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Originally published In Press as doi:10.1074/jbc.M500166200 on February 24, 2005

J. Biol. Chem., Vol. 280, Issue 17, 16891-16900, April 29, 2005
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Bach1 Competes with Nrf2 Leading to Negative Regulation of the Antioxidant Response Element (ARE)-mediated NAD(P)H:Quinone Oxidoreductase 1 Gene Expression and Induction in Response to Antioxidants*

Saravanakumar Dhakshinamoorthy{ddagger}§, Abhinav K. Jain{ddagger}, David A. Bloom, and Anil K. Jaiswal

From the Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030

Received for publication, January 5, 2005 , and in revised form, February 17, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The antioxidant response element (ARE) and Nrf2 are known to regulate the expression and coordinated induction of genes encoding detoxifying enzymes including NAD(P)H:quinone oxidoreductase1 (NQO1) in response to antioxidants. In this report, we demonstrate that overexpression of the transcription factor Bach1 in Hep-G2 cells negatively regulated NQO1 gene expression and induction in response to antioxidant t-BHQ. Bandshift and supershift assays revealed that Bach1 binds to the ARE as a heterodimer with small Maf proteins but not as a homodimer or heterodimer with Nrf2. The transfection and ChIP assays revealed that Bach1 and Nrf2 competed with each other to regulate ARE-mediated gene expression. Heme, a negative regulator of Bach1 relieved the Bach1 repression of NQO1 gene expression in transfected cells. The transcription of Bach1 and Nrf2 did not change in response to t-BHQ. Immunofluorescence assays and Western blot analysis revealed that both Bach1 and Nrf2 localized in the cytoplasm and nucleus of the untreated cells. The treatment of cells with t-BHQ resulted in the nuclear accumulation of both Bach1 and Nrf2. Interestingly, the t-BHQ-induced nuclear accumulation of Bach1 was significantly delayed over that of Nrf2. These results led to the conclusion that a balance of Nrf2 versus Bach1 inside the nucleus influences up- or down-regulation of ARE-mediated gene expression. The results further suggest that antioxidant-induced delayed accumulation of Bach1 contributes to the down-regulation of ARE-regulated genes, presumably to reduce the antioxidant enzymes to normal levels.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Antioxidant response element (ARE)1 is known to regulate the expression and coordinated induction of many detoxifying enzyme genes in response to antioxidants and xenobiotics (1, 2). These include NAD(P)H:quinone oxidoreductase1 (NQO1) that catalyzes the two-electron reductive metabolism and detoxification of quinones (3, 4); glutathione S-transferase Ya subunit (GST Ya), which conjugates electrophiles and ROS with glutathione (5); {gamma}-glutamylcysteine synthetase ({gamma}-GCS) that plays a role in glutathione metabolism (6); and heme oxygenase-1 (HO-1), which plays a role in heme catabolism (7). The coordinated induction of this battery of genes protects cells against free radical damage, oxidative stress, and neoplasia (1, 2). The ARE contains AP1/AP1-like elements arranged either as inverse or direct repeats followed by a GC box (1).

Several nuclear proteins have been shown to bind to the ARE either as homodimers or heterodimers (1, 2). Analysis of ARE-nuclear protein complexes have identified a number of nuclear transcription factors including c-Jun, Jun-B, Jun-D, c-Fos, Fra1, Nrf1, Nrf2, YABP, ARE-BP1, MafG, MafK, Ah (aromatic hydrocarbon) receptor, and the estrogen receptor (1, 2). Among these transcription factors, c-Jun, Jun-B, Jun-D, c-Fos, Fra1, Nrf1, and Nrf2 and small Maf proteins have been shown to bind to the human NQO1 gene ARE (1, 2, 8). More recently, a cytosolic inhibitor of Nrf2 (Keap1/INrf2) was identified (9, 10). Under normal conditions, Keap1/INrf2 retains Nrf2 in the cytoplasm. Exposure of cells to antioxidants leads to the release of Nrf2 from Keap1/INrf2. Nrf2 then translocates to the nucleus leading to the activation of ARE-mediated gene expression (9, 10). Small Maf (MafG, MafK, and MafF) proteins are b-Zip proteins that lack a transcriptional activation domain. They are known to form homodimers and heterodimers with other b-Zip proteins including Nrf2. Maf-Maf homodimers repressed, but Maf-Nrf2 heterodimers activated, the transcription of Maf recognition element (MARE)-regulated {beta}-globin gene (11). The role of small Maf proteins has also been investigated in ARE-mediated detoxifying enzyme gene regulation (8, 12, 13). The Maf-Maf homodimers repressed NQO1, GST Ya, and {gamma}-GCS gene expression and antioxidant induction (8, 12, 13). However, the role of Nrf2-Maf heterodimers in ARE-mediated gene regulation is controversial and requires further investigation (8, 12, 13).

The mammalian transcription factors Bach1 and Bach2 belong to the cap'n'collar (CNC), b-Zip family of proteins that include Nrf1, Nrf2, and Nrf3 (14). In addition to CNC and b-Zip domains, the Bach proteins also have a BTB domain at the N terminus. Bach proteins are the only transcription factors in which the BTB domains are associated with the b-Zip domains. The Bach proteins were originally identified as a heterodimeric partner molecule of MafK (14). Bach proteins are known to bind to the NF-E2 site and MARE as heterodimers with MafK (14, 15). A recent report has shown that in normal cells Bach1 heterodimerizes with MafK and is bound to the {beta}-globin gene MARE resulting in the repression of the gene. Upon induction, Bach1 is replaced by Nrf2, resulting in activation and suggesting competition between Bach1 and Nrf2 for the same DNA binding site in different cellular states (16). Bach1 is ubiquitously expressed, whereas Bach2 is specific for neural tissues and B lymphoid cells (14, 17). Recently, an alternatively spliced truncated form of Bach1, Bach1t, was cloned and sequenced (18). It was suggested that Bach1t recruits Bach1 to the nucleus through BTB domain-mediated interaction. Recent molecular investigation of Bach2 has revealed that the subcellular localization of Bach2 is regulated by oxidative stress-sensitive nuclear export, mediated by crm1/exportin1 protein. This process of nuclear export is directed by the cytoplasmic localization signal (CLS) found in the C-terminal region of Bach2 (17). The heavy metal cadmium induced the nuclear export of Bach1, a transcriptional repressor of the heme oxygenase-1 gene (19). Heme was identified as a negative regulator of Bach1 (20). Heme is known to bind directly with Bach1, inhibiting its binding to DNA and thus leading to increased MARE-mediated gene expression (20). The ChIP assays in a recent report have suggested the dynamic exchange of Bach1- and NF-E2-related factors in the Maf transcription factor network at the MARE site (16). A role of Bach1 protein in antioxidant regulation of detoxifying enzyme genes is expected because of its sensitivity to oxidative stress. However, the experimental evidence suggesting a role for Bach1 protein in the antioxidant regulation of ARE-mediated detoxifying enzyme genes is lacking.

