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Originally published In Press as doi:10.1074/jbc.M307597200 on August 26, 2003

J. Biol. Chem., Vol. 278, Issue 45, 44103-44112, November 7, 2003
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Oltipraz Inhibits 3-Methylcholanthrene Induction of CYP1A1 by CCAAT/Enhancer-binding Protein Activation*

IL Je Cho and Sang Geon Kim{ddagger}

From the National Research Laboratory, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Korea

Received for publication, July 15, 2003 , and in revised form, August 4, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Oltipraz, a cancer chemopreventive agent, induces CYP1A1 to a certain extent by transactivation of the gene via the Ah receptor (AhR)-xenobiotic response element (XRE) pathway. Previously, we showed that oltipraz promoted CCAAT/enhancer binding protein{beta} (C/EBP{beta}) activation, which leads to the induction of glutathione S-transferase. Given that oltipraz activates C/EBP{beta} for gene transactivation and that the putative C/EBP binding site is located in the CYP1A1 promoter region, this study investigated the effect of oltipraz on CYP1A1 induction by 3-methylcholanthrene (3-MC). 3-MC induced CYP1A1 in H4IIE cells in a time- and concentration-dependent manner. Gel shift analysis showed that 3-MC increased the band intensity of protein binding to the XRE. Immunocompetition analysis verified the specificity of AhR-XRE binding. Oltipraz (30 µM) induced CYP1A1 and the CYP1A1 promoter-luciferase gene and increased AhR DNA binding activity, which was 10–20% of those in 3-MC (100 nM)-treated cells. However, AhR-XRE binding was not increased after 10 µM oltipraz treatment. Oltipraz (10 µM) significantly inhibited CYP1A1 and CYP1A1-luciferase gene induction by 3-MC with no increase in AhR DNA binding. Oltipraz enhanced protein binding to the C/EBP binding site in the gene promoter and the binding complex comprised of C/EBP{beta} and partly C/EBP{delta}. Overexpression of dominant-negative mutant C/EBP significantly abolished the ability of oltipraz to suppress 3-MC-inducible CYP1A1 and the CYP1A1 reporter gene expression. Consistently, C/EBP{beta} overexpression blocked CYP1A1 reporter gene induction by 3-MC. These results provide evidence that oltipraz suppresses 3-MC induction of CYP1A1 gene expression and that activation of C/EBP{beta} by oltipraz contributes to suppression of 3-MC-inducible AhR-mediated CYP1A1 expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polycyclic aromatic hydrocarbons (PAHs)1 induce a battery of drug-metabolizing enzymes in hepatic and extrahepatic tissues (1). Among xenobiotic-metabolizing enzymes, CYP1A1, which possesses distinct substrate specificity, bioactivates a number of procarcinogens and toxicants (2). Exposure of animals or cells to PAHs leads to CYP1A1 induction. The substrates and inducers of CYP1A1 include 3-methylcholanthrene (3-MC) (3), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (4), benzo(a)pyrene (5), and nitropyrene (6). PAHs regulate gene expression via a specific aryl hydrocarbon receptor (AhR). The heterodimeric AhR and aryl hydrocarbon receptor nuclear translocator (ARNT) complex bind to the XREs located upstream of target genes and promote transactivation (7, 8). Epidemiology studies show that individuals with a high level of CYP1A1 gene expression were at greater risk for cancer incidence (9).

Oltipraz, a synthetic derivative of 1,2-dithiole-3-thione, serves as a cancer chemopreventive agent by inducing phase II detoxifying enzymes (10–12). The induction of glutathione S-transferases (GSTs) by oltipraz is associated with cancer chemopreventive and cytoprotective effects (14–17). The members of the CCAAT/enhancer-binding protein (C/EBP) family, which have roles in cell proliferation and differentiation, and regulation of tissue-specific genes, including C/EBP{beta} and C/EBP{delta} (18, 19). We recently found that activation of C/EBP{beta} and its binding to the C/EBP-response element play a critical role in the induction of the GSTA2 gene (11, 20). Oltipraz and flavonoid compounds promote phosphoinositide 3-kinase-mediated nuclear translocation of C/EBP{beta}, which leads to the induction of the GST gene by activating binding to the C/EBP binding site present in the GSTA2 gene. Signals activated by oxidative stress stimulate transduction of NF-E2-related factor-2 activity and activation of ARE (16, 21). Thus, the pathways involving both C/EBP and NF-E2-related factor-2 are essential and distinct for the phase II enzyme induction.

