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Nutrient-Gene Interactions

Thioredoxin Reductase in Human Hepatoma Cells Is Transcriptionally Regulated by Sulforaphane and Other Electrophiles via an Antioxidant Response Element¹–^,3

Korry J. Hintze, Karl A. Wald^*, Huawei Zeng^*, Elizabeth H. Jeffery and John W. Finley^*,4

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.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
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

Electrophilic substances such as sulforaphane (SF)⁴ have been demonstrated to upregulate multiple antioxidant and detoxification enzymes including quinone reductase (QR), -glutamylcysteine synthetase and glutathione-S-transferase (1). The induction is mediated by the antioxidant response element (ARE) found in the promoter regions of these genes (1). The ARE is a short cis-acting element with a consensus sequence of 5' TGAC-nnn-GC-3' (2).

Thioredoxin reductase (TR) is a selenoprotein that is the primary catalyst for the NADPH-dependent reduction of thioredoxin (3). Thioredoxin, the substrate of TR, is an ubiquitous small peptide with a redox active thiol group (4). It is essential for many biochemical reactions (e.g., ribonucleotide reduction, sulfate and disulfide reduction) that may be involved in the regulation of cell growth and/or differentiation (4). Post-transcriptional regulation of TR by dietary selenium (Se) is well documented; TR activity drops to <5% of normal controls in livers of Se-deficient rats (5) and in Se-depleted Ht-29 colon carcinoma cells (6).

We examined the possibility that TR is transcriptionally regulated by SF and other electrophilic compounds known to upregulate a battery of host-defense enzymes via an ARE-containing promoter (Fig. 1). Induction of a gene by the ARE is mediated through the Nrf2 transcription factor (1). In the unstimulated condition, Nrf2 is thought to bind to a cytosolic protein called keap1. When oxygen tension in the cell is high, (i.e., introduction of electrophilic compounds such as SF), Nrf2 is released from keap1, phosphorylated by protein kinase c and translocated to the nucleus where it binds as a heterodimer to the ARE and induces transcription (1).

View larger version (42K): [in this window] [in a new window]   FIGURE 1 Putative antioxidant response elements (ARE 1 and ARE 2) found in the human thioredoxin reductase (TR) promoter as sequenced by Rundlof and co-workers (10). Mutations made to the TR putative ARE regulatory area are listed. All mutations were produced using the QuickQuange XL mutagenesis kit (Stratagene). All mutations were sequenced to verify genotype. Deviations from the wild-type sequence are in bold.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Luciferase reporter gene constructs

    Thioredoxin reductase promoter constructs. The entire promoter region of TR (basepairs 1–827, accession number AF 247671) (10) was amplified by standard PCR using commercially available human genomic DNA (Clonetech, Palo Alto, CA). Primers were designed to incorporate a MluI restriction site on the 5' end of the amplified TR promoter sequence and a HindIII site on the 3' end (5'-GCC GAC GCG TCT GGA GTT AAA AGA CAC-3' and 5'-CTT ATA AGC TTC TGG GCT CGC GGC TTT G-3'). All primers were synthesized by Integrated DNA Technologies, Coralville, IA. The amplified sequence was blunt end cloned into pBCSK- (Stratagene, La Jolla, CA) using EcoR5 restriction endonuclease (New England Biolabs, Cambridge, MA). The pBCSK- plasmid was transformed into competent cells (Novagen, Madison, WI) and plated onto agar plates containing IPTG (200 g/L agar, Promega, Madison, WI) and X-Gal (50 g/L agar, Promega) to facilitate blue/white screening. Recombinants were identified, amplified and digested by MluI and HindIII (New England Biolabs). The insert was directionally cloned into pGL-3 basic (Promega). The same protocol, restriction enzymes and plasmids were used to generate the TR promoter insert in reverse orientation using the primers 5'-CGG CGA AGC TTC TGG AGT TAA AAG ACA C-3' and 5'-TTA TAC GCG TCT GGG CTC GCG GCT TTG-3'.