In the present report, we investigated the role of Bach1 in ARE-mediated gene expression and induction in response to antioxidants. We demonstrate that overexpression of Bach1 represses endogenous and transfected NQO1 gene expression. The studies also demonstrate that Bach1 forms heterodimers with small Maf to bind to ARE. The studies further suggest that Bach1 and Nrf2 compete with each other to regulate ARE-mediated gene expression in transfected cells. The Bach1 repression was reversed by heme, which is known to inhibit Bach1. The treatment of the cells with the antioxidant t-BHQ did not alter the Bach1 and Nrf2 mRNA transcript level. Bach1 and Nrf2 both were found localized in the cytoplasm and the nucleus under normal conditions. In response to t-BHQ, Bach1, and Nrf2 both accumulated in the nucleus. However, the rate of accumulation of Bach1 in the nucleus was significantly slower than Nrf2. These results suggest that Bach1 in association with small Maf proteins play a role in repression of ARE-mediated gene expression and induction presumably to participate in homeostatic control of ARE-regulated gene expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The enzymes used in this study, pcDNA3.1/V-5-His-TOPO, and anti-V5 antibodies were purchased from Invitrogen. {alpha}-minimum essential medium and Dulbecco's modified essential medium were purchased from Invitrogen. Effectene transfection reagent was purchased from Qiagen (Valencia, CA). The Nrf2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Actin antibodies, t-BHQ, and all other chemicals were purchased from Sigma. The pGL2 promoter plasmid containing firefly luciferase gene, internal control plasmid pRL-TK that encodes Renilla luciferase, the Dual Luciferase assay kit, and the TNT T7/T3-coupled rabbit reticulocyte lysate system were obtained from Promega (Madison, WI). The mouse liver Marathon ready cDNA library was obtained from Clontech Laboratories Inc, Palo Alto, CA. The Hybond ECL nitrocellulose membrane, ECL Western blot analysis kit, and Amplify NAMP1000 were purchased from Amersham Biosciences.

Plasmid Construction—The construction of the reporter plasmids pGL2-NQO1 ARE-Luc, pGL2-NQO1 mutant ARE-Luc, GST Ya ARE-Luc, and the expression plasmids pcDNA-Nrf2, pcDNA-MafG, pcDNA-MafK, pcDNA-MafG-V5, pcDNA-MafK-V5 are described (8). The mouse Bach1 cDNA was amplified from mouse liver marathon ready cDNA library using mouse Bach1-specific primers (forward, 5'-ACCATGTCTGTGAGTGAGAGTGCG and reverse, 5'-CTCGTCAGTAGTGCACTTGTCAGAC). The cDNA was cloned in pcDNA and designated as pcDNA-Bach1. The Bach1 cDNA was also subcloned inframe with the V5 epitope of the pcDNA expression plasmid. This plasmid encodes the V5-tagged Bach1 protein. The V5 epitope contains 14 amino acids in the sequence Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr. The various constructs were checked and confirmed by DNA sequencing.

Cell Culture, Transfection of Bach1 Expression Plasmid, and Analysis of Endogenous NQO1 Gene Expression—Human hepatoblastoma (Hep-G2) cells were grown in monolayer cultures containing {alpha}-minimum essential medium supplemented with fetal bovine serum. The Effectene transfection reagent kit was used to perform the transfections by previously described procedures (8). Briefly, varying concentrations of pcDNA-Bach1 expression plasmid were transfected into Hep-G2 cells. Forty-eight hours after transfection, Hep-G2 cells were washed with 1x PBS, scraped with a rubber policeman, and collected by centrifugation. The cells were homogenized briefly in 0.25 M sucrose supplemented with 0.1 mM phenylmethylsulfonyl fluoride to yield a homogenate with a protein concentration of 1–5 mg/ml. The homogenate was centrifuged at 14,000 rpm for 15 min at 4 °C, and the supernatant was removed and analyzed for NQO1 protein and activity. The NQO1 protein in the 14,000-rpm supernatant fraction was analyzed by 12% SDS/PAGE, Western blotting, and probing with NQO1 antibody as described (21). The NQO1 activity was determined by previously described procedures (21). The final reaction mixture contained 25 mM Tris/HCl, pH 7.4, 0.18 mg/ml bovine serum albumin, 5 µM FAD, 0.01% Tween 20, 200 µM NADH, and 50 µM 2,6-dichlorophenolindophenol. The reaction rate was monitored by measuring the decrease in absorption caused by reduction of 2,6-dichlorophenolindophenol at 600 nm. The activity was measured in the absence or presence of 20 µM dicoumarol, a specific inhibitor of NQO1 activity. The dicoumarol inhabitable NQO1 activity was determined by subtracting activity in the presence of dicoumarol from total activity.

Cotransfection of Reporter and Expression Plasmids—0.5 µg of reporter constructs (NQO1 ARE-Luc, mutant ARE-Luc, and GST Ya ARE-Luc) were mixed individually and in combination with different concentrations of the pcDNA alone or pcDNA expression plasmids (Bach1, MafK, and Nrf2) and transfected into Hep-G2 cells. The plasmid pRL-TK encoding Renilla luciferase was used as the internal control in each transfection. In related experiments, the NQO1 gene ARE-Luc was replaced with luciferase reporter plasmids in which 1.85 kb of the NQO1 promoter containing ARE or the NQO1 promoter containing an internal deletion of ARE-driving luciferase gene, and transfection assays were repeated. These NQO1 gene promoter plasmids were previously described (22). Forty-eight hours after transfection, the cells were washed with 1x PBS and lysed in 1x Passive lysis buffer from the Dual Luciferase reporter assay system kit. The same kit was used to assay samples for luciferase activity. The luciferase assay was performed by previously described procedures (8). Each set of transfections was repeated three times. For induction studies, the cells were treated with 50 µM t-BHQ, dissolved in Me2SO for 16 h, and analyzed for luciferase activity by procedures described above. The transfection experiments were performed using untagged and V5-tagged Bach1 and MafK expression constructs. Both sets of expression plasmids gave similar results. The presence or absence of V5 epitope tagged to the proteins had no effect on their activity/function. Therefore, we have shown transfection data only on proteins that do not have the V5 tag. The addition of the V5 tag to Maf and Bach1 proteins enabled us to use antibodies against V5 peptide in supershift assays (Ref. 8 and present report). This was necessary because the commercially available Bach1 antibodies are good only for Western and immunofluorescence assays, but not for supershift assays. In contrast, very good anti-V5 antibodies are commercially available from Invitrogen.