Oltipraz affects activities and the expression of cytochrome P450s. This agent inhibited CYP1A1 activity in vivo and in vitro (22). Other studies showed that oltipraz increased the expression of CYP1A in the rat liver, kidney, and lung following its inhibition of metabolic activity (23–25). It has also been shown that oltipraz is an inducer of the XRE-containing 5'-flanking region of the CYP1A1 gene (25). The proteins that bind to the XRE in the gene promoter have been found to be relevant to a member of the C/EBP family of transcription factors. C/EBP{alpha} showed overlapping DNA binding specificity to that of the AhR (26). Other studies provided evidence that several common transcriptional factors bind to the AhRE motif of the murine Cyp1a1 gene, indicated by competition studies with an excess of AhRE3, mutated AhRE3, and C/EBP{alpha} oligonucleotides (27).

In view of the fact that oltipraz activates C/EBP{beta} for gene transactivation and that the C/EBP protein is involved in the formation of the transcription complexes binding to the XRE, we investigated whether oltipraz alters AhR-mediated CYP1A1 induction by 3-MC and whether activating C/EBP{beta} inhibits AhR-bound XRE-mediated transcription of the gene. We now report that oltipraz suppresses 3-MC-inducible CYP1A1 expression and that activating C/EBP{beta} binding to the putative C/EBP binding site in the CYP1A1 gene promoter, which is promoted by oltipraz, is responsible for the inhibition of 3-MC-inducible AhR-mediated CYP1A1 expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—[{gamma}-32P]ATP (3000 mCi/mmol) was purchased from PerkinElmer Life Sciences (Arlington Heights, IL). Anti-CYP1A1/2 antibody was supplied from Oxford Biomedical Research, Inc. (Oxford, MI). Horseradish peroxidase-conjugated goat anti-mouse IgG was obtained from Zymed Laboratories Inc. laboratory (San Francisco, CA). Anti-AhR, anti-C/EBP{alpha}, anti-C/EBP{beta}, and anti-C/EBP{delta} antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). These antibodies specifically recognized their respective transcription factors without any cross-reactivity. Oltipraz was kindly provided by Aventis Pharma France (Virty-sur-Seine, France). 3-Methylcholanthrene and other reagents in the molecular studies were supplied from Sigma Chemical.

Cell Culture—H4IIE cells, a rat hepatocyte-derived cell line, were obtained from American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (FCS), 50 units/ml penicillin, and 50 µg/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO2.

Immunoblot Analysis—H4IIE cells were incubated with 1 nM to 10 µM 3-MC and/or 10–30 µM oltipraz for the indicated time period. After washing the cells twice with sterile phosphate-buffered saline, the cells were scraped and sonicated for disruption. Microsomal fractions were prepared by differential centrifugation. The microsomal fractions were stored at –70 °C until use. SDS-polyacrylamide gel electrophoresis and immunoblot analyses were performed according to previously published procedures (28). Microsomal proteins were separated by 7.5% gel electrophoresis and electrophoretically transferred to nitrocellulose paper. The nitrocellulose paper was incubated with anti-CYP1A1/2 antibody, followed by incubation with horseradish peroxidase-conjugated secondary antibody, and developed using ECL® chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, UK). Equal loading of proteins was verified by Coomassie staining. Changes in the levels of CYP1A1 were determined via scanning densitometry. At least three separate experiments were performed with different lysates to confirm changes in the protein levels.

Preparation of Nuclear Extracts—Nuclear extracts were prepared essentially according to the previously published method (29). Briefly, the cells in dishes were washed with ice-cold phosphate-buffered saline. Cells were then scraped, transferred to microtubes, and allowed to swell after the addition of 100 µl of hypotonic buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet P-40, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. The lysates were incubated for 10 min in ice and centrifuged at 7200 x g for 5 min at 4 °C. Pellets containing crude nuclei were resuspended in 50 µl of extraction buffer containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 10 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride and then incubated for 30 min in ice. The samples were centrifuged at 15,800 x g for 10 min to obtain supernatants containing nuclear fractions. Nuclear fractions were stored at –70 °C until use.

