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Department of Animal and Range Sciences, North Dakota State University, Fargo, ND 58105;
Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801; and
* U.S. Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, ND 58202-9034
4To whom correspondence should be addressed. E-mail: jfinley{at}gfhnrc.ars.usda.gov.
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 ß-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.
KEY WORDS: • thioredoxin reductase • antioxidant response element • selenium • sulforaphane
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