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The aryl hydrocarbon receptor (AHR) and NF-E2 p45-related factor (NRF2) are two distinct transcription factors involved in the regulation of drug-metabolizing enzymes. Increasing evidence from several studies implies that AHR and NRF2 have direct links, but the molecular mechanism remains unknown. In this work we demonstrate for the first time that Nrf2 gene transcription is directly modulated by AHR activation. DNA sequence analyses of the mouse Nrf2 promoter revealed one xenobiotic response element (XRE)-like element (XREL1) located at –712 and two additional XRE-like elements located at +755 (XREL2) and +850 (XREL3). Functional analysis using luciferase assay showed that XREL1, XREL2, and XREL3 are all inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment, with XREL2 being the most potent. The functionality of these XRE-like elements was further confirmed by mutagenesis and gel shift experiments. Finally, we used chromatin immunoprecipitation assay to show a direct binding of AHR to the Nrf2 promoter. Cells with silenced AHR expression using siRNA also lost NRF2 mRNA induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin. These new data position NRF2-antioxidant response element downstream in the AHR-XRE pathway. Moreover, direct regulation of NRF2 by AHR contributes to couple phase I and II enzymes into an integrated system facilitating more effective xenobiotic and carcinogen detoxification. The aryl hydrocarbon receptor (AHR) and NF-E2 p45-related factor (NRF2) are two distinct transcription factors involved in the regulation of drug-metabolizing enzymes. Increasing evidence from several studies implies that AHR and NRF2 have direct links, but the molecular mechanism remains unknown. In this work we demonstrate for the first time that Nrf2 gene transcription is directly modulated by AHR activation. DNA sequence analyses of the mouse Nrf2 promoter revealed one xenobiotic response element (XRE)-like element (XREL1) located at –712 and two additional XRE-like elements located at +755 (XREL2) and +850 (XREL3). Functional analysis using luciferase assay showed that XREL1, XREL2, and XREL3 are all inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment, with XREL2 being the most potent. The functionality of these XRE-like elements was further confirmed by mutagenesis and gel shift experiments. Finally, we used chromatin immunoprecipitation assay to show a direct binding of AHR to the Nrf2 promoter. Cells with silenced AHR expression using siRNA also lost NRF2 mRNA induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin. These new data position NRF2-antioxidant response element downstream in the AHR-XRE pathway. Moreover, direct regulation of NRF2 by AHR contributes to couple phase I and II enzymes into an integrated system facilitating more effective xenobiotic and carcinogen detoxification. Environmental exposure to carcinogens is an important risk factor for common cancers (1Lichtenstein P. Holm N.V. Verkasalo P.K. Iliadou A. Kaprio J. Koskenvuo M. Pukkala E. Skytthe A. Hemminki K. N. Engl. J. Med. 2000; 343: 78-85Crossref PubMed Scopus (3205) Google Scholar). Because carcinogen exposure is important in cancer risk, enhancing the body's carcinogen-detoxifying capability is a promising approach for cancer prevention (2Kensler T.W. Qian G.S. Chen J.G. Groopman J.D. Nat. Rev. Cancer. 2003; 3: 321-329Crossref PubMed Scopus (182) Google Scholar, 3Sudakin D.L. J. Toxicol. Clin. Toxicol. 2003; 41: 195-204Crossref PubMed Scopus (56) Google Scholar). Like antibody-mediated immunity, nature has evolved a flexible, comprehensive enzyme system to detoxify a range of environmental toxicants, mutagens, and potential carcinogens. The cellular detoxifying system is divided into phase I and II drug-metabolizing enzymes (DMEs). 1The abbreviations used are: DME, drug-metabolizing enzymes; CYP, cytochrome P450; GST, glutathione S-transferase; NQO1, NADP(H):quino oxidoreductase 1; ARE, antioxidant response element; XRE, xenobiotic response element; AHR, aryl hydrocarbon receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; NRF2, NF-E2 p45-related factor 2; EQ, ethoxyquin; siRNA, small interfering RNA; RT, reverse transcription. The phase I enzymes consist of a gene superfamily of cytochrome P450s (CYPs); phase II enzymes include detoxifying and antioxidant enzymes such as glutathione S-transferases, γ-glutamylcysteine synthetase, NADP(H):quino oxidoreductase 1 (NQO1), and UDP:glucuronosyl transferases (4Hayes J.D. McMahon M. Cancer Lett. 2001; 174: 103-113Crossref PubMed Scopus (306) Google Scholar). Critical DNA sequences are frequently found single or multiply in the promoters of these genes, including antioxidant response element (ARE) and xenobiotic response element (XRE). The ARE is present in many phase II genes, whereas the XRE is found in both phase I and II genes (5Rushmore T.H. Kong A.N.T. Curr. Drug Metab. 