Transcriptional Regulation of the Antioxidant Response Element

The antioxidant response element (ARE) mediates the transcriptional activation of many genes encoding phase II drug-metabolizing enzymes in response to oxidative stress. Recent studies using knockout mice suggest that NF-E2-related factor 2 (Nrf2), along with small Maf proteins, binds and activates the ARE. In this study, using in vitro binding assays, Nrf2/MafK heterodimers were found to interact with high affinity to the ARE. However, distinct differences were observed when this interaction was compared with that formed with nuclear proteins from H4II EC3 or HepG2 cells. Overexpression of Nrf2 activated ARE-mediated transcription in HepG2 cells, and this activation was further increased by tert-butylhydroquinone. In HeLa cells, overexpression of Nrf2 resulted in activation of the ARE, but this activation was no longer induced bytert-butylhydroquinone. Using ARE constructs with point mutations in the core sequence, we found that only mutations at the T or G nucleotides within the core (TGAC) render the ARE unresponsive to Nrf2. Overexpression of MafK led to dose-dependent repression of ARE activity. Activation of the ARE by Nrf2 was similarly antagonized by MafK. These data suggest that Nrf2 plays an important role mediating basal activity of the ARE but that small Maf proteins are repressors and not activators of ARE-mediated transcription. The antioxidant response element (ARE) mediates the transcriptional activation of many genes encoding phase II drug-metabolizing enzymes in response to oxidative stress. Recent studies using knockout mice suggest that NF-E2-related factor 2 (Nrf2), along with small Maf proteins, binds and activates the ARE. In this study, using in vitro binding assays, Nrf2/MafK heterodimers were found to interact with high affinity to the ARE. However, distinct differences were observed when this interaction was compared with that formed with nuclear proteins from H4II EC3 or HepG2 cells. Overexpression of Nrf2 activated ARE-mediated transcription in HepG2 cells, and this activation was further increased by tert-butylhydroquinone. In HeLa cells, overexpression of Nrf2 resulted in activation of the ARE, but this activation was no longer induced bytert-butylhydroquinone. Using ARE constructs with point mutations in the core sequence, we found that only mutations at the T or G nucleotides within the core (TGAC) render the ARE unresponsive to Nrf2. Overexpression of MafK led to dose-dependent repression of ARE activity. Activation of the ARE by Nrf2 was similarly antagonized by MafK. These data suggest that Nrf2 plays an important role mediating basal activity of the ARE but that small Maf proteins are repressors and not activators of ARE-mediated transcription. antioxidant response element glutathione S-transferase NF-E2-related factor 2 NAD(P)H:quinone oxidoreductase tert-butylhydroquinone chloramphenicol acetyltransferase electrophoretic mobility shift assay The antioxidant response element (ARE)1 is a uniquecis-acting regulatory sequence found in the 5′-regulatory region of a number of genes encoding enzymes involved in the phase II metabolism of xenobiotics. The induction of these enzymes by transcriptional activation through the ARE results from cellular exposure to a variety of chemical entities, including electrophilic compounds, antioxidants, and a host of other agents capable of generating radical species that alter the cellular redox state. This imbalanced cellular redox state, generally referred to as oxidative stress (1.Sies H. Eur. J. Biochem. 1993; 215: 213-219Crossref PubMed Scopus (1614) Google Scholar), appears to be the principal signal that acts, directly or indirectly, through a signaling pathway leading to the transcriptional activation of genes encoding these enzymes. The ARE was first identified on the promoter of the rat gene encoding the glutathioneS-transferase A2 subunit (GST A2) (2.Rushmore T.H. Pickett C.B. J. Biol. Chem. 1990; 265: 14648-14653Abstract Full Text PDF PubMed Google Scholar) and was found to be responsive to conditions of oxidative stress (3.Rushmore T.H. Morton M.R. Pickett C.B. J. Biol. Chem. 1991; 266: 11632-11639Abstract Full Text PDF PubMed Google Scholar). Functional analysis of this element by point mutations identified a core sequence, 5′-TGACNNNGC-3′, essential for mediating basal and/or inducible activity (3.Rushmore T.H. Morton M.R. Pickett C.B. J. Biol. Chem. 1991; 266: 11632-11639Abstract Full Text PDF PubMed Google Scholar). A functional ARE has also been identified in a number of other genes encoding phase II drug-metabolizing enzymes such as the mouse Gst A1 (4.Friling R.S. Bensimon A. Tichauer Y. Daniel V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6258-6262Crossref PubMed Scopus (426) Google Scholar) and the rat and human NAD(P)H:quinone oxidoreductase (QR) (5.Favreau L.V. Pickett C.B. J. Biol. Chem. 1991; 266: 4556-4561Abstract Full Text PDF PubMed Google Scholar, 6.Li Y. Jaiswal A.K. J. Biol. Chem. 1992; 267: 15097-15104Abstract Full Text PDF PubMed Google Scholar), as well as the γ-glutamylcysteine synthetase gene (7.Moinova H.R. Mulcahy R.T. J. Biol. Chem. 1998; 273: 14683-14689Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Through the use of bioinformatics, the ARE sequence has also been identified in the 5′-flanking region of a number of other genes, although its functional role has not as yet been experimentally demonstrated (8.Wasserman W.W. Fahl W.