Gel Shift/Supershift Assays—The in vitro transcription/translation of the plasmids encoding Bach1, Bach1-V5, Nrf2, MafK, and MafK-V5 were performed using the TNT-coupled rabbit reticulocyte lysate system by procedures suggested in the manufacturer's protocol. L-[35S]Methionine was substituted for methionine in the reactions. After the coupled transcription/translation assay, proteins were checked for their correct size on a 10% PAGE gel with Western blot analysis. Briefly, 5 µl of the translated proteins were resolved on a 10% PAGE gel, treated with Amplify solution (NAMP 100; Amersham Biosciences) to enhance the 35S signal, dried, and exposed to x-ray film. In similar experiments, the proteins were transferred onto a Hybond ECL nitrocellulose membrane and probed with V5 antibodies. V5 antibodies were used to detect the V5-tagged Bach1 and Maf proteins. All of the in vitro translated proteins gave the expected size products. The NQO1 gene ARE was end-labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase. The labeled ARE was incubated with the in vitro translated proteins. Bandshift and supershift assays were performed by previously described procedures (8). Equimolar concentrations of the in vitro translated proteins were used in the gel shift and supershift experiments. Two micrograms of anti-V5 antibody or IgG was used in the supershift assays.

Northern Analysis—Hep-G2 cells were treated for different time intervals with either Me2SO (control) or t-BHQ dissolved in Me2SO. The cells were washed three times with ice-cold Dulbecco's PBS without calcium and magnesium. The cells were scraped and RNA isolated using an RNeasy mini kit from Qiagen. Ten micrograms of total RNA were run on a 1% formaldehyde agarose gel and blotted by procedures as described (23). cDNAs encoding Bach1, NQO1, and GAPDH were labeled in separate reactions using a Prime-a-Gene labeling kit (Promega) or Nick translation kit (Amersham Biosciences) and used as probe to hybridize RNA blots. Prehybridization and hybridization were done according to a method described previously (23, 24).

Chromatin Immunoprecipitation (ChIP) Assay—Hep-G2 cells were grown in 100-mm dishes and transfected with pcDNA, pcDNA-Bach1, or pcDNA-Nrf2 using the Effectene transfection reagent. Twenty-four hours after transfection, the cells were fixed, and the ChIP assay was performed using the ChIP-IT kit from Active Motif, Carlsbad, CA, according to the manufacturer's instructions. Briefly, proteins and DNA were cross-linked with 1% formaldehyde in media for 20 min at room temperature. The cells were washed twice with ice-cold PBS and treated with glycine to stop fixation. The cells were scraped in PBS containing protease inhibitors, collected by centrifugation, lysed in SDS-lysis buffer supplemented with protease inhibitors, and sonicated for 20 cycles (20-s pulse and 30-s rest on ice). The sonication conditions were optimized by procedures as described in the manufacturer's instructions and examined by agarose gel electrophoresis to determine generation of DNA fragments between 200 and 1000 base pairs in length. Sheared chromatin was immunocleared with protein G-agarose slurry for 1 h at 4 °C. A portion of the precleared chromatin was stored and labeled as "input DNA." The remaining chromatin was immunoprecipitated with IgG (control) and Bach1 and Nrf2 antibodies. The immunoprecipitates were washed sequentially with wash buffers for 2 min each. Protein-DNA complexes were eluted from the antibody with freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3). Formaldehyde cross-links were reversed by addition of 200 mM NaCl and heating at 65 °C for 4 h. DNA was purified using proteinase K treatment followed by phenol-extraction and ethanol precipitation. PCR was performed using 1:10-diluted input DNA and 5 µl of immunoprecipitated DNA from a 100-µl DNA extraction with a primer pair spanning the human NQO1 gene ARE. The primers used were: forward, 5'-CAGTGGCATGCACCCAGGGAA-3' and reverse, 5'-GCATGCCCCTTTTAGCCTTGGCA-3'. A 2% agarose gel with ethidium bromide was used to separate and examine the PCR products. The PCR products were confirmed by Southern blotting and hybridization with {gamma}-32P-labeled human NQO1 gene ARE using the ExpressHyb hybridization protocol by procedures described in the manufacturer's instructions.

Effect of Heme on ARE-mediated Gene Expression—Hep-G2 cells were cotransfected with 0.5 µg of reporter plasmid hARE-Luc and Renilla luciferase as internal control, individually or in combination with pcDNA alone or pcDNA-Bach1 and/or pcDNA-Nrf2. After 36 h of transfection, heme was added to the culture medium at 10, 15, and 25 µM concentrations for 4 h as previously described (16). The cells were then washed with 1x PBS, and cell lysates were prepared using the Dual Luciferase reporter assay system kit (by procedures suggested in the manufacturer's protocol) and analyzed for luciferase activity. Each experiment was performed in triplicate. It is noteworthy that hemin (ferric protoporphyrin IX) is reduced inside the cells to heme (ferroprotoporphyrin IX).

Nrf2 and Bach1 Localization and Immunofluorescence—Mouse hepatoma (Hepa-1) cells were grown on Lab-Tek II chamber slides in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum. The cells were treated with 50 µM t-BHQ for 15 min, 1 h, and 2 h. Cells were then fixed in formalin from Polysciences, Inc. (Warrington, PA) and permeabilized with cold acetone from Fisher Scientific. The cells were probed with either Nrf2 (H-300) or Bach1 (C-20) antibodies from Santa Cruz Biotechnology followed by fluorescein isothiocyanate (FITC)-labeled secondary antibodies from Chemicon International (Temecula, CA) by procedures previously described (25). The cells were stained with Hoechst stain from Bio-Rad to visualize the nuclei. The fluorescent images were captured using appropriate filters in a Nikon eclipse TE 2000-U fluorescent microscope fitted with a Photometrics CoolSnap CF camera.

