt response of the cells. One of the aims of this response is of course that of preventing the possible harmful effects of the oxidative stress, through the scavenging of the ROS, before they reach concentrations sufficient to induce oxidative damages to cellular molecules, mainly to DNA. To do this, cells activate the transcription of more than 200 genes encoding “antioxidant proteins” (for a review, see Ref. 1Kobayashi M. Yamamoto M. Antioxid. Redox. Signal. 2005; 7: 385-394Crossref PubMed Scopus (894) Google Scholar). These proteins include, for example, those involved in the generation and metabolism of GSH, a very effective scavenger of ROS and electrophiles, such as heavy and light chains of γ-glutamylcysteine synthetase (2Wild A.C. Moinova H.R. Mulcahy R.T. J. Biol. Chem. 1999; 274: 33627-33636Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar), the x-CT (subunit of the cystine/glutamate transporter) component of the cystine/glutamate exchange transport system (3Sasaki H. Sato H. Kuriyama-Matsumura K. Sato K. Maebara K. Wang H. Tamba M. Itoh K. Yamamoto M. Bannai S. J. Biol. Chem. 2002; 277: 44765-44771Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar), the glutathione S-transferases (GSTs), and the glutathione peroxidase (4Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3191) Google Scholar, 5Ishii T. Itoh K. Takahashi S. Sato H. Yanagawa T. Katoh Y. Bannai S. Yamamoto M. J. Biol. Chem. 2000; 275: 16023-16029Abstract Full Text Full Text PDF PubMed Scopus (1235) Google Scholar). The expression of most of these antioxidant proteins is regulated through the interaction of the NF-E2-related factor (Nrf2) transcription factor with a cis-element present, often in multiple copies, in their cognate gene promoters, named ARE (antioxidant response element). Nrf2 is a potent transactivator, which is regulated through different mechanisms. The most studied mechanism concerns the regulation of the nuclear availability of Nrf2 by Keap1. Early results supported the hypothesis that Keap1 functions as an extranuclear anchor site for Nrf2, and more recently, experimental evidence suggested that Keap1 promotes Nrf2 degradation. In fact, Keap1 directly binds Nrf2 and the actin cytoskeleton, thus sequestering Nrf2 in the cytoplasm (6Kang M.-I. Kobayashi A. Wakabayashi N. Kim S.-G. Yamamoto M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2046-2051Crossref PubMed Scopus (429) Google Scholar) and, on the other hand, it controls Nrf2 ubiquitination, which marks this protein for degradation by the 26 S proteasome (7Zhang D.D. Hannink M. Mol. Cell Biol. 2003; 23: 8137-8151Crossref PubMed Scopus (1116) Google Scholar, 8Zhang D.D. Lo S.C. Cross J.V. Templeton D.J. Hannink M. Mol. Cell Biol. 2004; 24: 10941-10953Crossref PubMed Scopus (978) Google Scholar). Upon the addition of electrophiles, the Nrf2-Keap1 complex dissociates, the Keap1-dependent ubiquitination of Nrf2 is blocked (9Kobayashi A. Kang M.I. Watai Y. Tong K.I. Shibata T. Uchida K. Yamamoto M. Mol. Cell Biol. 2006; 26: 221-229Crossref PubMed Scopus (702) Google Scholar), and Nrf2 accumulates into the nucleus, where it forms transcriptionally active complexes on the AREs (10Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2791) Google Scholar). In parallel with the described “antioxidant approach,” cells counteract the consequences of an oxidative stress by attempting to repair the ROS- and/or electrophile-induced damages. A key role in this cellular response is played by p53. This is a transcription factor activated by the DNA damage, which regulates the expression of many target genes, leading to cell cycle arrest aimed to allow time for the repair of DNA damage (for a review, see Ref. 11Oren M. Cell Death Differ. 2003; 10: 431-442Crossref PubMed Scopus (910) Google Scholar). On the other hand, p53 also has a fundamental role in the induction of apoptosis of cells where DNA damage remains unrepaired. The apoptosis induced by p53 appears to be at least in part dependent on the accumulation of ROS (12Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2241) Google Scholar). In fact, a subset of the genes regulated by p53, named PIGs (p53-induced genes), encodes proteins that could collectively induce an increase in ROS concentration (12Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2241) Google Scholar). The crucial role of the p53-dependent ROS accumulation is supported by several observations; for example, antioxidants, such as N-acetylcysteine, diphenyleneiodium chloride, and pyrrolidine dithiocarbamate, counteract ROS accumulation caused by p53 and, at the same time, prevent p53-induced apoptosis (12Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2241) Google Scholar, 13Macip S. Igarashi M. Berggren P. Yu J. Lee S.W. Aaronson S.A. Mol. Cell Biol. 2003; 23: 8576-8585Crossref PubMed Scopus (271) Google Scholar). The possible coexistence of the Nrf2-mediated antioxidant response with the regulation of apoptosis by p53 raises an apparent paradox. At least