Gel Retardation Assay—Double-stranded DNA probes containing the consensus C/EBP binding oligonucleotide, the rat CYP1A1 XRE oligonucleotide, and the putative CYP1A1 C/EBP binding oligonucleotide were used for gel shift analyses after end-labeling of each probe with [{gamma}-32P]ATP and T4 polynucleotide kinase. The sequences of C/EBP consensus, CYP1A1 XRE and putative CYP1A1 C/EBP binding oligonucleotides were 5'-TGCAGATTGCGCAATCTGCA-3', 5'-GGAGTTGCGTGAGAAGAGCC-3' (30) and 5'-TGTAGCTTGCCTAAGGTGA-3', respectively. The reaction mixtures included 4 µl of 5 x binding buffer containing 20% glycerol, 5 mM MgCl2, 250 mM NaCl, 2.5 mM EDTA, 2.5 mM dithiothreitol, 0.25 mg/ml poly dI-dC, and 50 mM Tris-Cl (pH 7.5), 15 µg of nuclear extracts, and sterile water in a total volume of 20 µl. The reaction mixtures were preincubated for 10 min. DNA binding reactions were carried out at room temperature for 30 min after the addition of 1 µl of probe (106 cpm). Specificity of binding was determined by competition experiments, which were carried out by adding a 20-fold excess of unlabeled XRE, C/EBP, or SP-1 oligonucleotide to the reaction mixture before the DNA binding reaction. For the supershift assay, antibodies (1 µg each) were added to the reaction mixture, and additionally incubated for 1 h at 25 °C. Samples were loaded onto 4% polyacrylamide gels at 100 V. The gels were removed, fixed, and dried, followed by autoradiography.

Plasmid Construction—Firefly luciferase reporter gene construct, pGL-CYP1A1-1195 was generated by ligation of the 1195-bp promoter region of the rat CYP1A1 gene with the coding region of luciferase. Briefly, rat genomic DNA was prepared by using Wizard® SV genomic DNA purification system (Promega, Madison, WI). The flanking region of the CYP1A1 gene was generated by PCR amplification using specific primers. The amplified fragment was inserted into pGEM-T vector (Promega, Madison, WI) and subcloned into the MluI/BglII sites of the pGL3 reporter plasmid (Promega, Madison, WI). C/EBP-specific dominant-negative expression (AC/EBP) plasmid was a gift from Dr. C. Vinson (National Institutes of Health, Bethesda, MD) (31). pCDNA-C/EBP{beta}, which encodes C/EBP{beta}, was produced by PCR amplification of the coding region of the C/EBP{beta} gene using rat genomic DNA as a template (32) and the amplified DNA was then cloned into pCDNA3.1(+) (Invitrogen, Carlsbad, CA). The DNA sequences of the pGL-CYP1A1-1195 and pCDNA-C/EBP{beta} constructs were verified by sequence analysis using an ABI7700 DNA cycle sequencer.

CYP1A1 Promoter Luciferase Assay—The CYP1A1 promoter-luciferase reporter gene assay was carried out according to previously published methods with some modifications (11). Briefly, cells (7 x 105 cells/well) were replated in 6-well plates overnight, serum-starved for 6 h, and transiently transfected with the CYP1A1 promoter-luciferase construct (1 µg each) and pRL-SV plasmid (5 ng each, Renilla luciferase expression for normalization) (Promega, Madison, WI) using LipofectAMINE Plus® Reagent for 3 h (Invitrogen, Carlsbad, CA). Transfected cells were incubated in the medium containing 1% fetal calf serum for 13 h, and then exposed to 30 nM 3-MC in the presence or absence of oltipraz (10 µM) for 18 h or 24 h. Firefly and Renilla luciferase activities in cell lysates were measured using a luminometer (Luminoskan®, Thermo Labsystems, Helsinki, Finland). The relative luciferase activity was calculated by normalizing CYP1A1 promoter-driven firefly luciferase activity to that of Renilla luciferase. For some experiments, cells were co-transfected with pCMV-AC/EBP or pCDNA-C/EBP{beta} plasmid (1 µg each) in addition to CYP1A1 promoter-luciferase construct (1 µg), whereas pCMV500 or pCDNA3.1(+) (1 µg each) empty vector was used for mock-transfection.

AC/EBP or C/EBP{beta} Plasmid Transfection—H4IIE cells were transfected with the AC/EBP or pCDNA-C/EBP{beta} plasmid using LipofectAMINE plus® reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA), as described previously (11). Briefly, cells were re-plated 24 h before transfection at a density of 7 x 105 cells in 6-well plates. Cells were transfected by addition of 1 ml of minimal essential medium (MEM) containing 1 µg of each plasmid and 4 µl of LipofectAMINE, and then incubated at 37 °C in a humidified atmosphere of 5% CO2 for 3 h. After addition of 1 ml of MEM containing 1% fetal calf serum, cells were incubated for an additional 24 h at 37 °C. Control cells were transfected with an equal amount of pCMV500 or pCDNA3.1(+) (i.e. mock-transfection).