2002; 3: 481-490Crossref PubMed Scopus (376) Google Scholar). XRE- and ARE-driven regulation of DME genes has two pathways that are generally thought to function independently. The XRE motif is the ultimate target of a protein complex that includes a ligand-activated aryl hydrocarbon receptor (AHR) translocating to the nucleus after binding to a chaperone nuclear transporter ARNT. The AHR-ARNT complex binds to the XRE motif and activates a battery of genes (4Hayes J.D. McMahon M. Cancer Lett. 2001; 174: 103-113Crossref PubMed Scopus (306) Google Scholar, 5Rushmore T.H. Kong A.N.T. Curr. Drug Metab. 2002; 3: 481-490Crossref PubMed Scopus (376) Google Scholar). As well, a recent paper reported an alternate cis-acting element, called XRE II, which binds an as yet unknown protein factor with an AHR-ARNT heterodimer as a coactivator (6Sogawa K. Numayama-Tsuruta K. Takahashi T. Matsushita N. Miura C. Nikawa J. Gotoh O. Kikuchi Y. Fujii-Kuriyama Y. Biochem. Biophys. Res. Commun. 2004; 318: 746-755Crossref PubMed Scopus (95) Google Scholar). AHR is a ligand-activated transcription factor that mediates toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as well as induction of three cytochrome P450 enzymes and a number of phase II enzymes including several glutathione S-transferase isoforms. AHR is a basic helix-loop-helix transcription factor that binds both synthetic chemicals such as TCDD and naturally occurred phytochemicals, sterols, and heme breakdown products. Whereas persistent activation of the AHR signaling pathway is responsible for the spectrum of adverse effects produced by metabolically stable AHR ligands such as TCDD, activation of AHR signaling by TCDD at nontoxic concentrations or by less persistent AHR agonists has also been shown to have some beneficial antitumorigenic/antiestrogenic activities (7Safe S. McDougal A. Int. J. Oncol. 2002; 20: 1123-1128PubMed Google Scholar, 8Koliopanos A. Kleeff J. Xiao Y. Safe S. Zimmermann A. Buchler M.W. Friess H. Oncogene. 2002; 21: 6059-6070Crossref PubMed Scopus (117) Google Scholar). Binding of the ARE promoter by a number of proteins has been shown; however, studies in a mouse knock-out model have placed particular emphasis on the basic leucine zipper transcription factor NRF2 (NF-E2 p45-related factor 2) (9McMahon M. Itoh K. Yamamoto M. Chanas S.A. Henderson C.J. McLellan L.I. Wolf C.R. Cavin C. Hayes J.D. Cancer Res. 2001; 61: 3299-3307PubMed Google Scholar, 10Ramos-Gomez M. Kwak M.K. Dolan P.M. Itoh K. Yamamoto M. Talalay P. Kensler T.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3410-3415Crossref PubMed Scopus (990) Google Scholar). Electrophilic compounds stimulate the cytosolic protein KEAP1, activating and releasing the transcription factor NRF2 from the KEAP1·NRF2 complex. In the nucleus the activated NRF2 forms a complex with small Mafs (11Motohashi H. O'Connor T. Katsuoka F. Engel J.D. Yamamoto M. Gene (Amst.). 2002; 294: 1-12Crossref PubMed Scopus (385) Google Scholar, 12Motohashi H. Katsuoka F. Engel J.D. Yamamoto M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6379-6384Crossref PubMed Scopus (262) Google Scholar), and this protein complex binds to the ARE motif, activating the ARE gene battery. Exposure to a variety of inducers with specific KEAP1 sites releases NRF2 for nuclear localization in the ARE. Recent data show that NRF2 is degraded by the ubiquitin-dependent pathway (13McMahon M. Itoh K. Yamamoto M. Hayes J.D. J. Biol. 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Sci. 2003; 73: 124-134Crossref PubMed Scopus (213) Google Scholar). A range of enzymes involved in cellular detoxification are thus induced, including for example the rate-limiting enzyme in GSH synthesis γ-glutamylcysteine synthetase (20Moinova H.R. Mulcahy R.T. Biochem. Biophys. Res. Commun. 