in principle, the induction of a very efficient ROS scavenging machinery by the Nrf2-dependent response could hamper, or at least interfere with, the p53-induced apoptosis that on the contrary requires the accumulation of ROS. Therefore, it is expected that a cross-talk between the Nrf2- and p53-induced responses should exist. Here we demonstrate that p53 suppresses the Nrf2-dependent transcription of ARE-containing promoters. This result is achieved by a direct inhibitory effect of p53 on these promoters. Cells and Culture Conditions—The mouse hepatocarcinoma cell line Hepa1–6 was purchased from the American Type Culture Collection (ATCC, CRL-830, Manassas, VA) and was grown in Dulbecco's modified essential medium (Invitrogen) containing 10% fetal bovine serum (Cambrex, Bio Science Verviers, Belgium). Human lung carcinoma Calu-6 cell line (ATCC, HTB-56; ICLC, HTL97003) was cultured in Dulbecco's modified essential medium containing 10% fetal bovine serum with 2 mm l-glutamine and 0.1 mm non-essential amino acids (Invitrogen). Human osteosarcoma cell line Saos-2 (ATCC, HTB-85) and HEK293 cells were grown in Dulbecco's modified essential medium containing 10% fetal bovine serum. HCT116 cells (p53WT and p53–/–) were kindly provided by G. Blandino and were grown in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum. All the media were supplemented with penicillin 10 units/ml and streptomycin 10 mg/ml at 37 °C under 5% CO2 atmosphere. Before each experiment, cells were subcultured at a density of 3 × 106/100-mm diameter dish in complete medium and incubated overnight at 37 °C. Diethylmaleate (DEM), etoposide, and cisplatin (Sigma) were used at the indicated concentrations. Plasmids and Transfections—The expression vector for FLAG-Nrf2 was constructed by inserting into the EcoRV/BamHI-digested p3xFLAG-CMV 7-1 plasmid (Sigma) the cDNA encoding human Nrf2 (I.M.A.G.E.: 4548874). The insert was subcloned in-frame with FLAG epitope at the N terminus. The expression vector for p53 (pCMV-p53) contains the human wild-type full-length cDNAs inserted in pCMV-neo and was described S. Vogelstein B. PubMed Scopus Google Scholar). The of mouse x-CT gene from to and of the mouse glutathione from to were by from DNA from cells P. M. T. Antioxid. Redox. Signal. 2006; PubMed Scopus Google Scholar). These were into vector All the were by x-CT to as and in the and the was by and to on the of the from to or from to The were in The of human gene to S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) was by from DNA from HEK293 the and and in All plasmid were of cells were in before and each was in Cells were by the with and/or p53 and/or Nrf2 expression of in the of of encoding DNA concentration was with overnight with the the medium was and, where the cells were with etoposide, or the cells were and for The was with a system to the The was to control The (Invitrogen) was used to the in human HEK293 and Calu-6 the of and was by the cDNAs were in a system from of in a containing with mm 10 mm mm mm and 10 units/ml The was incubated at °C for 10 and at °C for 10 by °C for and °C for 3 of cDNA of transcription were used in was used to cDNA of cDNA were in an system in in The of the used with mouse of used as the and and and x-CT, and NQO1, and The of the used with human and NQO1, and and conditions °C for and of °C for and °C for were to as endogenous expression was as gene Scopus Google Scholar). and nuclear protein cells were with the indicated of or with the same concentration as the for with PBS, at °C with of mm mm mm mm mm phenylmethylsulfonyl mm and 10 and and at for at °C. The nuclear was with and in of the same This was to of with and and at for at °C. was in of a containing mm mm mm mm mm PMSF, and 10 mg/ml and were with three nuclear were by at for 10 at °C. of from each were at °C for by on 10% and to were and blocked with 5% in containing at for and Nrf2 were by and and at for 2 with at for the were on the cells were with and at °C for in containing 0.1 mm mm mm mm PMSF, and 10 each of and The cell were by at for 10 at °C. were as described with FLAG from p53 and mouse Cell and and the were by the Chromatin and Calu-6 cells were grown in Calu-6 cells were with FLAG-Nrf2 or p53 and/or with the of the x-CT gene by the the cells were in medium for 10 at The cells were with for to and with The cells were in PBS, by and in mm PIPES, mm mm supplemented with nuclear was in nuclear mm 10 mm mm supplemented with and for and on The conditions were to generation of DNA between and in was to and of were with protein for at °C. A of the was and as The was with p53 from FLAG and Nrf2 The were with A mm mm mm mm for with mm 10 mm and with mm mm complexes were in for of by at °C DNA was at °C for 3 by and was DNA and 3 of DNA from a The of p53 and Nrf2 with endogenous x-CT in and cells was by on the the x-CT DNA from the mouse and human x-CT genes at and at and at and at of the ARE were The The of p53 with the of the x-CT gene was by the in DNA to the