Statistical Analysis—Scanning densitometry of the immunoblots was performed with Image Scan & Analysis System (Alpha-Innotech Corp, San Leandro, CA). The area of each lane was integrated using the software AlphaEaseTM version 5.5, followed by background subtraction. One-way analysis of variance (ANOVA) procedures were used to assess significant differences among treatment groups. For each significant effect of treatment, the Newman-Keuls test was used for comparisons of multiple group means. The criterion for statistical significance was set at p < 0.05 or p < 0.01. All statistical tests were two-sided.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
CYP1A1 Induction by 3-MC—Western blot analysis was performed to confirm that 3-MC induced CYP1A1 in H4IIE cells. CYP1A1 was induced 24 h after 3-MC treatment at the concentration of 1 nM or above in a concentration-dependent manner (Fig. 1A). CYP1A1 induction plateaued at 1 µM. Because 10–100 nM 3-MC significantly induced CYP1A1, the concentrations were chosen in subsequent experiments. A time course study showed that 3-MC at the concentration of 100 nM induced CYP1A1 with the maximal induction being observed at 6 h or later (Fig. 1B).



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  		FIG. 1.
The effects of 3-MC on the expression of CYP1A1 and the activation of AhR. A, immunoblot analysis of CYP1A1 in cells treated with various concentrations of 3-MC (1 nM to 10 µM, 24 h). The relative CYP1A1 levels were assessed by scanning densitometry of immunoblots. Each lane was loaded with 10 µg of microsomal protein. Data represent the mean ± S.D. of three separate experiments (the level of CYP1A1 in cells treated with 100 nM 3-MC is 100%). B, immunoblot analyses of CYP1A1 in cells treated with 3-MC for 3–48 h. Representative immunoblots show the levels of CYP1A1 (and CYP1A2) protein(s) in H4IIE cells treated with 100 nM 3-MC for 3–48 h. Each lane was loaded with 10 or 20 µg of microsomal protein (the level of CYP1A1 in cells treated with 3-MC for 12 h is 100%). C, gel shift analysis of AhR binding to the XRE. Nuclear extracts were prepared from H4IIE cells incubated with 100 nM 3-MC for 3–12 h and subjected to gel shift analysis. All lanes contained 15 µg of nuclear extracts and 5 ng of radiolabeled XRE oligonucleotide. For immunoinhibition, the nuclear extract obtained from cells treated with 3-MC for 6 h was incubated with an anti-AhR antibody for 1 h. The immunodepleted extract was mixed with labeled probe. Competition experiments using 20-fold excess XRE or SP-1 oligonucleotide confirmed the specificity of protein binding to the XRE. Arrowhead indicates DNA bound with AhR.

 

We determined whether 3-MC induced ligand-activated AhR binding to the XRE. Gel shift analysis showed that 3-MC increased the band intensity of protein binding to the XRE consensus oligonucleotide (Fig. 1C). The band intensity of AhR binding to the XRE substantially increased from 3 to 6 h, followed by returning to control level at 12 h. The specificity of AhR binding to the XRE (6 h) was verified by immunocompetition analysis using an anti-AhR antibody (Fig. 1C). Competition experiments using a 20-fold excess of unlabeled XRE or SP-1 oligonucleotide confirmed the specificity of protein binding to the XRE (Fig. 1C).

Effect of Oltipraz on CYP1A1 Expression—Previous studies have shown that oltipraz (e.g. at 50–100 µM) induces CYP1A1 and activates transcription of the CYP1A1 gene through the AhR-XRE pathway (25). We compared the extent of CYP1A1 induction by oltipraz with that by 3-MC. The relative CYP1A1 protein level in H4IIE cells treated with 30 µM oltipraz was ~25% of that induced by 100 nM 3-MC (Fig. 2A). Oltipraz at the concentration of 10 µM was minimally active. Next, we tested whether oltipraz was capable of activating AhR binding to the XRE. Gel shift assay showed that oltipraz (30 µM, 3 h) increased protein binding to the XRE, which was much weaker than that induced by 3-MC (100 nM, 3 h)(Fig. 2B). AhR binding activity to a radiolabeled XRE oligonucleotide maximally increased 3 h after treatment of H4IIE cells with 30 µM oltipraz and returned toward that of control at 6 h or later time point (Fig. 2B). Oltipraz at the concentration of 10 µM did not activate AhR binding to the XRE (Fig. 2B). An increase in the band intensity of protein DNA binding by 30 µM oltipraz (3 h) was specific to the XRE, as evidenced by competition experiments using a 20-fold excess unlabeled XRE or SP-1 oligonucleotide (Fig. 2B).



