Identification of a novel Nrf2-regulated antioxidant response element (ARE) in the mouse NAD(P)H:quinone oxidoreductase 1 gene: reassessment of the ARE consensus sequence.

NQO1 [NAD(P)H:quinone oxidoreductase 1] has an integral role in cellular responses to oxidative stress. The expression of NQO1 is up-regulated in the mouse following challenge with electrophilic chemicals, in an Nrf2 (NF-E2 p45-related factor 2)-dependent fashion, but the molecular basis for this observation remains unexplained. Through characterization of the murine nqo1 5'-upstream region, we now show that Nrf2 regulates this gene directly via an ARE (antioxidant response element) that lies within a 24 bp region spanning nt -444 to -421. A comprehensive mutation study of this ARE revealed that it does not conform to the currently accepted ARE consensus sequence [(5'-TMAnnRTGAYnnnGCRwwww-3', with essential nucleotides shown in capitals); two cytosine residues (shown in bold in the following sequence) that have been designated 'n' previously because they were thought to be redundant (5'-gagTcA C aGTgAGt C ggCAaaatt-3') have now been found to be essential for enhancer activity; two guanines (also shown in bold) previously regarded as essential for ARE function (5'-gagTcACaGT g AGtCg g CAaaatt-3') have proven to be dispensable]. Examination of wild-type and nrf2 (-/-) mouse embryonic fibroblasts demonstrated that Nrf2 is essential for both constitutive expression of NQO1 and its induction by sulphoraphane. Electrophoretic mobility-shift and chromatin immunoprecipitation assays revealed that Nrf2 associates, in low amounts, with the nqo1 ARE under constitutive conditions, and following sulphoraphane challenge of cells, Nrf2 is recruited to the ARE in substantially greater quantities, as a heterodimer with the small Maf (musculoaponeurotic fibrosarcoma virus) protein, MafK. Also, MafK was found to bind the nqo1 ARE in an Nrf2-independent fashion, and may contribute to transcriptional repression of the oxidoreductase gene. These findings allow a model for transcriptional control of nqo1 through the ARE to be proposed. Furthermore, our results indicate that distinct AREs have differential sequence requirements, and a universally applicable consensus sequence cannot be derived.

[1]  C. B. Pickett,et al.  Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. , 2003, Annual review of pharmacology and toxicology.

[2]  S. Kaye,et al.  Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. , 1989, Cancer research.

[3]  Wild Ac,et al.  Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2. , 1999 .

[4]  Moinova Hr,et al.  An electrophile responsive element (EpRE) regulates beta-naphthoflavone induction of the human gamma-glutamylcysteine synthetase regulatory subunit gene. Constitutive expression is mediated by an adjacent AP-1 site. , 1998 .

[5]  S. Reddy,et al.  Role of NRF2 in protection against hyperoxic lung injury in mice. , 2002, American journal of respiratory cell and molecular biology.

[6]  R. Mulcahy,et al.  Identification of a Variant Antioxidant Response Element in the Promoter of the Human Glutamate-Cysteine Ligase Modifier Subunit Gene , 2002, The Journal of Biological Chemistry.

[7]  D. Harrison,et al.  Cellular response to cancer chemopreventive agents: contribution of the antioxidant responsive element to the adaptive response to oxidative and chemical stress. , 1999, Biochemical Society symposium.

[8]  P. Farnham,et al.  c-Myc target gene specificity is determined by a post-DNAbinding mechanism. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[9]  K. Itoh,et al.  An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. , 1997, Biochemical and biophysical research communications.

[10]  T. Kerppola,et al.  Asymmetric Recognition of Nonconsensus AP-1 Sites by Fos-Jun and Jun-Jun Influences Transcriptional Cooperativity with NFAT1 , 2003, Molecular and Cellular Biology.

[11]  Hiroshi Suzuki,et al.  Hemoprotein Bach1 regulates enhancer availability of heme oxygenase‐1 gene , 2002, The EMBO journal.

[12]  A. Dinkova-Kostova,et al.  Persuasive evidence that quinone reductase type 1 (DT diaphorase) protects cells against the toxicity of electrophiles and reactive forms of oxygen. , 2000, Free radical biology & medicine.

[13]  A. Dinkova-Kostova,et al.  Chemoprotective properties of phenylpropenoids, bis(benzylidene)cycloalkanones, and related Michael reaction acceptors: correlation of potencies as phase 2 enzyme inducers and radical scavengers. , 1998, Journal of medicinal chemistry.

[14]  J. Alam,et al.  The heme-responsive element of the mouse heme oxygenase-1 gene is an extended AP-1 binding site that resembles the recognition sequences for MAF and NF-E2 transcription factors. , 1996, Biochemical and biophysical research communications.

[15]  A. C. Wild,et al.  Overlapping antioxidant response element and PMA response element sequences mediate basal and beta-naphthoflavone-induced expression of the human gamma-glutamylcysteine synthetase catalytic subunit gene. , 1998, The Biochemical journal.

[16]  S. Bergelson,et al.  Two adjacent AP-1-like binding sites form the electrophile-responsive element of the murine glutathione S-transferase Ya subunit gene. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[17]  J. Alam,et al.  Degradation of Transcription Factor Nrf2 via the Ubiquitin-Proteasome Pathway and Stabilization by Cadmium* , 2003, The Journal of Biological Chemistry.