gene are at and of the The were on and by the of apoptosis by HCT116 cells were with different concentrations of for Cells were and for 10 at The cell was in of containing mm mm 10 mm mm and 10 of was with p53 with the Nrf2-mediated of ARE-containing x-CT gene transcription is activated by the electrophilic DEM, through a mechanism based on the of Nrf2 transcription factor on the AREs in the of the transcription site (3Sasaki H. Sato H. Kuriyama-Matsumura K. Sato K. Maebara K. Wang H. Tamba M. Itoh K. Yamamoto M. Bannai S. J. Biol. Chem. 2002; 277: 44765-44771Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar) of vector in cells induced the activation of the x-CT gene A effect was in cells to DEM, an electrophile that also provokes an accumulation of ROS by the exposure to of cells with FLAG-Nrf2 to a induction of the This was in cells but in and in which the exposure to the response of the to the of Nrf2 The most between and and the other cell is that and are J. G. M. B. M. Vogelstein B. Mol. Cell Biol. 10: PubMed Scopus Google Scholar, J. B. S. A. J. Google Scholar). Therefore, we explored the overexpression of p53 the Nrf2-dependent activation of the x-CT gene in the of Nrf2 with of a vector a of the Nrf2-dependent activation. This was in cells and in which the of a very of the p53 vector the transcription from the x-CT gene To that other than the of active p53, and cells were for their different we the same in in HCT116 cells in which p53 is and in HCT116 cells in which p53 gene is by gene T. Y. J. Kinzler K.W. Vogelstein B. J. 1999; PubMed Scopus Google Scholar). in in the cells and in the the Nrf2-dependent induction of the x-CT gene explored p53 overexpression also suppresses the Nrf2-mediated activation of other ARE-containing promoters, those of GST-α1 and genes A. Y. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) A and suppresses the Nrf2-dependent activation of the GST-α1 and gene promoters. and cells were with in which the gene is under the control of of the transcription site of the mouse GST-α1 were also with Nrf2 expression vector and/or with of p53 vector or with an of the the cells were for 3 with 200 in or with as was as described under and cells were and as described in with in which the gene is under the control of of the transcription site of the human All the are as the of as the in control is The the of FLAG-Nrf2 and of p53 in a were as a control of To the effects of Nrf2 and p53 on the transcription of endogenous x-CT and genes, HEK293 cells were with Nrf2 or with Nrf2 and p53, and the of these genes were by in Nrf2 overexpression the x-CT and and the of p53 the effects of was as a control and, as were by On the p53 induced the accumulation of and this induction was by DNA the Nrf2-mediated through a p53-dependent results in be by that ROS induced by provokes the DNA activation of p53. we the activation of endogenous p53, induced by DNA damage, has the same effects of p53 overexpression by To this with an vector or with the vector the expression of Nrf2, were with or 10 cisplatin for to induce DNA that and cisplatin a of the activation of the x-CT gene induced by the is dependent on p53 activation is by the that the DNA was in cells This was by the results in and in in which cisplatin or the Nrf2-dependent transcription activation. results were by the the gene under the control of GST-α1 and gene promoters these results an response of the cells to the activation of p53 counteracts the Nrf2-mediated activation of antioxidant response genes. To results by the overexpression of Nrf2, we cells with to induce the activation of p53, and the same cells were for to to induce the Nrf2-dependent antioxidant In these conditions, p53 was activated by etoposide, as by the accumulation of the and of p53 and induces a accumulation of Nrf2 in the In these we explored the transcription from the x-CT gene The results that activated the transcription of the On the the activation of the transcription induced by was in cells to The response of the endogenous genes was explored in the same experimental x-CT and were induced by but the with the response to On the effects in cells on the transcription of the and on the endogenous of the x-CT thus the role of p53 in the explored the of and cells to in cells with etoposide, cisplatin induced the activation of p53 in and in the nuclear of Nrf2 in and cells In these conditions, the transcription of the and the accumulation of endogenous x-CT induced by DEM, were in and in cells and p53 the Nrf2-dependent by with and the x-CT mechanism the could be based on the direct interaction of p53 with the promoters we To this we explored the possible of this protein with the x-CT in cells with the x-CT is with The same is also with in cells with p53, thus that this with the transcription the cells are with Nrf2 and p53, the with p53, a was with the To the of the x-CT gene involved in the interaction with p53, we several of the and their interaction with p53 in cells. that the the AREs