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  		FIG. 2.
The effects of oltipraz on the expression of CYP1A1 and the AhR binding to the XRE. A, expression of CYP1A1. The levels of CYP1A1 protein were determined by immunoblot analyses in H4IIE cells treated with 100 nM 3-MC or 10–30 µM oltipraz for 24 h. Data represent the mean ± S.D. with three separate experiments (significant as compared with 3-MC; **, p < 0.01; the level of CYP1A1 in cells treated with 3-MC is 100%). B, gel shift analysis of AhR-XRE complex. The band intensity of AhR-XRE binding in cells treated with oltipraz (10–30 µM, 3 h) was compared with that in 3-MC-treated cells (100 nM, 3 h). Nuclear extracts were also prepared from cells incubated with 10–30 µM oltipraz for 3–12 h and subjected to gel shift analyses. The specificity of protein binding to the XRE was verified as described in the legend to Fig. 1C.

 

We then examined the effect of oltipraz on transactivation of the CYP1A1 gene by using the CYP1A1 promoter reporter gene assay. H4IIE cells were transfected with a reporter vector pGL-CYP1A1-1195, which contained the luciferase structural gene and the –1.2-kb rat CYP1A1 promoter (Fig. 3A). Exposure of H4IIE cells, transiently transfected with the plasmid, to 10 or 30 µM oltipraz resulted in increases in luciferase activity, which was 15 and 25% of that induced by 100 nM 3-MC, respectively (Fig. 3B).



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  		FIG. 3.
Induction of CYP1A1 promoter-luciferase activity by 3-MC or oltipraz. A, scheme showing the XRE and NRE sites present in the chimeric gene construct pGL-CYP1A1-1195 that contained the promoter region of CYP1A1 and the coding region of luciferase. NRE, negative regulatory element. B, luciferase activity was measured in H4IIE cells transiently transfected with pGL-CYP1A1-1195. Dual luciferase reporter assay was performed with the lysates obtained from cells co-transfected with the CYP1A1-luciferase gene construct (firefly luciferase) and pRL-SV (Renilla luciferase)(a ratio of 200:1) after exposure to 3-MC (100 nM) or oltipraz (10–30 µM) for 18 h. Activation of the reporter gene was calculated as a relative change to Renilla luciferase activity. Data represented the mean ± S.D. of three separate experiments (significant as compared with control; **, p < 0.01; *, p < 0.05; vehicle-treated control is 1).

 

Inhibition of 3-MC-Inducible CYP1A1 Expression by Oltipraz—We previously showed that oltipraz promotes activation of C/EBP{beta}, which contributes to GSTA2 induction via activating C/EBP{beta} binding to the C/EBP binding site present within the XRE in the GSTA2 gene (11, 21). Because the putative C/EBP binding site is located in proximity to the XRE in the promoter region of the CYP1A1 gene, we were interested in whether oltipraz might affect 3-MC-inducible CYP1A1 expression via activation of C/EBP protein(s). We determined the extents of CYP1A1 induction in H4IIE cells treated with 10–100 nM 3-MC in combination with 10 µM oltipraz. Western blot analysis revealed that oltipraz (10 µM) significantly blocked the induction of CYP1A1 by 10 or 30 nM 3-MC, whereas CYP1A1 induction by 100 nM 3-MC was not significantly decreased by concomitant treatment with oltipraz (Fig. 4A). To verify the inhibition by oltipraz of 3-MC induction of CYP1A1, luciferase reporter gene analyses were performed. The luciferase induction by 3-MC (30 nM) in pGL-CYP1A1-1195-transfected cells was markedly decreased by 10 µM oltipraz treatment (Fig. 4B). These results demonstrated that oltipraz was capable of suppressing 3-MC induction of the CYP1A1 gene.