Subcellular Fractionation and Western Blot Analysis—HepG-2 cells were grown in 100-mm tissue culture plates and treated with Me2SO or t-BHQ for 15 min or 2 h. At the end of treatment, the cells were washed twice with ice-cold phosphate-buffered saline, scraped in PBS using a rubber policeman, and centrifuged at 500 rpm for 5 min. Biochemical fractionation of the cells was done using the Nuclear Extract kit (Active Motif) following the manufacturer's protocol. Briefly, the cell pellet was suspended in 1x hypotonic buffer (cytoplasmic buffer) supplemented with Complete protease inhibitor mixture (Roche Applied Science), incubated for 15 min at 4 °C, vortexed in the presence of detergents and centrifuged briefly. The supernatant (cytoplasmic fraction) was collected into a prechilled microcentrifuge tube; the nuclear pellet remained. The nuclear pellet was washed twice with cytoplasmic buffer followed by resuspension in lysis buffer supplemented with 1 mM dithiothreitol and protease inhibitors. The suspension was incubated on a rocking platform at 4 °C for 30 min. The suspension was then vortexed briefly and centrifuged for 10 min at 14,000 x g at 4 °C. The supernatant (nuclear fraction) was collected. The protein concentration was determined using protein assay reagent (Bio-Rad). 75 µg of the cell fractions were resolved on a 10% SDS-PAGE gel, Western-blotted, and probed with antibodies against Nrf2, Bach1, and {beta}-actin (Sigma). To confirm the purity of subcellular fractionations the extracts were immunoblotted with cytoplasmic-specific anti-lactate dehydrogenase (LDH) antibody (Chemicon International) and nuclear-specific anti-LaminB antibody (Santa Cruz Biotechnology).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The nucleotide sequences of the human NQO1, NQO1 mutant, and rat GST Ya AREs are shown in Fig. 1A. These AREs contain two TRE and TRE-like elements followed by a GC box. The AREs are more distinct elements, than TRE even though they contain TRE and TRE-like elements (1). This is because it is ARE and not TRE that is responsive to antioxidants. The mutant NQO1 gene ARE contains the mutated 3'-TRE element. This mutation is known to result in the loss of ARE-mediated gene expression and induction in response to antioxidants (26). The structure and the various domains of Bach1, Nrf2, and MafK are shown in Fig. 1B. All these factors are b-Zip proteins that contain leucine zipper (LZ) domains similar to Jun and Fos. Nrf2 and Bach1 also contain a CNC domain that is conserved in the Nrf and Bach family of proteins (1, 2). In addition to the leucine zipper domain, Nrf2 also contains a transcriptional activation (TA) domain rich in acidic residues (1, 2, 27, 28). Small Maf proteins, including MafK, lack the acidic transcriptional activation domain (8, 12). The transcriptional activation domain has not been identified in Bach1.



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  		FIG. 1.
Effect of overexpression of Bach1 on endogenous NQO1 gene expression. A, nucleotide sequences of NQO1 and GST Ya genes ARE. The nucleotide sequence of the human NQO1 gene ARE, NQO1 mutant ARE, and rat GST Ya gene ARE are shown. The NQO1 gene ARE contains one perfect and one imperfect TRE arranged as inverted repeats separated by three base pairs followed by a GC box (1–2). The perfect TRE element in the NQO1 gene ARE was mutated to generate NQO1 gene mutant ARE. This mutation was previously shown to result in the loss of ARE-mediated gene expression and induction in response to antioxidants (1). The GST Ya gene ARE contains two TRE-like elements arranged as direct repeats followed by a GC box (1). B, schematic map of Bach1, Nrf2, and MafK showing the various protein domains. BTB, broad complex, tramtrack, bric-a-brac domain; H, hydrophobic region; TA, transcriptional activation domain; B, basic region; LZ, leucine zipper region. C and D, effect of Bach1 overexpression on the in vivo expression of the NQO1 gene. Hep-G2 cells were transfected with varying concentrations of pcDNA-Bach1 expression plasmid. Forty-eight hours after transfection the cells were scraped, homogenized in 50 mM Tris-HCl, pH 7.0 containing 0.25 M sucrose, and centrifuged to collect nuclear and cytosolic fractions by standard procedures. The nuclei were lysed to prepare nuclear extract. Western blotting was performed to analyze nuclear fractions for Bach1 and nuclear actin and cytosolic fractions for NQO1 and {beta}-actin proteins. The supernatant was also analyzed for NQO1 activity. C, Western blot analysis of Bach1 and NQO1 proteins. One hundred micrograms of proteins were resolved on 12% SDS/PAGE gels, Western-blotted, and probed with respective antibodies. D, NQO1 activity. The cytosolic fraction was analyzed for dicoumarol inhibitable NQO1 activity by methods described under "Experimental Procedures." One unit of NQO1 activity is the amount of activity that reduced 1 µmol of 2,6-dichlorophenolindophenol in 1 min. The values represent mean ± S.E. of three independent transfection experiments.

 
The overexpression of varying concentrations of Bach1 in Hep-G2 cells led to concentration-dependent increases in Bach1 and decreases in NQO1 protein as determined by Western blot analysis (Fig. 1C). The overexpression of Bach1 also repressed the NQO1 activity in Hep-G2 cells (Fig. 1D). The magnitude of repression in NQO1 activity was comparable to the decrease in NQO1 protein in Bach1-overexpressing Hep-G2 cells (compare Fig. 1, C with D).

The transfection of Hep-G2 cells with pcDNA-Bach1 resulted in concentration-dependent increases in Bach1 protein levels and repression of NQO1 ARE-mediated luciferase gene expression (Fig. 2, A and B). The Bach1 repression of NQO1 ARE-mediated expression was highly significant and Bach1 concentration-dependent (Fig. 2B). The transfection of Hep-G2 cells with 0.5 µg of Bach1 plasmid resulted in ~50% inhibition of NQO1 ARE-mediated luciferase gene expression (p < 0.001). Overexpression of Bach1 also repressed the t-BHQ induction of NQO1 ARE-mediated luciferase gene expression. Interestingly, the repression of induction was in same proportion as repression of basal expression. Thus the fold induction of NQO1 ARE-mediated luciferase gene expression remained unchanged in cells overexpressing Bach1. In other words, the Hep-G2 cells overexpressing Bach1 could still be induced ~2-fold by t-BHQ. The mutation of 3'-TRE in NQO1 ARE led to the loss of basal expression, induction in response to t-BHQ, and Bach1-mediated repression (Fig. 2C). Replacement of NQO1 ARE with GST Ya ARE also showed Bach1 repression of GST Ya ARE-mediated luciferase gene expression (Fig. 2D). The replacement of pcDNA-Bach1 with pcDNA-Bach1-V5 or NQO1 gene ARE-Luc with the 1.85-kb wild-type NQO1 gene promoter containing ARE showed similar results to the NQO1 gene ARE-Luc as seen in Fig. 2E. However, when the 1.85-kb NQO1 gene promoter lacking the ARE was used, it resulted in the loss of basal and induced expression, similar to what is shown in Fig. 2C.