    Quinone reductase. Quinone reductase (QR) luciferase promoter constructs were generated by amplification of the entire rat QR regulatory sequence using pDTD1097-CAT as a template (accession number M58495) (11) and primers engineered to incorporate MluI and HindIII restriction sites (5'-CTT ATA CGC GTT GGA GGT CAG ATG AGG-3' and 5'-TCA CCA TAA GCT TAG CTG TAC TGA GCA C-3'). The amplified sequence was then inserted into pBCSK-. Recombinants were selected by blue/white screening, amplified and digested by MluI and HindIII. The insert was then directionally cloned into pGL-3 basic. All constructs were sequenced (Davis Sequencing, Davis, CA) to verify homology to published sequences (10,11).

    Mutation constructs. Mutations to the TR promoter construct (see Fig. 1) were made using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) following the manufacturer protocols. The following mutations were created:

Mutation 1: change of the core consensus sequence of the putative ARE 1, mutation of -47 to -50 sequence of 5'-TGAC-3' to 5'-GACA-3' of the noncoding strand using the mutagenic primers: 5'-GGA TTT CTG CTT TTG TCT TCT GAC TCT GGC AGT TAG CCC G-3' and 5'-CGG GCT AAC TGC CAG AGT CAG AAG ACA AAA GCA GAA ATC-C-3'.

Mutation 2: change of the core consensus sequence of the putative ARE 2, mutation of -45 to -42 sequence of 5'-TGAC-3' to 5'-GAGT-3' using the mutagenic primers: 5'-GCT TTG TCA TTC GAG TTC TGG CAG TTA GCC CGC CCG CTC G-3' and 5'-CGA GCG GGC GGG CTA ACT GCC AGA ACT CGA ATG ACA AAG G-3'.

Mutation 3: revertant of Mutation 2 to wild-type, -45 to -42 5'-GAGT-3' to 5'-TGAC-3' using the mutagenic primers: 5'-GCT TTG TCA TTC TGA CTC TGG CAG TTA GCC CGC CCG CTC G-3' and '-CGA GCG GGC GGG CTA ACT GCC AGA GTC AGA ATG ACA AAG G-3'.

Mutation 4: mutation of "wobble" base pairs within ARE 2, change of -40 to -38 5'-TCT-3' to 5'-AGA-3' using the mutagenic primers: 5'-GCT TTG TCA TTC TGA CAG AGG CAG TTA GCC CGC CCG CTC G-3' and 5'-CGA GCG GGC GGG CTA ACT GCC TCT GTC AGA ATG ACA AAG C-3'.

Mutation 5: mutation 500 bp upstream of the putative TR ARE regulatory region, change of -545 to -542 5'-CATT-3' to 5'-GTAA-3' using the mutagenic primers: 5'-GGT GTG TTC CTA AGA ATT TGT GTA ATC TTT TCA GGA TTA GTC AGG-3' and 5'-CCT GAC TAA TCC TGA AAA GAT TAC ACA AAT TCT TAG GAA CAC ACC-3'.

    Electrophoretic mobility shift assay. T4 polynucleotide kinase was obtained from Promega. ATP (-³²P) and CTP (a-³²P) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Oligonucleotides were synthesized by Gibco BRL (Rockville, MD). Control and 2 mol/L SF-treated HepG2 cells were washed twice with ice-cold PBS and subjected to centrifugation at 300 x g for 5 min at 4°C. The cells were lysed for 15 min on ice in extraction buffer A (20 mmol/L HEPES pH 7.6, 20% glycerol, 10 mmol/L NaCl, 1.5 mmol/L MgCl₂, 0.2 mmol/L EDTA, 1 mmol/L dithiothreitol, 0.1% Nonidet P-40 with protease inhibitors). After centrifugation at 300 x g for 5 min at 4°C, the supernatant was designated as cytoplasmic protein and kept at -80°C, and nuclei (pellets) were collected and lysed for 30 min on ice in extraction buffer B (same as buffer A but with 0.5 mol/L instead of 10 mmol/L NaCl), and then centrifuged at 15000 x g for 15 min at 4°C. The supernatant was designated nuclear protein and kept at -80°C. Protein concentrations were determined by the Bio-Rad protein assay.