1999; 261: 661-668Crossref PubMed Scopus (217) Google Scholar). It is generally considered that the ARE and XRE pathways are distinct. The conventional explanation of how bi-functional agents activate both phase I and II DMEs is a sequential mechanism whereby XRE-driven enzymes metabolize the inducer to an intermediary capable of ARE activation. However, Jaiswal and Radjendirane (21Radjendirane V. Jaiswal A.K. Biochem. Pharmacol. 1999; 58: 1649-1655Crossref PubMed Scopus (42) Google Scholar) showed that TCDD, a metabolically stable AHR inducer, can induce the human NQO1 gene expression through ARE activation. Moreover, using our cell-based system for XRE/ARE-activating drugs, we showed that some phase II-activating agents (ARE inducers) require the presence of AHR, suggesting a more direct cross-talk between the ARE and XRE pathways (22Miao W. Hu L. Kandouz M. Hamilton D. Batist G. Biochem. Pharmacol. 2004; 67: 1897-1905Crossref PubMed Scopus (15) Google Scholar). A more recent paper showed that induction of NQO1 by TCDD requires NRF2, implicating a link between AHR and NRF2 (23Ma Q. Kinneer K. Bi Y. Chan J.Y. Kan Y.W. Biochem. J. 2004; 377: 205-213Crossref PubMed Scopus (176) Google Scholar). In this work, we provide the first evidence that NRF2, the master transcriptional factor in the ARE pathway, can be directly modulated by the AHR-XRE activation, representing a novel molecular pathway in modulating drug-metabolizing enzymes. Chemicals and Antibodies—TCDD was from Wellington laboratories Inc. (ON, Canada). Anti-NRF2 antibody (h-300) was bought from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-AHR antibody (RPT9) was from Abcam Inc. (Cambridge, MA). Cell Lines and Tissue Culture—AHR-deficient cell line tao, CYP1A1-deficient cell line c37, and their parent cell line mouse hepatoma 1c1c7 were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium. Western Blot—Mouse hepatoma cells 1c1c7, tao, and c37 were treated with TCDD for indicated times before harvesting. 30 μg of cell lysate per each sample was loaded and run through a 7.5% SDS-PAGE gel before being transferred electrophoretically onto a nitrocellulose membrane. The membrane was blocked with 5% fat-free milk solution and then sequentially incubated with primary antibody and enzyme-conjugated secondary antibody. The results were documented on x-ray film with ECL detection and autophotography. Quantitative RT-PCR—Mouse hepatoma cells were treated with test compounds for an indicated time, and total RNA was with was in two the mRNA was into using reverse the was used as the for detection using the DNA I The were as for and of for for and for by and The for mouse NRF2 were and The for mouse were and RNA sequence of mouse AHR was with the AHR 1c1c7 cells were in a and in the presence of a of siRNA or RNA in a with the of AHR was by Western detection of AHR protein and Nrf2 promoter were by DNA from 1c1c7 cells was used as the promoter to the downstream promoter to and the promoter to were and then into the and sites of a luciferase A of of the Nrf2 promoter was also by and into The and XREL1, XREL2, and XREL3 were by The were to the and sites of a promoter and a luciferase were to the of mutagenesis were with a mutagenesis the sequences of and were by of and were sequentially with the to were were to the of The is to the is to the is to the sequences of the ARE or XRE, and the and were at per well using and in The cells were using with luciferase The a luciferase was as cells were incubated with for after which were and in cells were treated with test compounds for before for luciferase activities were in cell with the luciferase assay on a The luciferase activities reported are as a of the activity to that of the induction is as the of induction from treated cells the of three analysis was by of treated and cells shift were as J. Biol. Chem. Full Text PDF PubMed Google Scholar). cells were treated with TCDD for before of nuclear binding μg of nuclear protein was with 1 μg of in a 30 1 1 and The was for at after which the DNA was was for at were the with the of or 1 μg of antibody. were through a 5% gel using The gel was then and to with an at chromatin immunoprecipitation assay was using a from Biotechnology hepatoma 1c1c7 cells were to TCDD for and then cells were treated with for The chromatin of these cells was to the of the chromatin solution was as the total of the chromatin solution was incubated with an antibody or was at with and DNA was with and and then in of of