[18]  A. Jaiswal,et al.  Human NAD(P)H:quinone oxidoreductase (NQO1) gene structure and induction by dioxin. , 1991, Biochemistry.

[19]  S. Biswal,et al.  Identification of Nrf2-regulated genes induced by the chemopreventive agent sulforaphane by oligonucleotide microarray. , 2002, Cancer research.

[20]  James Douglas Engel,et al.  Integration and diversity of the regulatory network composed of Maf and CNC families of transcription factors. , 2002, Gene.

[21]  C. Cho,et al.  A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[22]  C. B. Pickett,et al.  Transcriptional Regulation of the Antioxidant Response Element , 2000, The Journal of Biological Chemistry.

[23]  W. Wasserman,et al.  Functional antioxidant responsive elements. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[24]  S. Dhakshinamoorthy,et al.  Small Maf (MafG and MafK) Proteins Negatively Regulate Antioxidant Response Element-mediated Expression and Antioxidant Induction of the NAD(P)H:Quinone Oxidoreductase1 Gene* , 2000, The Journal of Biological Chemistry.

[25]  P. D. de Jong,et al.  Bacterial artificial chromosome libraries for mouse sequencing and functional analysis. , 2000, Genome research.

[26]  T. Rushmore,et al.  Regulation of glutathione S-transferase Ya subunit gene expression: identification of a unique xenobiotic-responsive element controlling inducible expression by planar aromatic compounds. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[27]  C. B. Pickett,et al.  Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Identification of regulatory elements controlling basal level expression and inducible expression by planar aromatic compounds and phenolic antioxidants. , 1991, The Journal of biological chemistry.

[28]  R. Mulcahy,et al.  Constitutive and β-Naphthoflavone-induced Expression of the Human γ-Glutamylcysteine Synthetase Heavy Subunit Gene Is Regulated by a Distal Antioxidant Response Element/TRE Sequence* , 1997, The Journal of Biological Chemistry.

[29]  C. Wolf,et al.  The Cap'n'Collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes. , 2001, Cancer research.

[30]  Ken Itoh,et al.  Modulation of Gene Expression by Cancer Chemopreventive Dithiolethiones through the Keap1-Nrf2 Pathway , 2003, The Journal of Biological Chemistry.

[31]  W. Deppert,et al.  Immortalization of BALB/c mouse embryo fibroblasts alters SV40 large T-antigen interactions with the tumor suppressor p53 and results in a reduced SV40 transformation-efficiency. , 1994, Oncogene.

[32]  Y. Kan,et al.  Identification of the NF-E2-related Factor-2-dependent Genes Conferring Protection against Oxidative Stress in Primary Cortical Astrocytes Using Oligonucleotide Microarray Analysis* , 2003, The Journal of Biological Chemistry.

[33]  D. Roop,et al.  NAD(P)H:quinone oxidoreductase 1 deficiency increases susceptibility to benzo(a)pyrene-induced mouse skin carcinogenesis. , 2000, Cancer research.

[34]  E Yoshida,et al.  Two domains of Nrf2 cooperatively bind CBP, a CREB binding protein, and synergistically activate transcription , 2001, Genes to cells : devoted to molecular & cellular mechanisms.

[35]  C. Wolf,et al.  Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice. , 2002, The Biochemical journal.

[36]  C. B. Pickett,et al.  The Rat Quinone Reductase Antioxidant Response Element , 1995, The Journal of Biological Chemistry.

[37]  K. Itoh,et al.  Keap1-dependent Proteasomal Degradation of Transcription Factor Nrf2 Contributes to the Negative Regulation of Antioxidant Response Element-driven Gene Expression* , 2003, Journal of Biological Chemistry.

[38]  T. Rushmore,et al.  The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity. , 1991, The Journal of biological chemistry.

[39]  K. Itoh,et al.  Keap1 regulates both cytoplasmic‐nuclear shuttling and degradation of Nrf2 in response to electrophiles , 2003, Genes to cells : devoted to molecular & cellular mechanisms.

[40]  H. Huang,et al.  Increased Protein Stability as a Mechanism That Enhances Nrf2-mediated Transcriptional Activation of the Antioxidant Response Element , 2003, The Journal of Biological Chemistry.

[41]  F. Torti,et al.  Response to Oxidative Stress Translational Regulation of Ferritin in Coordinate Transcriptional and , 2000 .

[42]  D. Ross,et al.  NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms. , 2000, Chemico-biological interactions.

[43]  Y. Kan,et al.  The CNC Basic Leucine Zipper Factor, Nrf1, Is Essential for Cell Survival in Response to Oxidative Stress-inducing Agents , 1999, The Journal of Biological Chemistry.

[44]  A. Santamaria,et al.  Rapid detection of inducers of enzymes that protect against carcinogens. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[45]  J. Hayes,et al.  Chemoprevention of aflatoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin B1-aldehyde reductase, the glutathione S-transferase A5 and P1 subunits, and NAD(P)H:quinone oxidoreductase in rat liver. , 2000, Cancer research.

[46]  A. Jaiswal,et al.  Disruption of the DT Diaphorase (NQO1) Gene in Mice Leads to Increased Menadione Toxicity* , 1998, The Journal of Biological Chemistry.