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5361-5366Crossref PubMed Scopus (633) Google Scholar). Because the ARE core sequence is similar to the sequence recognized by AP-1 proteins, the possibility exists that members of the Jun and Fos families of transcriptional factors might be involved in the transcriptional activation of the rat GST A2 andQR genes. This hypothesis was strengthened by the observation that Jun and Fos family members can be activated by oxidative stress (9.Devary Y. Gottlieb R.A. Lau L.F. Karin M. Mol. Cell. Biol. 1991; 11: 2804-2811Crossref PubMed Scopus (602) Google Scholar, 10.Abate C. Patel L. Rauscher III, F.J. Curran T. Science. 1990; 249: 1157-1161Crossref PubMed Scopus (1375) Google Scholar). Nevertheless, data from a number of studies have suggested that transcriptional factors distinct from Jun and Fos are involved in the transcriptional activation of the ARE (11.Nguyen T. Rushmore T.H. Pickett C.B. J. Biol. Chem. 1994; 269: 13656-13662Abstract Full Text PDF PubMed Google Scholar, 12.Yoshioka K. Deng T. Cavigelli M. Karin M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4972-4976Crossref PubMed Scopus (210) Google Scholar, 13.Prestera T. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8965-8969Crossref PubMed Scopus (219) Google Scholar). In fact, a recent study using knockout mice indicated that the NF-E2-related factor 2, Nrf2, was part of a transcriptional complex directly involved in mediating the transcriptional regulation of the mouse Gst A1 gene (14.Itoh 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). Furthermore, using in vitro gel shift assays, these investigators found that Nrf2/small Maf heterodimers bind with high affinity to the ARE sequence and regulate transcription of the GST andQR genes. The role of Nrf2 in regulating the ARE of the human QR gene has also been demonstrated recently (15.Venugopal R. Jaiswal A.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14960-14965Crossref PubMed Scopus (929) Google Scholar,16.Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (486) Google Scholar). In the present study, we have examined the binding specificities of Nrf2/MafK heterodimers to the ARE derived from the rat GST A2 and QR genes using gel mobility shift and DNase I protection assays. Furthermore, we utilized co-transfection assays to explore the role these transcription factors play in basal and inducible expression of the rat GST A2 and QRgenes. We found that Nrf2/small Maf heterodimers bind with high affinity to the rat QR ARE but with low affinity to the ratGST A2 ARE. Our data suggest that Nrf2 is involved in regulating basal level expression of the rat GST A2 andQR genes; however, small Maf acts as a transcriptional repressor rather than activator of ARE-mediated transcription. The cDNA encoding the rat Nrf2 and rat MafK proteins was cloned from a rat liver cDNA library (Stratagene) by polymerase chain reaction methodologies. Primers used in the polymerase chain reaction experiments were synthesized based on cDNA sequences obtained from the GenBankTM data base for rat Nrf2 (accession number AF037350) and for mouse MafK (accession number D42124) (17.Igarashi K. Itoh K. Motohashi H. Hayashi N. Matuzaki Y. Nakauchi H. Nishizawa M. Yamamoto M. J. Biol. Chem. 1995; 270: 7615-7624Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The cDNA thus obtained was ligated to the plasmid vector pcDNA3 (Invitrogen), which contains the T7 promoter and the cytomegalovirus promoter for expression in mammalian cells. The recombinant plasmids were propagated in Escherichia coli DH5αF′ cells. The electrophoretic mobility shift assays (EMSA) to assess DNA-protein interactions were performed essentially as described (11.Nguyen T. Rushmore T.H. Pickett C.B. J. Biol. Chem. 1994; 269: 13656-13662Abstract Full Text PDF PubMed Google Scholar), using a 43-base, double-stranded oligonucleotide containing the ARE sequence of the ratQR gene as probe. The oligonucleotides were labeled at the 5′-end using γ-32PATP and T4 polynucleotide kinase. Nuclear extracts were prepared according to published procedures (18.Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar) from rat H4II EC3 cells or from human HepG2 cells treated withtert-butylhydroquinone (tBHQ) for 6 h. Rat Nrf2 and MafK proteins were synthesized in vitro using the TNT-T7 coupled wheat germ extract system (Promega Corp.). To generate Nrf2/MafK dimers, equal volumes of translation mixtures containing Nrf2 and MafK were mixed and incubated at 37 °C for 30 min. In the competition experiments, unlabeled DNA at approximately 50-fold molar excess was preincubated with protein samples for 20 min at 25 °C prior to addition of the labeled probe. The reaction mixtures were incubated for an additional 30 min with the labeled probe (0.1 ng, 10,000 dpm) and fractionated on non-denaturing 6% polyacrylamide gels in Tris borate-EDTA (TBE) buffer. DNA fragments containing the ARE sequence encompassing nucleotides −771 to −606 of the rat GST A2 gene and −479 to −302 of the rat QR gene were used as templates in the DNase I protection assays. These fragments were prepared by polymerase chain reaction, purified on agarose gel, and end-labeled with α-32PdATP and α-32PdCTP by fill-in reactions using the Klenow fragment. Binding reactions and DNase I were as described T. Pickett C.B. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). were purified by with and on 6% Rat H4II HeLa and HepG2 cells were and as described (2.Rushmore T.H. Pickett C.B. J. Biol. Chem. 1990; 265: 14648-14653Abstract Full Text PDF PubMed Google Scholar). experiments, cells were in at approximately and incubated for 20 at 37 were using according to the by the cells were to for 20 at 37 to or and incubated for an additional h. were with and for chloramphenicol acetyltransferase activity. Recent studies suggest that NF-E2-related proteins be involved in regulating the transcriptional activity of the ARE (14.Itoh 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, R. Jaiswal A.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14960-14965Crossref PubMed Scopus (929) Google Scholar, R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (486) Google Scholar). Furthermore, was suggested that a of Nrf2/small Maf is involved in the activation (14.Itoh 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). We this hypothesis by vitro to these proteins interact with the ARE with high of in vitro Nrf2 or MafK with the labeled ARE not to of complex (14.Itoh 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), H. C. and C. that Nrf2 and MafK not interact with the ARE with high affinity as or as a the Nrf2/MafK complex was found to interact with the ARE derived from the rat QR gene in a The of this DNA-protein complex was by a 50-fold molar excess of the ARE but was by the molar excess ARE oligonucleotides containing a within the core sequence 2 A similar of competition was using nuclear extracts from H4II EC3 cells The mobility of the however, was when compared with that formed with nuclear extract from rat H4II EC3 cells. In an excess of the unlabeled ARE sequence derived from the rat GST A2 gene only the interaction as compared with a when rat nuclear extract was used and However, the ARE interaction was the ARE of the GST A2 gene was to a core sequence with to and used as unlabeled DNA These data suggest that a sequence within the DNA binding is essential for high affinity binding by the Nrf2/MafK of the binding affinity for the ARE of the Nrf2/MafK with that of a nuclear protein complex from HepG2 cells the differences with to mobility as well as the competition observed with rat nuclear In this we also additional and as unlabeled and are response recognized by K. M. Nishizawa M. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar), a of the protein the a core sequence, with the ARE for binding to the Nrf2/MafK and to the nuclear protein from HepG2 cells and In the the with the ARE for binding to the HepG2 nuclear However, the oligonucleotide not with binding of the Nrf2/MafK to the ARE 6 These data are with the that a sequence a high affinity for the Nrf2/MafK similar conditions in the binding assays, we not high affinity interactions recombinant Nrf2/MafK and the GST A2 ARE, the interactions are to be observed by We further the DNA binding of Nrf2/MafK by in vitro DNase I protection assays. the results of these A DNA −479 to −302 of the QR gene was to a complex with the Nrf2/MafK or with nuclear proteins prepared from or rat H4II EC3 cells, and the complex was to with DNase I. region of from to was observed with or nuclear extracts and In the of an excess of the unlabeled ARE sequence, the labeled DNA was no longer from DNase I 2 6 and the labeled DNA was incubated with the Nrf2/MafK the region was an additional at the 5′-end 2 and was an excess of unlabeled ARE sequence was in the 2 In binding of the protein to the labeled DNA induced a DNase at the by an We also performed DNase I protection assays using