and the in the are to with p53, the the of the transcription site which also contains the p53 cis-elements are in this To the of endogenous Nrf2 and p53 to the x-CT gene we immunoprecipitation experiments in or to This to that in conditions, p53 is in complexes with the x-CT In cells with DEM, the of with endogenous Nrf2 that with p53 the of the Cells to that the antioxidant response of the cells by Nrf2 is by p53 that in the of this p53-dependent the of the cells to apoptosis should be To this we the apoptosis induced by concentrations of in and cells. in the of p53 the of the cells to the oxidative The cells respond to the accumulation of ROS in two The is aimed at the antioxidant scavenging to of the of ROS and is based on the activation of ARE-containing “antioxidant by the transcription factor the second the cells respond to the DNA damages by cell cycle and, in the of by cell apoptosis. p53 is the of this In this we these two responses to the oxidative stress are that the Nrf2-dependent activation of antioxidant genes, such as x-CT, and NQO1, is by p53. These results demonstrate the of a cross-talk between the two mechanisms. The of p53 in the regulation of the has suggested by numerous in fact, the transcription of several genes have pro-oxidant or anti-oxidant functions (12Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2241) Google Scholar, M. S. M. K. Y. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, P. P. A. S. I. M. S. S. M. M. Res. 2004; PubMed Scopus Google Scholar, Y. H. J. 2004; PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar). of p53 to be with the activation of several antioxidant genes and with the of intracellular ROS 2005; PubMed Scopus Google Scholar). On the DNA activation of p53 induces a accumulation of ROS that is crucial for the p53-dependent apoptosis (12Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2241) Google Scholar, 13Macip S. Igarashi M. Berggren P. Yu J. Lee S.W. Aaronson S.A. Mol. Cell Biol. 2003; 23: 8576-8585Crossref PubMed Scopus (271) Google Scholar). the of a mechanism through which p53 intracellular p53 could the ARE-containing promoters by the expression of key proteins that control these genes. Nrf2 and Keap1 expression is by p53 overexpression or by the activation of endogenous p53 induced by DNA damage On the results that p53 could these genes by as a transcription are numerous that have for the p53-dependent trans-repression (for a review, see Ref. Cell Death Differ. 2006; 13: PubMed Scopus Google Scholar), but this remains to be the genes expression is by p53, of genes encodes proteins regulated in the of the cell cycle K. M. U. J. K. Res. 2000; PubMed Scopus Google Scholar, I. G. A. U. K. K. A. S. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Y. T. T. H. H. Itoh M. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). p53 is with these promoters in the of p53 cis-elements by the protein nuclear factor as an anchor Upon DNA damage, p53 is and this to the transcription of the genes A. S. M. M. G. G. Mol. Cell Biol. 2005; PubMed Scopus Google Scholar). Therefore, in these p53 the promoters containing the The three promoters we do it is that p53 their through the nuclear factor these promoters do p53 in the of the x-CT gene results show that p53 is in complexes also in the of DNA damage, as for other gene promoters 2004; 23: PubMed Scopus Google Scholar). The of the x-CT gene to demonstrate that the involved in the interaction of p53 is of the transcription where p53 cis-elements are This that p53 could directly with DNA cis-elements through In cells to p53 to these promoters to the of In fact, with the of active promoters, such as that of the transcription of the three promoters we studied is very in the cell the p53 This as in the of the promoters p53 these promoters regulatory and/or induced by the DNA The results in that p53 the transcription by Nrf2 to the x-CT gene the immunoprecipitation experiments the endogenous proteins do support this In fact, endogenous p53 was to be with the in the interaction of Nrf2 with the induced by the oxidative stress, appears to be with the of the by p53. These results support the that the p53-dependent is due to the activation of p53, as a consequence of and/or and to the of the protein on the The results that from the of the x-CT gene that the involved in the interaction with p53 also contains the support the that activated p53 could by with the of transcription other be such as the of or of other by activated p53. The we in this support to the hypothesis that the antioxidant machinery could have a role in was that of the Keap1 gene are with lung B. Tong K.I. T. Y. M. M. Kang M.I. Kobayashi A. S. Yamamoto M. Mol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The amino acids by these in the involved in the of Keap1 to in the Keap1 is and this results in a activation of this to the of the is the that p53, is also for the of the Nrf2 that the antioxidant response of the cell is under a control of possible harmful