DNA-binding activities of putative ARE elements were analyzed by electrophoretic mobility shift assay (EMSA) as described previously (12). Sequences of double-stranded oligonucleotide probes used in these assays were as followings (mutations in bold): wild-type putative TR ARE regulatory region -57 to -34, 5'-CTG CTT TGT CAT TCT GAC TCT GGC A-3'; Mutation 1, 5'-CTG CTT TGT CAT TCG AGT TCT GGC A-3'; Mutation 2, 5'-CTG CTT TTG TCT TCT GAC TCT GGC A-3'; Combined Mutation 1 and 2, 5'-CTG CTT TTG TCT TCG AGT TCT GGC A-3'.

Cell culture

    Cells and conditions. Human hepatoma HepG2 cells were obtained from the American Tissue Culture Collection (Rockville, MD) and seeded at 8.0 x 10⁵ cells/dish. Cells were grown at 37°C in 95% ambient air and 5% CO₂ on 60 x 15 mm collagen-coated culture dishes (Corning, Corning, NY). Cells were grown for 24 h in control medium, MEM (Sigma, St. Louis, MO) with 2.2 mol/L sodium bicarbonate, 1 mmol/L sodium pyruvate and 10% fetal bovine serum. The Se concentration of the basal medium was 17.6 nmol/L.

    Promoter construct transfection. After 24 h of growth, cells were transfected with the TR, QR or mutated TR promoter constructs along with pRL-SV40, an expression vector for renilla luciferase (Promega). Transfections were performed using FuGene6 according to the manufacturer’s instructions (Roche Biochemicals, Indianapolis, IN).

    Experimental design. Twenty-four hours after transfection, Hep-G2 cells were exposed to media containing different concentrations of the test chemical. All experiments used 3 or 4 culture dishes/treatment and were replicated twice. Experiment 1 used 0, 0.5, 1.0 or 2.0 µmol/L SF dissolved in dimethyl sulfoxide (DMSO, Sigma). All media contained a final DMSO concentration of 0.1%. Sulforaphane was purified from broccoli seeds by the method of Matusheski et al., (13). Purity was determined to be >99% by GC analysis. After 24 h of exposure to experimental treatments, cells were washed with PBS and lysed in 1.8 mL Passive Lysis Buffer (Promega) for 15 min at room temperature, transferred to 2-mL tubes and frozen at -80°C.

Experiment 2 was similar to Experiment 1 except that cells were exposed to 0, 2, 5 or 10 µmol/L TBHQ (Aldrich, Milwaukee, WI) dissolved in DMSO. Experiment 3 used cells exposed to 0, 0.5, 1.0 or 2.0 µmol/L ß-napthoflavone (BNF, Sigma) dissolved in DMSO. Experiment 4 used cells transfected with wild-type or mutated TR constructs exposed to 0 or 2 µmol/L SF. Nuclear extracts were isolated from an untransfected cohort exposed to the same treatments for use in EMSA assays. Experiment 5 was similar to Experiment 4 except that only wild-type TR and mutations 1 and 2 were transfected, and cells were exposed to 2 µmol/L SF, 0.1 µmol/L phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma) in 0.1% DMSO, both 2 µmol/L SF and 0.1 µmol/L TPA, or control medium. Experiment 6 examined the interaction of Se and SF on TR induction in a 2 x 2 factorial design. Cells were exposed to media with 0 or 2 µmol/L SF and 0 or 2 µmol/L Se as sodium selenite (Sigma); all media contained 0.1% DMSO. The effects of treatment on TR enzyme activity were determined in a cohort group of untransfected cells subjected to the same media and conditions. Cells were exposed to experimental media for 24 h, trypsinized, washed with PBS, sonicated and frozen at -80° until TR enzyme activity was determined.