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  		FIG. 2.
Effect of overexpression of Bach1 on NQO1 and GST Ya gene ARE-mediated luciferase expression and induction in response to antioxidant. A, overexpression of Bach1 in Hep-G2 cells. Hep-G2 cells were transfected with pcDNA-Bach1 plasmid in concentrations as shown. The transfected cells were lysed, and 100 µg of lysate proteins analyzed for Bach1 by Western blotting and probing with Bach1 antibodies followed by {beta}-actin antibodies. B–D, effect of Bach1 overexpression on NQO1 gene ARE and GST Ya gene ARE-mediated luciferase expression and induction by tBHQ. B, Hep-G2 cells were cotransfected with 0.5 µg of reporter plasmid NQO1 gene ARE-Luc and expression plasmid pcDNA-Bach1 in concentrations as shown. C, reporter plasmid NQO1 gene ARE-Luc was replaced with mutant ARE-Luc. D, reporter plasmid NQO1 gene ARE-Luc was replaced with GST Ya ARE-Luc. E, reporter plasmid NQO1 gene ARE-Luc was replaced with 1.85-kb wild-type NQO1 gene promoter-Luc. 0.01 µg of plasmid pRL-TK encoding Renilla luciferase was used as the internal control in each transfection. The transfected cells were either untreated or treated with Me2SO or 50 µM t-BHQ 32 h after transfection. The cells were harvested 16 h after treatment and analyzed for luciferase activity. The values represent mean ± S.E. of three independent transfection experiments.

 
The heterodimerization and the ARE binding of Bach1 were analyzed by bandshift and supershift assays. The pcDNA-Bach1, pcDNA-Bach1-V5, pcDNA-MafK, and pcDNA-MafK-V5 plasmids were in vitro transcribed and translated with the rabbit reticulocyte lysate system. SDS-PAGE analysis of in vitro transcribed/translated Bach1-V5 and Bach1 revealed that both proteins migrated at ~75 kDa. Therefore, data are shown only for the Bach1-V5 protein (Fig. 3A). The in vitro transcribed/translated Bach1-V5 protein was also confirmed by Western blot analysis using anti-V5 antibodies (data not shown). The in vitro translated Nrf2 protein migrated at ~110 kDa (8) and MafK protein migrated at ~18 kDa as previously observed (Fig. 3A; Ref. 8). The results of the bandshift and supershift assays with NQO1 gene ARE and in vitro translated Bach1-V5, Nrf2, and MafK proteins are shown in Fig. 3. Bach1-V5 failed to bind to the NQO1 gene ARE as a homodimer (Fig. 3B). Nrf2 alone and Bach1+Nrf2 also failed to bind to NQO1 gene ARE (Fig. 3C). These results were clearly evident from the absence of a shifted band(s) in Fig. 3, B and C. The asterisk is a nonspecific band observed with the rabbit reticulocyte lysate (8). However, Bach1-V5 bound to the NQO1 gene ARE as a heterodimer with MafK that was supershifted with V5 antibody (Fig. 3D). Similarly, MafK-V5 bound NQO1 gene ARE as a heterodimer with Bach1 and was supershifted with V5 antibody (Fig. 3E). In a related experiment, the supershifted band with NQO1 gene ARE was specifically competed with cold ARE but not with mutant ARE (Fig. 3F). Because Bach1 bound to the NQO1 gene ARE as Bach1-MafK heterodimers, we determined the effect of coexpression of Bach1 with MafK on ARE-mediated gene expression and induction in response to t-BHQ. The results are shown in Fig. 3G. A comparison of 0.05 µg of Bach1, MafK, and Bach1 + MafK demonstrated a cooperative effect of repression by Bach1 and MafK (p < 0.001) (Fig. 3G).



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  		FIG. 3.
Bandshift and supershift assays. A, in vitro transcription/translation of Bach1-V5 and MafK proteins. The in vitro transcription/translation of the pcDNA plasmids encoding Bach1-V5 and MafK were performed using the TNT-coupled Reticulocyte Lysate system by procedures suggested in the manufacturer's protocol. 10 µl of the translated proteins were resolved on a 10% PAGE gel, treated with Amplify solution (NAMP 10) to enhance the 35S signal, dried, and autoradiographed. B–E, bandshift assays. NQO1 gene ARE was end-labeled with [{gamma}-32P]ATP. 50,000 cpm of the labeled ARE were incubated with in vitro translated Bach1-V5 either alone (B) or in combination with in vitro translated Nrf2 (C) or in combination with in vitro translated small Maf proteins (D and E) as shown. The Bach1-V5, Nrf2, and small Maf proteins alone and in combination were preincubated at 37 °C for 15 min before incubation with the labeled NQO1 gene ARE. The bandshift experiment was performed at room temperature. The bandshift reaction mixture was incubated with anti-V5 antibody, Nrf2 antibody or IgG for 2 h at 4 °C. The bandshift and supershift mixtures were analyzed on 5% non-denaturing polyacrylamide gel. The gel was dried and autoradiographed. Double closed arrows indicate shifted and triple closed arrows indicate supershifted bands. Asterisk denotes the nonspecific band from rabbit reticulocyte lysate. F, in vitro translated Bach1 and MafK-V5 proteins were mixed and preincubated without and with cold ARE and mutant ARE. The numbers in parentheses denote the amount in nanograms of cold competitor used. The bandshift assay with labeled NQO1 gene ARE was performed. The bandshift reaction mixture was incubated with anti-V5 antibody for 2 h at 4 °C and analyzed on 5% non-denaturing polyacrylamide gel. The gel was dried and autoradiographed. Asterisk denotes the nonspecific band from rabbit reticulocyte lysate. The arrow indicated a supershifted band with anti-V5 antibody. G, effect of coexpression of Bach1 and MafK on NQO1 gene ARE-mediated luciferase gene expression and induction by tBHQ. The Hep-G2 cells were cotransfected with 0.5 µg of reporter plasmid NQO1 gene ARE-Luc and 0.05 µg of expression plasmids pcDNA-Bach1 and pcDNA-MafK either alone and or combined. 0.01 µg of plasmid pRL-TK encoding Renilla luciferase was used as the internal control in each transfection. The transfected cells were either treated with Me2SO or 50 µM of t-BHQ at 32 h after transfection. The cells were harvested 16 h after the treatment and analyzed for luciferase activity. The values represent mean ± S.E. of three independent transfection experiments.