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  		FIG. 4.
The effect of oltipraz on the induction of CYP1A1 by 3-MC. A, inhibition of 3-MC-inducible CYP1A1 expression by oltipraz. The expression of CYP1A1 was measured in cells treated with 10–100 nM 3-MC in the presence or absence of 10 µM oltipraz for 24 h. Western blot analysis was performed, as described in the legend to Fig. 1A. Relative levels of CYP1A1 protein were assessed by scanning densitometry of immunoblots. Data represent the mean ± S.D. of three separate experiments (significant as compared with 100 nM 3-MC; **, p < 0.01; *, p < 0.05; significant as compared with the respective treatment with 3-MC alone; {dagger}{dagger}, p < 0.01; CYP1A1 level in cells treated with 100 nM 3-MC is 100%). B, inhibition of 3-MC-inducible pGL-CYP1A1-1195 gene activation by oltipraz. Cells were co-transfected with pGL-CYP1A1-1195/pRL-SV (200:1), and then the luciferase activity was measured 18 h after oltipraz (10 µM), 3-MC (30 nM) or 3-MC + oltipraz. The experimental value for luciferase activity was expressed as the relative luciferase unit of cell lysates and represented the mean ± S.D. of three separate experiments (significant as compared with 3-MC alone; **, p < 0.01; CYP1A1 level in cells treated with 30 nM 3-MC is 100%).

 

Next, we assessed whether oltipraz altered the extent of ligand-activated AhR binding to the XRE by gel shift analysis. AhR binding to the XRE in cells treated with 30 nM 3-MC was completely abolished by concomitant treatment with oltipraz (10 µM, 6 h)(Fig. 5).



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  		FIG. 5.
The effect of oltipraz on 3-MC-inducible AhR binding to the XRE. Gel shift analysis was performed with the nuclear fraction prepared from cells treated with oltipraz (10 µM), 3-MC (30 nM) or 3-MC + oltipraz for 6 h. Results were confirmed in four separate experiments. Arrowhead indicates DNA bound with AhR.

 

Activation of the C/EBP Binding Site in the CYP1A1 Promoter by Oltipraz—To test whether C/EBP{beta} was involved in alteration of CYP1A1 gene expression, gel shift analyses were conducted with the putative C/EBP binding site present in the CYP1A1 gene promoter (Fig. 6A), with nuclear fractions. Protein binding to the putative C/EBP binding site was increased 6 h after oltipraz treatment (30 µM), which was in parallel with the previous observation (11). Competition experiments using excess amounts of unlabeled C/EBP or SP-1 oligonucleotides (20-fold) confirmed the specificity of C/EBP DNA binding. Supershift analysis with the highly specific antibody directed against C/EBP{beta}, C/EBP{alpha}, C/EBP{delta}, or AhR indicated that the C/EBP binding complex was comprised of C/EBP{beta} and partly C/EBP{delta}, but not AhR (Fig. 6B). Similar activation of C/EBP DNA binding was observed in cells treated with 10 µM oltipraz (data not shown). Activation of C/EBP was also confirmed by increases in band intensities of C/EBP protein complex with the consensus C/EBP binding oligonucleotide (Fig. 6C).



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  		FIG. 6.
C/EBP binding to the putative C/EBP binding site in the CYP1A1 promoter by oltipraz. A, putative C/EBP binding site present in the promoter region of CYP1A1 and the sequence comparison of the C/EBP binding site with the C/EBP consensus sequence. B, gel shift analysis of C/EBP protein binding to the putative CYP1A1 C/EBP binding site. Nuclear extracts were prepared from cells treated with 30 µM oltipraz for 3–12 h. Competition experiments using 20-fold excess C/EBP or SP-1 binding oligonucleotide confirmed the specificity of C/EBP binding. All lanes contained 15 µg of nuclear extracts and 5 ng of radiolabeled putative CYP1A1 C/EBP binding oligonucleotide. Immunodepletion experiments were carried out by incubating the nuclear extracts (30 µM oltipraz, 6 h) with the specific polyclonal antibody directed against C/EBP{beta}, C/EBP{alpha}, C/EBP{delta}, or AhR. The closed and open arrowheads indicate shifted and supershifted DNA bound with C/EBP protein, respectively. Supershift analysis was carried out by preincubating the nuclear extracts with the respective antibody for 1 h. C, gel shift analysis of C/EBP protein binding to the consensus C/EBP oligonucleotide. Gel shift analysis was performed, as described in the legend to Fig. 1C. Results were confirmed with three repeated experiments.

 

Effect of C/EBP on 3-MC-inducible CYP1A1 Transactivation—To correlate the activation of C/EBP with the inhibition of 3-MC induction of CYP1A1 by oltipraz, constitutively active C/EBP-specific dominant-negative mutant (AC/EBP) was expressed in cells treated with 3-MC in the presence or absence of oltipraz. Expression of AC/EBP significantly abolished the ability of oltipraz to suppress 3-MC induction of CYP1A1 (Fig. 7A). Transfection with pCMV500, which was used as a control vector, allowed oltipraz to inhibit CYP1A1 induction by 3-MC (mock-transfection).