Biochemical analyses

    Luciferase assay. Cell lysates were analyzed for firefly and renilla luciferase activities using the Dual-Luciferase Reporter Assay System (Promega), following the manufacturer’s protocol. Luminescence was measured on a Turner Designs TD 20/20 luminometer (Sunnyvale, CA). To normalize data, all values are reported as the ratio of firefly luminescence (experimental plasmid) to renilla luminescence (pRL-SV40, noninducible plasmid).

    Thioredoxin reductase assay. Thioredoxin reductase activity was determined spectrophotometrically using the method of Holmgren and Bjornstedt (14) as modified by Hill et al. (15). Enzymatic activity was determined by subtracting the time-dependent increase in absorbance at 412 nm in the presence of the TR activity inhibitor aurothioglucose from total activity. One unit of activity was defined as 1µmol TNB formed/(min · mg protein). Protein concentrations were determined by the BioRad assay (Hercules, CA).

Statistical analysis

Treatment effects were determined using analysis of variance (ANOVA). Experiments 1–4 were analyzed by 1-way ANOVA, and Experiments 5 and 6 were analyzed by 2-way ANOVA. When a significant effect (P < 0.05) was found, Tukey’s Studentized Range was used to determine differences between means (16).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Experiment. 1.

Sulforaphane dose-dependently increased TR (Fig. 2A) and QR (Fig. 2B) transcription (P < 0.0001). The patterns of induction were similar in the TR and QR constructs, except that the TR construct responded in a dose-dependent manner up to 2 µmol/L SF (P < 0.0001), whereas the QR construct reached a plateau at 1 µmol/L SF. Both constructs showed approximately two-fold increases in luminescence between the 0 and 2 µmol/L SF treatments. The negative controls (reverse-oriented TR constructs) had a basal luminescence of <5% of comparable constructs in the correct orientation; in addition, SF had no effect on the parent vector pGL3 luminescence compared with controls (data not shown).

View larger version (42K): [in this window] [in a new window]   FIGURE 2 Sulforaphane (SF) induction of the human thioredoxin reductase luciferase promoter construct (panel A) and the rat quinone reductase luciferase promoter construct (panel B) transfected into HepG2 cells. Twenty-four h after transfection, cells were exposed to MEM medium containing 0, 0.5, 1.0 or 2 µmol/L SF dissolved in dimethyl sulfoxide (DMSO). All media were formulated to contain 0.1% DMSO. Luciferase activities were determined 24 h after exposure to experimental media. Data are the mean ratio of firefly luminescence to renilla luminescence. Values are means ± SD, n = 3. Means without a common letter differ (P < 0.05).

Thioredoxin reductase and QR constructs dose dependently increased luminescence when exposed to TBHQ (P < 0.0001, Table 1). Quinone reductase exhibited a dose-response from 2.5 to 10 µmol/L TBHQ (P < 0.0001) and TR from 2.5 to 5 µmol/L.

BNF dose dependently increased luminescence for both TR and QR over the entire range of concentrations (0 to 2 µmol/L, P < 0.0001 Table 1). The TR construct was induced a maximum of 2.7-fold, whereas QR had an almost fivefold range of induction.

Experiment. 4.

Mutations of the core sequences 5'-TGAC-3' of either putative ARE (Mutation 1, mutated ARE 1; Mutation 2, mutated ARE 2, Fig. 1) caused a significant loss of SF inducibility compared with the wild-type TR promoter genotype (P < 0.0001, Fig. 3). However, when Mutation construct 2 was reverted to the original genotype, inducibility by SF was restored (P < 0.0001, Fig. 3). Mutation of the nonconsensus "wobble" basepairs of ARE 2 (Mutation 4) and the mutation outside both of the putative ARE (Mutation 5) did not affect SF inducibility (P < 0.0001, Fig. 3).

