Nrf3 negatively regulates antioxidant-response element-mediated expression and antioxidant induction of NAD(P)H:quinone oxidoreductase1 gene.
Antioxidant-response element (ARE) and nuclear factor Nrf2-mediated expression and coordinated induction of genes encoding chemopreventive proteins, including NQO1, are critical mechanisms in chemoprotection. Recently, Nrf3, a new member of the Nrf family with substantial homology to Nrf2, was identified and cloned. In this report, we have investigated the role of Nrf3 in ARE-mediated gene expression and induction of NQO1 in response to antioxidants. Overexpression of Nrf3 in Hep-G2 cells led to a concentration-dependent decrease in transfected and endogenous NQO1 gene expression and induction in response to antioxidant tert-butylhydroquinone (t-BHQ). Deletion mutation analysis revealed that Nrf3 repression of NQO1 gene expression required heterodimerization and DNA binding domains but not transcriptional activation domain of Nrf3. Bandshift and supershift assays with in vitro transcribed and translated proteins and nuclear extracts from Hep-G2 cells treated with Me2SO and t-BHQ and immunoprecipitation assays demonstrated that Nrf3 associates with small Maf proteins to bind to the ARE. RNA interference specific to Nrf3 reduced intracellular Nrf3 leading to increased expression and induction of transfected and endogenous NQO1 gene expression in response to t-BHQ. These results combined suggest that Nrf3 is a negative regulator of ARE-mediated gene expression.
Sankaranarayanan
Kannan
K
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030, USA.
Jaiswal
Anil K
AK
D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D047468:Immunoprecipitation; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry* / Q000378:metabolism*; D051267:NF-E2-Related Factor 2; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection
D000975:Antioxidants; D050976:Basic-Leucine Zipper Transcription Factors; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D006873:Hydroquinones; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; C117963:NFE2L3 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; C018855:2-tert-butylhydroquinone; D008156:Luciferases; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human