DNA solution was used as for of with were used to the promoter of and Nrf2 The sequences for were and The sequences for were and The for were and The gene was used as The sequences for were and protein by TCDD 1c1c7 cells were treated with and 1 and TCDD for and NRF2 protein expression was by Western analyses using indicated that induction is from the of three analysis was by of treated and cells NRF2 by AHR hepatoma 1c1c7 cells were treated with TCDD for and In our Western NRF2 protein was as a at by with a NRF2 protein and the Nrf2 protein expression was at and The revealed that the NRF2 mRNA was to by TCDD at and was at These data that at the gene transcription further AHR is for this the cell line and CYP1A1-deficient cell line c37 were each treated with TCDD for and their NRF2 mRNA were The data showed that NRF2 mRNA was in 1c1c7 and c37 cell but the induction was in cell line suggesting an the that the cell have that for the we a cell AHR at by of siRNA studies were in 1c1c7 with siRNA or the the cells were treated with or TCDD for before were The data showed that the induction of NRF2 mRNA by TCDD in cells is in the cell these data that NRF2 expression be through AHR activation at the gene transcription XRE-like in mouse Nrf2 transcriptional regulation of sequences mouse Nrf2 promoter were from the mouse data analyses showed that in the of the Nrf2 in to the two elements at and reported is also one XRE-like sequence (XREL1) located at The downstream which is important for transcriptional was also additional XRE-like elements were found located at +755 (XREL2) and at the first of the gene of Nrf2 mouse DNA as was used to the promoter to the downstream promoter to and the promoter The were then into which were used in The luciferase showed that in 1c1c7 the promoter was the downstream promoter was and the promoter was in response to of TCDD but all were or in the cell line functionality of XRE-like elements in the a of were for both and downstream promoter The luciferase data showed that the which both and ARE elements is at however, the the ARE elements but is less and at The a response In with the downstream promoter the both XREL2 and XREL3 is most and at TCDD the XREL2 is also and at TCDD treatment, whereas the XREL2 XREL3 has response further of the XRE and ARE elements in the mutagenesis was The luciferase assay showed that promoter to to both TCDD or of two ARE elements induction by TCDD but the response to at the the response to TCDD but the response to of XREL1, and sequences the response to both TCDD and These data that ARE elements are responsible for however, both XRE and ARE elements to TCDD of XRE-like of Nrf2 the of three XRE XREL1, XREL2, and XREL3 were into the promoter which a promoter. showed that XREL1, XREL2, and XRE element found in were and in response to TCDD The XREL2 is more and XREL3 and is in the range as shift were also to XRE elements found in the Nrf2 promoter. The results show that and XREL3 a by an which can be by assay showed that the was by or AHR antibody but by antibody A and In the of XREL2 we but one of which by the was by showed that all can be by XREL2 but the indicated can be by or AHR antibody The that the indicated is specific to the XRE sequence and AHR and we that be to the sequences in the of TCDD on of XREL2 were with and used as gel shift were from cells that been treated with was as are but one of by the was by TCDD In all can be by XREL2 however, the indicated by an can be by or AHR antibody but by cells were treated with TCDD for and chromatin were used for immunoprecipitation with AHR antibody. The results show that the sequences specific to the found in the Nrf2 promoter as well as from the promoter can be from AHR but from Moreover, more Nrf2 promoter sequences were from AHR after with 1 or TCDD with A of on and that of the body's DME provide an effective approach for cancer prevention (2Kensler T.W. Qian G.S. Chen J.G. Groopman J.D. Nat. Rev. Cancer. 2003; 3: 321-329Crossref PubMed Scopus (182) Google Scholar, Curr. Med. Chem. 2001; PubMed Scopus Google Scholar). the molecular mechanism of DME is important in cancer many naturally or synthetic compounds have shown effects on DME the molecular are It has been that all bi-functional inducers can activate XRE and ARE pathways The most common explanation is an bi-functional compounds first activate the AHR-XRE pathway and induce phase I enzymes including which in metabolize these The intermediary further activate the pathway. A of studies by and A. S. Biol. Med. 2002; PubMed Scopus Google Scholar, A. M. D. K. E. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar) showed that TCDD can induce and that the induction is on the AHR, which approach of activating phase II enzymes through gel shift and V. A. M.W. Biochem. Pharmacol. PubMed Scopus Google Scholar) reported that AHR to both XRE and ARE suggesting that one transcription factor activate two distinct cis-acting elements V. A. M.W. Biochem. Pharmacol. PubMed Scopus Google Scholar). direct between the two pathways to the of but further In Jaiswal and Radjendirane (21Radjendirane V. Jaiswal A.K. Biochem. Pharmacol. 1999; 58: 1649-1655Crossref PubMed Scopus (42) Google Scholar) reported that TCDD, a metabolically stable AHR inducer, can induce NQO1 an ARE element (21Radjendirane V. Jaiswal A.K. Biochem. Pharmacol. 1999; 58: 1649-1655Crossref PubMed Scopus (42) Google Scholar). (23Ma Q. Kinneer K. Bi Y. Chan J.Y. Kan Y.W. Biochem. J. 2004; 377: 205-213Crossref PubMed Scopus (176) Google Scholar) showed that NRF2 is involved in the activation of NQO1 by TCDD, but the molecular mechanism was have also shown that some bi-functional compounds including TCDD can activate the ARE element in the presence of AHR (22Miao W. Hu L. Kandouz M. Hamilton D. Batist G. Biochem. Pharmacol. 2004; 67: 1897-1905Crossref PubMed Scopus (15) Google Scholar). on the evidence from we that NRF2, the master transcription factor for the ARE pathway, be downstream target of regulation by AHR activation. that NRF2 expression is by TCDD at the transcriptional with cells showed that AHR is an factor involved in the induction of NRF2 by These to the Nrf2 promoter The mouse Nrf2 promoter was in K. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), with further analysis in two elements in the at one of which is activated by Nrf2 binding M.K. Itoh K. Yamamoto M. Kensler T.W. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). Because the Nrf2 promoter has been in we the mouse promoter sequence of Nrf2 from the data we found that one XRE-like element is located at which is to the It has been reported that XRE and ARE elements in the in promoters of several important detoxifying genes including T.H. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 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Full Text Full Text PDF PubMed Scopus Google Scholar). data also showed that of these two is responsible for Nrf2 promoter response to a the single XRE Finally, a chromatin immunoprecipitation assay showed that AHR binds the the DNA sequence of mouse Nrf2 promoter with of the and human Nrf2 The showed that mouse and have a of in promoter and both have three of XRE-like elements in of the promoter. the human promoter sequence has a of with of of XRE-like elements in the of the the and sequence of these human XRE-like elements are distinct from of The of in Nrf2 promoters from all three an important in Nrf2 gene These novel demonstrate that Nrf2 gene expression is at by AHR inducers by activating XRE elements in its promoter. molecular a direct between AHR and NRF2 and the pathway downstream of AHR-XRE activation in found pathway important in the molecular mechanism of DME regulation It also The DME system is of phase I and II which have generally been thought to and at times in on of drug-metabolizing enzymes for is also considered that phase II enzymes are involved in detoxifying and and this has to at NRF2 and agents phase I enzymes such as are considered to have the one detoxify some and and on the can also metabolically activate some into carcinogens and the risk of cancer A number of recent studies using J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus (990) Google Scholar) that phase I enzyme are at as more in detoxification as in activation. further by our work reported is that the pathway in is and be NRF2 agents for generally at releasing NRF2 from KEAP1, a additional approach to NRF2 expression In this work, our of direct of NRF2 expression by AHR activation a molecular mechanism whereby phase I and II enzymes are and their are at the promoter transcription In of these is that the induction of phase II enzymes in both enzyme In this I and II work to a and system of detoxification. for NRF2 protein and for in the in this
Miao et al. (Fri,) studied this question.