View larger version (52K): [in this window] [in a new window]   FIGURE 3 Sulforaphane (SF)-mediated inducibility of thioredoxin reductase (TR) mutation constructs transfected into HepG2 cells (see Fig. 1). Twenty-four hours after transfection of mutation constructs, cells were exposed to media containing 0 or 2.0 µmol/L SF dissolved in dimethyl sulfoxide (DMSO). All media were formulated to contain 0.1% DMSO. Luciferase activities were determined 24 h after exposure to experimental media. Fold induction for each construct was determined as the firefly/renilla luminescence of 2 µmol/L SF treated cells divided by the firefly/renilla luminescence of control cells from 3 separate experiments. Values are means ± SD, n = 3. Means without a common letter differ (P < 0.05).

Only nuclear extracts bound to the labeled TR probe (Fig. 4). Treatment of cells with SF did not produce a discernible change in transcription factor binding to the labeled probe compared with the control. Transcription factors preferentially bound to the 100 fmol unlabeled TR probe when competing with the 2 fmol labeled TR probe. However, 100 fmol of unlabeled probe with the ARE 2 sequence mutated (mutation 2, see Fig. 1) had reduced ability to compete with the labeled wild-type probe. Conversely, unlabeled ARE 1 mutant probes (mutation 1, Fig. 1) were able to outcompete the labeled wild-type probes. When both mutations were combined into a single, unlabeled probe, the ability to compete with the labeled wild-type probe was reduced.

View larger version (83K): [in this window] [in a new window]   FIGURE 4 Effect of antioxidant response element (ARE) sequence on HepG2 nuclear protein DNA binding. Electrophoretic mobility shift assay of either cytoplasmic or nuclear protein (6 µg) bound to 2 fmol 32P labeled ARE probe. Lane 1 represents the binding activities of cytoplasmic proteins of control cells to 2 fmol 32P labeled wild-type ARE probe. Lane 2 represents the binding activities of cytoplasmic proteins of sulforaphane (SF)-treated cells to 2 fmol 32P labeled wild-type ARE probe. Lane 3 represents the binding activity of nuclear proteins of control cells to 2 fmol 32P labeled wild-type ARE probe. Lane 4 represents the binding activity of nuclear proteins of SF-treated cells to 2 fmol 32P labeled wild-type ARE probe. Lane 5 represents the binding activity of nuclear proteins of SF-treated cells to 2 fmol 32P labeled wild-type ARE probe in competition with 100 fmol of unlabeled wild-type ARE probe. Lane 6 represents the binding activity of nuclear proteins of SF-treated cells to 2 fmol 32P labeled wild-type ARE probe in competition with 100 fmol of unlabeled mutation 2 ARE probe (Fig. 1). Lane 7 represents the binding activity of nuclear proteins of SF-treated cells to 2 fmol 32P labeled wild-type ARE probe in competition with 100 fmol of unlabeled mutation 1 ARE probe. Lane 8 represents the binding activity of nuclear proteins of SF- treated cells to 2 fmol 32P labeled wild-type ARE probe in competition with 100 fmol of an unlabeled ARE probe that contained both mutation 1and 2. NS stands for nonspecific DNA binding. No ARE binding signals for Lanes 1 and 2, and much weaker ARE binding signals for Lanes 5 and 7 were consistently seen in all four different cytoplasmic and nuclear protein preparations.

The wild-type TR promoter was significantly induced by TPA but this induction was less than the induction by SF (P < 0.0001, Fig. 5). When SF and TPA were combined, induction was not different from TPA treatment alone. Cells transfected with mutation constructs 1 and 2 had a reduced response to SF treatment similar to Experiment 4. However, TPA treatment induced luciferase in these mutated constructs (P < 0.0001, Fig. 5).