DNA nucleotides −771 to −606 of the GST A2 gene as a binding Nuclear extracts prepared from or rat H4II EC3 cells an region of encompassing to 2 of an excess of the unlabeled ARE this protection 2 6 In the Nrf2/MafK to sequence on this DNA from DNase I These results are with obtained by described which that the ARE derived from the GST A2 gene not with that derived from the QR gene for binding to this protein These data suggest that the interaction of the Nrf2/MafK with the ARE sequence of the GST at a affinity than with the ARE sequence from the QR The role of Nrf2 in regulating the transcriptional activity of the ARE was further by experiments in HepG2 and HeLa cells. HepG2 cells along with the ARE derived from the GST A2 Nrf2 induced expression of activity in a expression was observed at of plasmid DNA used in the with an approximately activation The level of activation as the of plasmid DNA was to The ARE-mediated activation of activity by Nrf2 was induced further when the cells were treated with Because Nrf2 has been to be at high in HeLa cells by analysis P. K. I. A. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), we also these cells with the ARE constructs along with an plasmid vector as or with the Nrf2 expression vector and the cells to HepG2 cells, HeLa cells low of basal activity when with the ARE and plasmid of these cells with not in in activity of HeLa cells with the Nrf2 expression however, led to a DNA in but no further induction by was observed with of the DNA used in These suggest that Nrf2 be in regulating the basal activity of the ARE. To this possibility in we performed similar experiments using plasmid constructs containing point mutations within the core sequence for the basal and/or inducible activity of the ARE (3.Rushmore T.H. Morton M.R. Pickett C.B. J. Biol. Chem. 1991; 266: 11632-11639Abstract Full Text PDF PubMed Google Scholar). an ARE containing a point at the T or G within the was HepG2 cells, the basal activity was Overexpression of Nrf2 no on the level of activity in these cells. In when ARE constructs containing point mutations at other within the core sequence were used in the experiments, expression of Nrf2 although than with the ARE of these cells with not to a further in activity not These results that the T and G nucleotides are for Nrf2 interaction and that other nucleotides within the ARE core sequence also have but essential in this of Nrf2 overexpression on the transcriptional activity of the ARE and point The procedures were performed as described in the to HepG2 cells were with of and of ARE constructs from the GST A2 gene or with of and of ARE constructs from the QR gene cells were to for and for assays. The sequences of the and ARE are indicated with the nucleotides We similar studies with Nrf2 to its transcriptional activity on the ARE derived from the ratQR the GST A2 ARE, Nrf2 the QR ARE with the DNA used in the of HepG2 or HeLa cells However, the level of activity was at a DNA of in HepG2 cells for a activation In HeLa cells, a activation was observed with the DNA used in the The level of activation by Nrf2 in HepG2 cells for the QR ARE as compared with that of A2 ARE is to the level of basal activity by the which differences in DNA sequences the core We also performed experiments using constructs with point mutations within the ARE sequence L.V. Pickett C.B. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) to nucleotides for activation by Nrf2. in a of the T within the activation to a Overexpression of Nrf2 in cells with this ARE not in activation of activity. Because the contains that are to and are essential for ARE we cells with ARE with a point at the G within the on the with this ARE a basal level activity of approximately of the Nrf2 was in these cells, gene expression was increased as compared with the activation observed with the ARE. The level of activity activated by Nrf2 for these ARE constructs however, similar of HepG2 cells with an ARE containing a point at the of the the basal level expression of ARE to of that of the Overexpression of Nrf2 led to a activation of activity. A of the also led to a of the basal but in this no activation by Nrf2 was This that the is also essential for activation by Nrf2, in with the competition data of the in vitro binding studies that a of this results in a sequence with affinity for the Nrf2/MafK protein indicated has been suggested that small Maf proteins, which a a complex with Nrf2 to regulate the transcriptional activity of the ARE (14.Itoh 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). We this possibility by HepG2 cells with a MafK expression plasmid along with the GST A2 ARE In a MafK the ARE-mediated gene This however, only the basal level expression was to activity in these cells We also performed experiments in which Nrf2 and MafK