 
The coexpression of increasing concentrations of Nrf2 along with a single concentration of Bach1 plasmid rescued the Bach1 repression of NQO1 ARE-mediated luciferase gene expression and induction in response to t-BHQ (Fig. 4A). In a similar experiment, increasing concentrations of Bach1 with a 0.5-µg constant concentration of Nrf2 failed to repress NQO1 ARE-mediated gene expression and induction (Fig. 4B). However, increasing the concentration of pcDNA-Bach1 to 2.0 µgor more in cotransfection with 0.5 µg of pcDNA-Nrf2 resulted in less than 20% repression of ARE-mediated luciferase gene expression (data not shown). Reducing the concentration of pcDNA-Nrf2 from 0.5 to 0.1 µg for cotransfection, with varying concentrations of pcDNA-Bach1, resulted in a Bach1-dependent decrease in NQO1 gene ARE-mediated luciferase activity (Fig. 4C; p < 0.01, p < 0.005, and p < 0.001 with 0.1, 0.2, and 0.5 µg of Bach1, respectively).



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  		FIG. 4.
Effect of coexpression of Bach1 and Nrf2 on NQO1 gene ARE-mediated luciferase gene expression and induction by tBHQ. A, Hep-G2 cells were cotransfected with 0.5 µg of reporter plasmid NQO1 gene ARE-Luc and 0.5 µg of the expression plasmid pcDNA-Bach1 and varying concentrations of pcDNA-Nrf2 as shown. B, a constant (0.5 µg) concentration of pcDNA-Nrf2 was used with varying concentrations of pcDNA-Bach1 in experiments as described in A. C, 0.1 µg of pcDNA-Nrf2 was cotransfected with varying concentrations of pcDNA-Bach1. 0.01 µg of plasmid pRL-TK encoding Renilla luciferase was used as the internal control in each transfection. The transfected cells were either treated with Me2SO4 or 50 µM t-BHQ at 32 h after transfection. The cells were harvested 16 h after the treatment and analyzed for luciferase activity. The values represent mean ± S.E. of three independent transfection experiments.

 
The human NQO1 gene promoter and the PCR-amplified region of 227 base pairs containing the ARE region is shown (Fig. 5A). The ChIP assays showed a significant increase in the amount of amplified PCR product from both Bach1 and Nrf2 antibody-immunoprecipitated chromatin compared with IgG (control)-immunoprecipitated chromatin from Hep-G2 cells (Fig. 5B). The amplified PCR products hybridized strongly with the 32P-labeled human NQO1 gene ARE probe (Fig. 5C). These results together supported the bandshift and supershift data that indicate both Bach1 and Nrf2 bind to the NQO1 gene ARE. The ChIP assays were extended to determine the competition between Bach1 and Nrf2 for binding to ARE. The cells were either transfected with pcDNA or increasing concentrations of pcDNA-Bach1 or pcDNA-Nrf2 plasmids. Western blot analysis of transfected cells revealed plasmid concentration-dependent overexpression of Bach1 or Nrf2, compared with the pcDNA vector control (Fig. 5D). The ChIP assays revealed that overexpression of Bach1 in Hep-G2 cells led to concentration-dependent increases in Bach1 binding and decreases in Nrf2 binding with the NQO1 gene ARE (Fig. 5E). Similarly, the overexpression of Nrf2 showed concentration-dependent increases in Nrf2 binding with decreasing binding of Bach1 with ARE (Fig. 5F).



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  		FIG. 5.
ChIP assay to determine competition between Bach1 and Nrf2 for binding to the NQO1 gene ARE. A, schematic representation of the forward and reverse primer design for PCR amplification of the chromatin immunoprecipitates. Forward (F) and reverse (R) primers were designed flanking the human NQO1 ARE region so as to produce a 227-bp PCR product. B, ChIP assay analysis for binding of Bach1 and Nrf2 to the NQO1 gene ARE. Hep-G2 cells were cultured, fixed in formaldehyde, and ChIP analysis was performed using rabbit IgG (control), Bach1, and Nrf2 antibodies by procedures recommended in the manufacturer's protocol. The immunoprecipitated chromatin was PCR-amplified, run on agarose gel, and photographed. C, Southern blotting of PCR-amplified products and hybridization with the NQO1 gene ARE. The immunoprecipitated chromatin was PCR-amplified, run on an agarose gel, Southern blotted, and hybridized with 32P-labeled human NQO1 gene ARE. D, overexpression of Bach1 and Nrf2 in Hep-G2 cells. Hep-G2 cells were transfected with either pcDNA or pcDNA-Bach1 or pcDNA-Nrf2 in concentrations as shown. One hundred micrograms of proteins were separated on SDS-PAGE gels, Western-blotted, and probed with Bach1, Nrf2, and {beta}-actin antibodies. The expression of Nrf2 in pcDNA-Bach1 and Bach1 in pcDNA-Nrf2-transfected cells did not demonstrate any alterations and are not shown. E and F, ChIP analysis to determine the competition between Bach1 and Nrf2 for binding with ARE. Hep-G2 cells were transfected with pcDNA (control) or pcDNA-Bach1 or pcDNA-Nrf2 (0.5 and 1.0 µg). The chromatin was immunoprecipitated with rabbit IgG, anti-Bach1, and anti-Nrf2 antibody. Immunoprecipitated DNA were used in the PCR reaction to amplify the ARE region. Input DNA shows that equal amounts of chromatin were used to immunoprecipitate with respective antibodies.

 
The Hep-G2 cells transfected with hARE-Luc alone or in combination with pcDNA-Nrf2 and/or pcDNA-Bach1 were either untreated or treated with hemin for 4 h and analyzed for luciferase activity to determine the effect of heme, an inhibitor of Bach1, on Bach1 repression of ARE-mediated gene expression. The results are shown in Fig. 6. The treatment of cells with 10 µM hemin increased the ARE-mediated luciferase gene expression in cells expressing endogenous Nrf2 (Fig. 6A, compare columns 1 between the hemin– and hemin + panels) and in cells overexpressing Nrf2 (Fig. 6A, compare columns 2 between the hemin– and hemin + panels). Hemin (10 µM) also partially relieved Bach1 repression of hARE-mediated gene expression in cells expressing endogenous and overexpressed Nrf2 (Fig. 6A, compare columns 3 and 4 between the hemin– and hemin + panels). Increasing the hemin concentration from 10 to 15 µM further relieved Bach1 repression of hARE-mediated luciferase gene expression (Fig. 6B, p < 0.005). The treatment with 15 µM hemin relieved more than 75% of Bach1 repression (Fig. 6B). Increasing the concentration of hemin to 25 µM showed results more or less similar to 15 µM hemin (Fig. 6B).



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  		FIG. 6.
Effect of heme on ARE-mediated luciferase expression. Hep-G2 cells were cotransfected with 0.5 µg of hARE-Luc and 0.01 µg of Renilla luciferase either alone or in combination with 0.2 µg of pcDNA-Nrf2 and/or 0.5 µg of pcDNA-Bach1. Thirty-two hours after transfection, the cells were treated with hemin for 4 h and analyzed for luciferase activity by procedures described under "Experimental Procedures."