View larger version (32K): [in this window] [in a new window]   FIGURE 5 Sulforaphane (SF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) induction of human wild-type thioredoxin reductase (TR) luciferase promoter construct, mutation construct #1 (see Fig. 1) and mutation construct #2 transfected into HepG2 cells. Twenty-four hours after transfection, cells were exposed to MEM medium containing 2 µmol/L SF, 0.1 µmol/L TPA, 2 µmol/L SF + 0.1 µmol/L TPA or control. SF and TPA were dissolved in dimethyl sulfoxide (DMSO). All media were formulated to contain 0.1% DMSO. Luciferase activities were determined 24 h after exposure to experimental media. Data are the mean ratio of firefly luminescence to renilla luminescence. Values are means ± SD, n = 3. Columns within the same plasmid treatment without a common letters differ (P < 0.05).

When HepG2 cells containing the wild-type TR construct were incubated with SF and Se alone or together, 2 µmol/L SF increased luminescence similarly to that reported in Experiment 1 (P < 0.001), whereas 2 µmol/L selenite had no effect on luminescence (P = 0.29) and there was no interaction between Se and SF (P = 0.81, Fig. 6A). Conversely, in untransfected HepG2 cells subjected to the same treatments, both Se and SF increased TR activity (P < 0.0001), and when combined, synergistically increased TR activity (P < 0.05, Fig. 6B).

View larger version (38K): [in this window] [in a new window]   FIGURE 6 Sulforaphane (SF) and/or selenium (Se) induction of human thioredoxin reductase luciferase promoter constructs transfected into HepG2 cells (panel A) and thioredoxin reductase activity mU/(mg protein · min) of human hepatoma HepG2 cells (panel B). Twenty-four hours after transfection, cells were exposed to experimental media as follows: Se as selenite, SF dissolved in dimethyl sulfoxide (DMSO), both Se and SF, or control media for 24 h. All media were formulated to contain 0.1% DMSO. (A) Luciferase activities were determined 24 h after exposure to experimental media. Data are the mean ratio of firefly luminescence to renilla luminescence. Values are means ± SD, n = 3. ANOVA: effect of SF (P < 0.0001), effect of Se (P = 0.29), SF x Se (P = 0.81). (B) Data are mean TR activity ± SD, n = 3. ANOVA: effect of SF (P < 0.0001), effect of Se (P < 0.0001), SF x Se (P = 0.036).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

By building TR-luciferase promoter constructs we isolated regulatory mechanisms specific to the promoter region of the gene. We demonstrated that these TR promoter constructs are induced by electrophilic compounds and the pattern of induction is similar to that seen with QR promoter constructs. This is important because the ARE-dependent induction of quinone reductase is well characterized (2). We also characterized the sequences responsible for this induction by mutation analysis and found that the TR ARE requires the same core consensus sequence as other reported ARE sequences (1). Because of the similarities of induction of TR and QR constructs, we suggest that both are part of a coordinated antioxidant defense system upregulated via the ARE by oxidative stress.

Promoter elements such as the ARE allow concerted eukaryotic gene expression for multiple related genes. Thioredoxin reductase reduces thioredoxin and Kim et al. (19) reported that thioredoxin had a functional ARE that responds to the Nrf2 transcription factor. Thioredoxin reductase obtains its reducing equivalents from NADPH; a recent report described the induction of NADPH-generating enzymes glucose 6 phosphate dehydrogenase and malic enzyme by SF treatment (20). Therefore it is logical that these genes are regulated by a similar mechanism such as the ARE.

Analysis of the TR promoter sequence (10) (accession number AF 247671) revealed two possible ARE; one is on the noncoding strand from bp -57 to -49 and the other is on the coding strand from bp -45 to -36 (Fig. 1). The -57 to -49 sequence is identical to the ARE found in GST-A2 (21). The flanking sequences of the TR ARE match the consensus ARE sequence and have homology to other ARE flanking sequences such as human and rat QR (22) (Fig. 7). Mutation of the core sequence of this ARE abolished SF inducibility, and EMSA analysis revealed a change in nuclear protein binding to DNA (Fig. 4).