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  		FIG. 7.
The role of C/EBP in suppressing 3-MC-inducible CYP1A1 gene expression. A, reversal by AC/EBP of oltipraz inhibition of 3-MC-inducible CYP1A1 expression. H4IIE cells were transfected with the plasmid encoding AC/EBP for 3 h and further incubated in the medium containing 1% fetal calf serum for 13 h. The cells were then treated with oltipraz (10 µM), 3-MC (30 nM) or 3-MC + oltipraz for 24 h. Data represented the mean ± S.D. of three separate experiments (significant as compared with mock-transfected cells treated with 3-MC alone; **, p < 0.01; significant as compared with mock-transfected cells treated with 3-MC + oltipraz; {dagger}{dagger}, p < 0.01). B, reversal of oltipraz inhibition of 3-MC-inducible pGL-CYP1A1-1195 reporter gene activation by AC/EBP. H4IIE cells were co-transfected with pGL-CYP1A1-1195/pRL-SV (200:1) in combination with pCMV-AC/EBP at a ratio of 1:1, and luciferase activity was measured at 24 h after transfection. Luciferase activity was expressed as firefly luciferase units of cell lysate relative to that of lysed cells transfected with pCMV500. Data represented the mean ± S.D. of three separate experiments (significant as compared with 3-MC alone = 100%; **, p < 0.01; *, p < 0.05) NS, not significant. C, inhibition by transfection with the plasmid encoding C/EBP{beta} of 3-MC induction of pGL-CYP1A1-1195 reporter gene. The luciferase reporter gene assay was performed in cells transiently transfected with the pCDNA-C/EBP{beta}. Control cells were transfected with an empty plasmid pCDNA3.1(+) (mock transfection). Data represented the mean ± S.D. of three separate experiments (significant as compared with control; **, p < 0.01; control level is 1).

 

To further verify the role of C/EBP activation and suppression of AhR-mediated CYP1A1 induction, AC/EBP was expressed in combination with the pGL-CYP1A1-1195 luciferase reporter in H4IIE cells. Expression of AC/EBP completely blocked the ability of oltipraz to inhibit reporter gene expression from the pGL-CYP1A1-1195 plasmid (Fig. 7B). Consistent with this, transfection of cells with the pCDNA-C/EBP{beta} plasmid that encodes C/EBP{beta} blocked 3-MC induction of luciferase activity (Fig. 7C). These results provided evidence that C/EBP{beta} activated by oltipraz was indeed responsible for the suppression of CYP1A1 induction by a PAH compound 3-MC.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Oltipraz affects the activities and the expression of major cytochrome P450s (33). The metabolic activities of major cytochrome P450s including CYP1A and CYP2B responsible for aflatoxin B metabolism were inhibited by oltipraz in hepatocytes (22). Oltipraz also strongly inhibited the activities of CYP1A2 and CYP3A4 in primary hepatocyte culture (34). On the contrary, oltipraz resulted in CYP1A1 and CYP2B induction in animals (22).

The mechanism responsible for an increase of CYP1A1 transcript level primarily involves activation of AhR and activating AhR binding to the XRE in the promoter region of the gene (7, 8). The CBP/p300 protein, and ERAP140 and NcoA/SRC-1/p160 family of transcriptional coactivators have been implicated in AhR/ARNT-mediated transcriptional activation (35–37). It is apparent that oltipraz at relatively high concentrations utilizes the AhR-mediated signal transduction pathway, as does PAH, for the induction of CYP1A1. Nonetheless, the effectiveness of oltipraz for AhR activation differs from that of PAH. The relative potency and efficacy of oltipraz for CYP1A1 induction were much lower than those induced by PAHs.

The physiologically obtainable concentrations of PAHs including 3-MC would be in the nanomolar or subnanomolar range by taking PAH-contaminated foods. This and other research laboratories (25) showed that CYP1A1 was induced by treatment of cells with increasing concentrations of oltipraz (10–100 µM) (25). The levels of CYP1A1 mRNA were also augmented by exposure of H4IIE cells to increasing concentrations of 3-MC or oltipraz (50–100 µM). We found that the physiologically obtainable concentrations of oltipraz in animals and humans (e.g. 30 mg/kg body weight) are within the micromolar range (e.g. 3–30 µM). These observations led us to examine the extents of 3-MC-bound AhR binding to the XRE and CYP1A1 gene transactivation in cells treated with 3-MC (30–50 nM) in combination with oltipraz at physiologically relevant concentrations (i.e. 10–30 µM).