 
Northern analysis of Hep-G2 cells treated with Me2SO and t-BHQ demonstrated that the Bach1 and Nrf2 mRNA remained unchanged up to 16 h (Fig. 7). MafK mRNA was also unchanged until 4 h after t-BHQ treatment. However, MafK mRNA increased to 2-fold after 8 h of t-BHQ treatment (Fig. 7). The NQO1 mRNA increased exponentially starting at 4 h of the tBHQ treatment (Fig. 7). Two RNA transcripts were observed with NQO1 because of two different polyadenylation signals (29).



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  		FIG. 7.
Northern analysis. The total RNA from the HepG2 cells treated with Me2SO4 or 50 µM t-BHQ for different time intervals were prepared using the RNAeasy minikit. 20 µg of total RNA were run on a 1% formaldehyde agarose gel, transferred to the membrane, and hybridized with nick-translated full-length Bach1 cDNA. The membrane was washed and exposed to x-ray film for 48 h. The probe was removed, and the membrane was probed again with Nrf2 (A), MafK, and GAPDH cDNA. The RNA from the same experiment as described above was run on a second gel, Northern-blotted, and hybridized with NQO1 cDNA followed by GAPDH cDNA.

 
The results of cellular localization of endogenous Bach1 in untreated and t-BHQ treated Hepa-1 cells are shown in Fig. 8. Bach1 was found localized in the cytoplasm as well as nucleus in the untreated and Me2SO-treated cells (Fig. 8, Me2SO). The treatment of cells with t-BHQ for 15 min and for 1 h had no effect on the localization of Bach1 (Fig. 8, t-BHQ, 15 min and 1 h). However, the treatment of cells with t-BHQ for 2 h led to nuclear accumulation of Bach1 (Fig. 8, t-BHQ, 2 h). Next, we compared the t-BHQ induced nuclear localization of endogenous Bach1 protein with t-BHQ induced nuclear localization of Nrf2 (Fig. 8). The results indicated that t-BHQ-induced nuclear accumulation of Nrf2 was significantly rapid compared with the time taken for accumulation of Bach1 in the nucleus. The nuclear localization of Nrf2 was observed within 15 min after t-BHQ treatment. This was in contrast to Bach1 nuclear accumulation that was observed only at around 2 h after t-BHQ treatment. Western blot analysis of cytosolic and nuclear fractions with Nrf2 and Bach1 antibodies showed a similar time difference in t-BHQ-induced nuclear translocation of Nrf2 and Bach1 as observed with immunofluorescence studies (Fig. 9). The purity of cytosolic and nuclear fractions was checked by anti-LDH and anti-LaminB antibodies (Fig. 9).



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  		FIG. 8.
Kinetics of nuclear accumulation of Nrf2 and Bach1. Hepa-1 cells were treated with Me2SO4 or 50 µM t-BHQ for different time intervals. The cells were fixed, then probed with either Bach1 or Nrf2 antibodies, followed by FITC-tagged secondary antibody by procedures as described (25). The cells were washed twice with 1x PBS and were processed and visualized for fluorescence under the Nikon eclipse TE2000-U fluorescent microscope. Each experiment was repeated three times. Representative results are shown.

 



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  		FIG. 9.
Effect of t-BHQ on subcellular localization of Nrf2 and Bach1. HepG-2 cells were seeded in 100-mm plates and treated with either Me2SO or t-BHQ (50 µM) for 15 min or 2 h. Cells were then harvested, and cytosol and nuclear fractions prepared and immunoblotted against anti-Nrf2 or anti-Bach1 antibodies as described under "Experimental Procedures." The immunoblot was stripped and reprobed with anti-LaminB and anti-LDH antibodies to confirm the purity of nuclear and cytosolic fractions, respectively. The Western blot was also reprobed with anti-actin antibody to show equal loading. A, Western blot analysis. B, densitometric measurement of Nrf2 and Bach1 bands versus LaminB bands.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It is fairly well established that a balance between phase I enzymes that activate carcinogens and phase II enzymes that detoxify them significantly contributes to the development of chemically induced neoplasia (1–3). Therefore, the expression and antioxidant induction of enzymes that catalyze metabolic detoxification of xenobiotics, drugs, and carcinogens play an important role in reducing the risk of cancer in animals and human (1, 2). Recent studies have shown that ARE and ARE-binding transcription factors including Nrf1, Nrf2, c-Jun, c-Fos, Fra1, and small Maf (MafK and MafG) regulate the expression and induction of detoxifying enzyme genes in response to antioxidants (1, 2). Interestingly, genes encoding many other chemopreventive proteins that include p53 are also shown to contain ARE-like element and may be coordinately up-regulated along with detoxifying enzyme genes (30). Nrf1 and Nrf2 positively and c-Fos, Fra1, and small Maf proteins negatively regulate ARE-mediated NQO1 and other detoxifying enzyme gene expression and antioxidant induction (1, 25, 31–33, 12, 8, 10). Small Maf proteins are known to heterodimerize with Nrf2 and bind to the ARE. However, the role of Nrf2-small Maf heterodimers in ARE-mediated gene expression and induction is controversial and requires further investigation (8, 12, 33).

The mechanism of signal transduction from antioxidants to Nrf2 and Nrf1 that up-regulate ARE-mediated gene expression is complex and largely unknown (1, 2). Nrf2 is a much stronger activator than Nrf1 in the activation of ARE-mediated gene expression (1, 2). A third member of the Nrf family of proteins designated as Nrf3 has also been reported (34). Recently, Nrf3 was found to repress ARE-mediated gene expression (35). Nrf2, but not Nrf1 or Nrf3, is retained in the cytosol by an anchoring protein Keap1/INrf2 (9, 10). Antioxidants antagonize this interaction leading to the release of Nrf2 from INrf2 that translocates into the nucleus, leading to the activation of ARE-mediated gene expression. Phosphorylation of Nrf2 has been shown to cause the release of Nrf2 from INrf2 (25, 36). The studies in our laboratory and that of others continue to identify new ARE-regulated genes (and transcription factors that bind to the ARE) and contribute to the regulation of ARE-mediated expression and induction of detoxifying/chemopreventive proteins.

