Thioredoxin reductase in human hepatoma cells is transcriptionally regulated by sulforaphane and other electrophiles via an antioxidant response element.
We previously reported the in vitro and in vivo induction of thioredoxin reductase (TR) by sulforaphane (SF) purified from broccoli. The present study was designed to determine whether this induction is mediated by putative antioxidant response elements (ARE) found in the promoter. Luciferase reporter constructs were built using the TR promoter sequence. Sulforaphane, tert-butylhydroquinone and beta-napthoflavone, as well as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), increased luciferase activity in HepG2 cells transfected with the reporter construct (P < 0.0001). Quinone reductase (QR) is an enzyme with a well-characterized ARE, and QR reporter constructs built as positive controls showed similar patterns of induction. Mutation of the core sequence of a putative ARE in the TR promoter drastically decreased inducibility by SF, but mutations in nonconsensus areas of the ARE and outside of the ARE did not affect inducibility. Results from electrophoretic mobility shift assay analysis corroborated mutated reporter gene findings. Induction by TPA was not affected by mutation of the putative ARE. Se plus SF, and SF alone were equally effective for induction of TR reporter luciferase activity (P < 0.0001); Se alone had no effect. Se and SF independently increased TR activity (P < 0.0001) and when combined, increased TR activity synergistically (P = 0.036). These data suggest that TR is transcriptionally regulated by electrophilic compounds via an ARE in the 5' region of the gene, and that this mechanism is unrelated to the established Se-dependent induction of selenoproteins.
Hintze
Korry J
KJ
Department of Animal and Range Sciences, North Dakota State University, Fargo, ND 58105, USA.
Wald
Karl A
KA
Zeng
Huawei
H
Jeffery
Elizabeth H
EH
Finley
John W
JW
D000975:Antioxidants / Q000378:metabolism*; D001483:Base Sequence / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D006801:Humans; D017879:Isothiocyanates; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D020218:Response Elements / Q000235:genetics / Q000502:physiology*; D012643:Selenium / Q000494:pharmacology; D013454:Sulfoxides; D013861:Thiocyanates / Q000008:administration & dosage / Q000494:pharmacology*; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects* / Q000502:physiology*; D014407:Tumor Cells, Cultured
D000975:Antioxidants; D004338:Drug Combinations; D017879:Isothiocyanates; D013454:Sulfoxides; D013861:Thiocyanates; D016660:NAD(P)H Dehydrogenase (Quinone); D013880:Thioredoxin-Disulfide Reductase; C016766:sulforaphane; D012643:Selenium