View larger version (32K): [in this window] [in a new window]   FIGURE 7 Homology comparison of putative antioxidant response elements (ARE) found in the thioredoxin reductase promoter to ARE found in other genes and to the consensus ARE sequence including flanking regions. Abbreviations follow standard IUPAC nomenclature (M = A or C; R = A or G; Y = C or T; W = A or T;S = G or C). Adapted from Wasserman and Fahl (22).

The present data are consistent with our understanding of selenoprotein synthesis in that Se had no effect on TR transcription (24). However, when Se and SF were added to the medium of nontransfected cells, TR activity was increased by each compound, and was increased synergistically when both were added simultaneously. Thus modulation of TR activity by SF and Se occurs independently; SF works transcriptionally, whereas Se increases TR activity by a post-transcriptional mechanism. Thioredoxin reductase activity is increased by dietary Se; Hill et al. (5) reported that TR activity in Se-deficient rat liver was 4.5% that of Se-adequate controls. Berggren et al. (25) found that supranutritional concentrations of Se in rat diets increased TR activity but had little effect on TR protein concentrations, possibly by increasing the specific activity of TR. Similarly, Hadley and Sunde (26) found that Se deficiency decreased TR activity with only modest decreases in TR mRNA. Thus Se had little, if any, effect on TR transcription, a conclusion supported by the present data. These data provide initial evidence that the regulation of TR activity is accomplished by multiple mechanisms, by Se and by a classical upstream promoter sequence.

Supranutritional Se and cruciferous vegetable intake have separately been implicated as protective against certain cancers. Selenium supplementation significantly decreased prostate cancer as well as total cancer incidence in a long-term, double blind study (27). However, it is unclear what role, if any, TR plays in cancer. Thioredoxin reductase is the primary enzyme that reduces thioredoxin, and reduced thioredoxin is associated with increased cell growth and decreased apoptosis, conditions that promote the growth of cancerous cells [for a review, see (4)], suggesting that increased TR activity may favor the growth of cancerous cells. Yet TR functions as an antioxidant, and antioxidants are considered to reduce the risk of cancer. The present data suggest that TR is regulated like other endogenous antioxidants such as QR, glutathione and the glutathione-S-transferases all of which are positively associated with cancer prevention. Epidemiologic studies also have identified a correlation between cruciferous vegetable intake and the prevention of certain cancers (28). The mechanism is thought involve the upregulation of antioxidant and detoxification enzymes through an ARE-dependent mechanism, triggered by bioactive components such as SF (28). The present data suggest that TR may belong to this family of antioxidant and detoxification enzymes. The complete role of TR in carcinogenesis remains to be clarified. The data from the present study suggest that TR may be regulated by both an ARE-containing promoter and by Se. Further research is warranted to determine in vivo whether these pathways are coordinately regulated, or whether they are synergistic, antagonistic or independent of each other. Nevertheless, the existence of these two regulatory pathways has implications for how nutrient-nutrient interactions in food may alter potential chemoprotective pathways in the body.

	FOOTNOTES

 
¹ Supported by USDA/IFAFS grants # 00–04766 and 00–52102-9636.

² The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination.

³ Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.

⁵ Abbreviations used: AP-1, activator protein-1; ARE, antioxidant response element; BNF, ß-napthoflavone; DMSO, dimethyl sulfoxide; EMSA, electrophoretic mobility shift assay; QR, quinone reductase; SF, sulforaphane; TBHQ, tert-butylhydroquinone; TPA, 12-O-tetradecanoylphorbol-13-acetate; TR, thioredoxin reductase; TRE, TPA response element.

Manuscript received 1 April 2003. Initial review completed 27 May 2003. Revision accepted 25 June 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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