The Aryl Hydrocarbon Receptor Interacts With Nuclear Factor Erythroid2-Related Factor2to Mediate Induction Of NAD(P)H:Quinone Oxidoreductase1by2,3,7,8-Tetrachlorodibenzo-p-dioxin

TCDD (2,3.7.8-tetrachlorodibenzo-p-dioxin), a ubiquitous environmental contaminant,elicits diverse species-specific xenobiotic metabolism effects, including tumor promotion, teratogenesis, hepatotoxicity, immunotoxicity and modulation of endocrine systems. TCDD is a potent agonist of the AhR (aryl hydrocarbon receptor). Induction of gene transcription by TCDD is best studied for induction of CYP1A1. These ana-lyses have revealed an’AhR/DRE (dioxin response element) paradigm" for the induction, in which AhR is activated by a ligand, binds to DRE sequences located in the enhancer of the gene with partner protein Arnt (aryl hydrocarbon receptor nuclear translocator), and thereby mediates transcription. NQO1[NAD(P)H:quinone oxidoreductase; EC1.6.99.2; DT-diaphorase] catalyses the obligatory two-electron reduction of a wide range of endogenous and environmental quinones and quinoid compounds such as benz[a]pyrene-3,6-quinone, vitamin K, vitamin Eα-tocopherol, benzene quinones and anthraquinone-based anti-tumour drugs such as mitomycin C. Epidemiological and genetic studies reveal that a loss or reduction of NQO1activity is associated with increased risks for a number of pathological lesions, including benzene-induced haematotoxicity, acute leukaemia in adults and children, secondary leukaemia after chemotherapy in cancer patients, increased myelogenous hyperplasia and decreased therapeutic effect of chemotherapy in patients with disseminated peritoneal cancer. On the other hand, increased activities of NQO1are found to contribute to chemoprotection against cancer and chemical toxicity by natural or synthetic compounds.NQO1is broadly expressed in mammalian tissues and cell types (i.e. constitutive expression). Moreover, NQO1is highly inducible by AhR agonists such as TCDD and polycyclic aromatic hydrocarbon or by phenolic antioxidants such as tBHQ (2-t-butylbenzene-1,4-diol). Induction of NQO1is considered as a model for analyzing transcriptional regulation of many cytoprotective enzymes and proteins. AhR mediates the induction of NQO1by TCDD and benzo[a]pyrene (Bap); whereas, the antioxidant-activated transcription factor Nrf2is critical for induction by antioxidants such as tBHQ. We previously reported a cross-interaction between AhR and Nrf2signal transduction is required for induction of NQO1by TCDD. In this study, we analyzed the interaction between AhR and Nrf2at the promoter of NQO1. Part1AhR agonist TCDD induced interaction of AhR and Nrf2,and stabilization, nuclear accumulation and delayed kinetics of Nrf2Object:The aim of the present study is to elucidate AhR agonist TCDD induced NQO1is Nrf2independent and TCDD induced stabilization, nuclear accumulation.and delayed kinetics of Nrf2compared with AhR. Methods:Wild type (Wt, hepalclc7), AhR-deficient (AhR-D) or Arnt-deficient (Arnt-D) variant, and AhR-D or Arnt-D variant hepatoma cells reconstituted with AhR, Arnt, or their mutants were established before. PCR were performed to measure TCDD induced CYP1A1and NQO1mRNA in Wild type,(AhR-D) or (Arnt-D) variant, and AhR-D or Arnt-D variant hepatoma cells reconstituted with AhR, Arnt, or their mutants. Nrf2knockout (KO) mice in which the Nrf2gene was disrupted. Nrf2and wild type control (Nrf2+/+) mice in C57BL/6background (male,2months old) were bred properly.Mice were treated with B[a]p by intraperitoneal injection at80mg/kg body weight once, or BHA (butyl hydroxyanisol) by intragastric administration at400mg/kg body weight on day1and day3. Corn oil was used as the solvent control. Liver was taken for CYP1A1and NQO1mRNA expression24h after B[a]p injection or4days after BHA treatment.Western Blotting were used to measure TCDD induced Nrf2protein Stabilization and nuclear accumulation. Results:NQO1induction was not observed in AhR or Arnt-deficient cells but induction was restored upon reconstitution of the variant cells with functional AhR or Arnt, respectively. Induction also required Nrf2as induction by benzo[a]pyrene was lost in the liver of Nrf2knockout mice similarly to induction by BHA, demonstrating a cross-interaction between the AhR and Nrf2pathways for NQO1induction in vivo. Consistent with the activation of Nrf2, TCDD treatment inhibits Keapl-dependent ubiquitination and proteasomal degradation of Nrf2resulting in the stabilization and nuclear accumulation of Nrf2with delayed kinetics compared with activation of AhR. Conclusions: Interaction of AhR and Nrf2mediate TCDD induced NQO1.TCDD also induced stabilization, nuclear accumulation and delayed kinetics of Nrf2. Part2AhR agonist TCDD induce interaction of Ahr and Nrf2by AhR binding with Nrf2Object:To investigate TCDD induce interaction of Ahr and Nrf2by AhR binding with Nrf2.Methods:Chromatin immunoprecipitation were carried out to analyse TCDD induce both AhR and Nrf2binding with ARE motif in NQO1promoter region. RT-PCR were used to measure TCDD induced Ho-1mRNA expression. Co-immunoprecipitation experiments were performed to observe Ahr and Nrf2binding and Ahr and keapl binding.Other methods, including cell culture, Western Blotting assay, and so on, are also used in this section with similar procedure to Part1. Results:Chromatin immunoprecipitation analyses revealed that treatment with TCDD recruits both AhR and Nrf2to the promoter region where a DRE and an ARE locate; the finding is in agreement with the result from genetic studies in which induction by TCDD or Bap was shown to require both AhR and Nrf2. TCDD-induced binding of AhR and Nrf2to DNA is time-dependent and TCDD-induced Nrf2and ARE binding with delayed kinetics compared with activation of Ahr. RT-PCR results reveal TCDD failed to stimulate the expression of Ho-1and significant oxidative stress in the cells. Co-immunoprecipitation experiments revealed that, in addition to AhR-Arnt binding, TCDD induced an interaction between AhR and Nrf2as well as Keapl. Conclusions:The study demonstrates that TCDD induces multi-protein complexes to mediate the cross-interaction between the AhR and Nrf2pathways, thus uncovering a novel mechanistic aspect of gene regulation by environmental chemicals through AhR and Nrf2. and provide a molecular model for studying the induction of NQO1, GSTs, and UGTs by AhR agonists.