In the present report, we demonstrated that overexpression of transcription factor Bach1 negatively regulated endogenous and transfected NQO1 gene expression in Hep-G2 cells. Bach1 failed to bind to the ARE by itself and required a small Maf (MafK) protein to heterodimerize and bind to the ARE. This was in agreement with previous reports of Bach1 heterodimerization with MafK and their binding to the NF-E2 site (14, 15). Furthermore, with this study, the pivotal roles of small Maf proteins in ARE-mediated gene regulation becomes more evident as they bind and regulate ARE in three different combinations: as homodimers (8) and as heterodimers with Nrf2 (8) and Bach1 (present report). We further demonstrated that Bach1 also inhibited antioxidant induction of ARE-mediated NQO1 and GST Ya gene expression and induction in response to antioxidant t-BHQ. However, Bach1-mediated repression of the induction was in a similar proportion to basal repression. In other words, ARE-mediated expression could still be induced by t-BHQ in cells overexpressing Bach1. The results also suggested that positive and negative regulation of ARE-mediated gene expression depend on the critical balance between Nrf2 and Bach1 in the nucleus. This was clearly evident from the observation that Bach1 repression of ARE-mediated gene expression was relieved by coexpression of Nrf2 with Bach1, and Bach1 failed to repress the ARE in cells overexpressing Nrf2. However, Bach1 repressed the ARE activation in cells expressing moderate levels of Nrf2. In addition, the ChIP assays clearly demonstrated competition between Bach1 and Nrf2 for binding to the NQO1 gene ARE. The similar balance of factors would also account for the observation that ARE-mediated gene expression was inducible in response to t-BHQ in cells overexpressing Bach1.

Heme is a negative regulator of Bach1 (18–20). In the present studies, heme relieved Bach1 repression of ARE-mediated gene expression. This effect of heme was observed in Hep-G2 cells expressing endogenous and also in Hep-G2 cells overexpressing cDNA-derived Nrf2. The observation, that heme significantly (p > 0.01) increased ARE-mediated basal gene expression along with the observation of binding of Bach1 with endogenous NQO1 gene ARE in ChIP assays, showed that Bach1 repression contributed to basal levels of ARE-mediated gene expression. The treatment with heme relieved this effect leading to increased expression of ARE-luciferase. The results from heme studies, therefore, also support that Nrf2 (positive) and Bach1 (negative) compete with each other to regulate ARE-mediated gene expression.

The present studies also demonstrated that Bach1 and the Nrf2 RNA transcript did not increase in t-BHQ-treated cells. However, an increase in small Maf RNA was observed 4 h after t-BHQ treatment. These results indicated that post-translational modifications and/or nuclear accumulation presumably contributed to Bach1 and Nrf2 competition for ARE binding and regulation of gene expression. Bach1 was localized both in the cytoplasm as well as in the nucleus in untreated cells. The Bach1 contains a cytoplasmic localization signal (CLS) that exports Bach1 from nucleus to cytosol (18). The Crm1/exportin1 and extracellular signal-regulated kinase1/2 (ERK1/2) are known to regulate the export of Bach1 from nucleus to the cytosol (18). The treatment of cells with antioxidant t-BHQ led to the nuclear accumulation of Bach1. This result with Bach1 was similar as reported earlier for Nrf2 (Refs. 10, 25, and present report). This raised questions regarding the role of nuclear accumulation of both positive (Nrf2) and negative (Bach1) factors following antioxidant treatment in the regulation of ARE-mediated gene expression. The studies in the present report revealed a significant difference between time taken for nuclear accumulation of Bach1 and Nrf2 following treatment of cells with antioxidants. The t-BHQ-induced nuclear accumulation of Bach1 was significantly slower than that of Nrf2. Nrf2 accumulated in the nucleus within 15 min as compared with Bach1, which took almost 2 h after t-BHQ treatment for the accumulation in the nucleus. This difference in nuclear accumulation also indicated that initial response of t-BHQ treatment is to activate Nrf2-regulated ARE-mediated gene expression.

The delayed nuclear accumulation of Bach1 along with transcriptional activation of MafK may be required to check the activation and/or rapidly bring down the activated levels of ARE-mediated gene expression to normal levels and maintain it at normal levels. The above hypothesis is consistent with previous reports that small amounts of free radicals including superoxide and related reactive species are consistently required to keep cellular defenses active (37). Because activation of detoxifying enzymes and other defensive proteins leads to a significant reduction in the levels of free radicals, the cell may require negative regulatory factors such as Bach1 to keep the levels of free radicals from falling below a critical threshold. Therefore, the ARE-binding negative factors may work in parallel to positive factors and play an important role in maintaining the basal expression of NQO1 and other detoxifying enzyme genes. The reasons for slow accumulation of Bach1 in the nucleus in response to antioxidant remain unknown. It is possible that t-BHQ might inhibit the Crm1/Exportin1 and/or ERK1/2, which mediate export of Bach1 from the nucleus to the cytosol. However, this remains to be determined.

In conclusion, Bach1 forms heterodimers with small Maf (MafK and MafG) proteins that bind to the ARE leading to the repression of ARE-mediated gene expression and induction. The results also led to the conclusion that ARE-mediated regulation of genes encoding detoxifying/chemopreventive proteins is controlled by a balance of positive (Nrf2) and negative (Bach1) factors. Future studies are required to study the kinetics of the balance between positive and negative factors in the regulation of ARE-mediated gene expression and induction and the mechanism of significantly delayed accumulation of Bach1 in the nucleus in response to antioxidants.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM47466. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Both authors contributed equally to this work. Back

§ Present address: Cell Death and Human Disease Group, Institute of Molecular and Cell Biology, 61 Biopolis Dr., Singapore 138673. Back

To whom correspondence should be addressed: Dept. of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-7691; Fax: 713-798-3145; E-mail: ajaiswal{at}bcm.tmc.edu.

1 The abbreviations used are: ARE, antioxidant response element; NQO1, NAD(P)H:quinone oxidoreductase1; GST Ya, glutathione S-transferase Ya subunit; INrf2, inhibitor of Nrf2; t-BHQ, tert-butyl hydroquinone; Hep-G2, human hepatoblastoma cells; Hepa-1, mouse hepatoma; Me2SO4, dimethylsulfoxide; TPA, 12-O-tetradecanoylphorbol-13-acetate; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; ChIP, chromatin immunoprecipitation assay; TRE, TPA response element; MARE, Maf recognition element; GCS, glutamylcysteine synthetase; CNC, cap'n'collar; LDH, lactate dehydrogenase. Back


   ACKNOWLEDGMENTS
 
We thank Namphuong Tran for technical assistance. We are grateful to Drs. Jefferson Y. Chan and Yuet W. Kan, both from the University of California, San Francisco for providing us the cDNA encoding Nrf2. We are also thankful to Dr. Makoto Nishizawa from The Scripps Research Institute, La Jolla, CA for providing us with the small Maf proteins cDNA.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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