expression plasmids were HepG2 cells with the ARE. The results of these experiments are in 6 MafK was found to transcriptional This increased with the of MafK plasmid DNA used in the experiments 6 results were obtained when the QR ARE was HepG2 cells with the MafK expression plasmid or with Nrf2 expression in MafK has a on the ARE its basal level with the GST A2 ARE, was to activity in these cells. the transcriptional activation of this ARE by Nrf2 was also antagonized by the expression of MafK in a Recent studies suggest that Nrf2/small Maf be the transcriptional complex involved in the transcriptional activation by the ARE of the mouse Gst A1 and by (14.Itoh 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). In this study, we in vitro binding assays and co-transfection assays Nrf2 directly interact with the ARE. Our results that the in vitro rat Nrf2/MafK with high and affinity with the ARE of the ratQR This interaction results in a complex that has a mobility in as compared with that formed the ARE and a nuclear protein from rat H4II EC3 or human HepG2 cells. appears that a sequence within the of the QR ARE is essential for high affinity binding by the Nrf2/MafK This the observation that the ARE derived from the rat GST A2 which not have within the sequence, to with the QR ARE for binding to the Nrf2/MafK complex binding by nuclear proteins from rat H4II EC3 or human HepG2 A that a within the GST A2 ARE core sequence, however, to this element the to with ARE for Nrf2/MafK the data from HepG2 and HeLa cells that overexpression of Nrf2 activates gene expression through the ARE. Nrf2 acts as or in with to the ARE in these studies is not However, an Nrf2 not interact with the ARE in in vitro binding assays not and 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). Nevertheless, Nrf2 can the ARE in a in experiments in HepG2 and HeLa cells. are at to Nrf2 acts in this Nrf2 with an protein that at a high level in the cells to for the in these overexpression assays Nrf2 might as a with affinity for the ARE. Because Nrf2 is at a high level in HeLa cells P. K. I. A. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), the observation that this not to induction by that Nrf2 have an important role in mediating the basal activity with the ARE. The role Nrf2 plays in basal level expression with the observation that the QR ARE mediates a basal activity than the GST the QR ARE a sequence that is for interaction with Nrf2. Nrf2 be the factor involved in mediating the basal activity of the ARE. However, Nrf2 an factor that exists in HepG2 and not in HeLa cells to generate a response to through the ARE. This factor not be MafK or other small Maf proteins these proteins have a repressor on the ARE activity. The repression the basal activity an inducible response be by in HepG2 cells MafK. These data are with published that small Maf proteins generally as transcriptional repressors as or as heterodimers with or Nrf2 proteins K. K. Itoh K. Hayashi N. Nishizawa M. Yamamoto M. 1994; PubMed Scopus Google Scholar, N. L. Nishizawa M. H. Nucleic Acids Res. 1996; PubMed Scopus Google Scholar, K. L. A. P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Itoh J. J. K. K. J. Igarashi K. N. M. Yamamoto M. Oncogene. 1997; PubMed Scopus Google Scholar). These also the mobility by Nrf2/MafK and nuclear observed in the gel mobility shift assays. is that the nuclear protein complex of Nrf2 in with a factor distinct from small Maf proteins that on This when with Nrf2, a complex that with the GST A2 andQR with high affinity and in a to transcriptional activation in response to oxidative stress. The and of this factor through the use of to important this signaling A recent study described the of a protein with the as part of a pathway mediating the response to oxidative stress through the ARE K. N. Katoh Y. Ishii T. Igarashi K. J.D. Yamamoto M. PubMed Scopus Google Scholar). was suggested that Nrf2 is in the through with and that this is in cells oxidative thus Nrf2 for the to gene transcription. These data for a pathway of signal in response to oxidative stress the ARE of the well be of to is in HeLa cells, these cells to the ARE when treated with the high level of Nrf2 expression observed in these cells. HeLa cells other in the signaling pathway that the interaction and Nrf2. The signaling pathway leading to the activation of gene expression the ARE has recently been to the protein R. T.H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, R. S. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In these were used to the role of distinct protein in regulating the transcriptional activity of the ARE. was suggested that the of and the 2 are important for ARE activity. studies are to members of the protein family are involved in regulating the interaction in response to oxidative stress.