In the present study, we found the intriguing inhibitory effect of oltipraz on CYP1A1 induction by PAH. In contrast to the AhR-mediated induction of CYP1A1 by 30 µM oltipraz, oltipraz at the concentration of 10 µM, at which AhR binding to the XRE was not detectable, inhibited 3-MC (30–50 nM)-inducible CYP1A1 expression. The band intensity of AhR binding to the XRE was attenuated by treatment of cells with oltipraz (10 µM), suggesting that oltipraz directly inhibit PAH-bound AhR activation of the XRE or interfere with AhR DNA binding through activation of other proteins (e.g. transcription factors or corepressors)(Fig. 8). AhR repressor (AhRR), a member of the superfamily bHLH/PAS transcription factors, has recently been identified as a negative factor that suppresses AhR-mediated gene expression (38). The AhRR was shown to compete with AhR for ARNT by forming AhRR/ARNT complex (39). Oltipraz may inhibit CYP1A1 gene transactivation by AhRR activation, which remains to be established.



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  		FIG. 8.
Schematic diagram illustrating the inhibitory effect of oltipraz on the induction of CYP1A1 by 3-MC. PI3-kinase, phosphoinositide 3-kinase.

 

Previous study from our laboratory showed that oltipraz induces nuclear translocation of C/EBP{beta}, but not C/EBP{alpha}, and stimulates C/EBP{beta} binding to the C/EBP-response element in the GSTA2 gene (11). In the present study, we demonstrated for the first time that C/EBP{beta} activation by a physiologically obtainable concentration of oltipraz (i.e. 10 µM) is associated with the negative regulation of PAH-inducible CYP1A1 gene transactivation (Fig. 8). This was further supported by suppression of CYP1A1 promoter-luciferase reporter gene induction. The negative role of C/EBP protein activation by its binding to the C/EBP binding site in the CYP1A1 promoter was further corroborated by reversal of oltipraz inhibition of CYP1A1 induction by AC/EBP transfection. AhR-mediated gene transactivation by physiologically obtainable PAH (i.e. 10–50 nM) is likely to be balanced with C/EBP{beta}-mediated negative regulation by oltipraz for CYP1A1 expression. Gel shift analysis in this study also provided evidence that oltipraz serves as a negative regulator of CYP1A1 expression involving PAH-bound AhR activation of the XRE. When we increased the concentrations of 3-MC to 100 nM or above, the inhibitory effect of oltipraz for CYP1A1 induction by 3-MC was gradually abolished. The pathway of AhR activation and AhR/ARNT binding to the XRE by PAH (at the relatively high concentrations) for CYP1A1 induction was apparently very efficacious probably because the core binding motif of the XRE is present in multiple copies upstream of the CYP1A1 gene promoter (40), whereas a single C/EBP binding site is present within the –1.2-kb gene promoter as shown in the current study. The binding of ligand-activated AhR-ARNT heterodimer to the XRE seems to be more dominating at the high PAH concentrations than the antagonistic pathway involving C/EBP{beta} activation and its binding to the C/EBP binding site.

In the present study, oltipraz at 10 µM, which is a concentration effective for C/EBP{beta} activation, induced CYP1A1 without noticeable AhR binding to the XRE. Transfection of H4IIE cells with the plasmid encoding for C/EBP{beta} caused a moderate increase in the basal CYP1A1-reporter gene expression. Thus, activation of the C/EBP binding site in the CYP1A1 gene by activating C/EBP{beta} (presumably homodimers) binding may transactivate the gene via recruitment of coactivators (e.g. CBP/p300). It is likely that CYP1A1 induction by 10 µM oltipraz may have resulted from C/EBP{beta}-mediated transactivation of the CYP1A1 gene. The differential intrinsic activities between oltipraz and PAH for CYP1A1 induction at the physiologically relevant concentrations seem to be associated with the distinct signaling pathways involving C/EBP{beta} and AhR activation, respectively.

AhR-mediated CYP1A1 transcription was down-regulated by oxidative stress (e.g. hydrogen peroxide) via nuclear factor-1 site in the gene promoter (41). The C/EBP-mediated negative regulation by oltipraz of the AhR-induced CYP1A1 gene transcription in the present study constitutes a distinct pathway, which differs from oxidative stress-induced down-regulation of the CYP1A1 gene. In the study from our laboratory, we revealed that the signaling pathway of phosphoinositide 3-kinase regulated C/EBP{beta}-mediated GSTA2 induction by PD98059 or flavone (20). The potential cancer chemoprevention through dietary flavonoids may result from C/EBP{beta}-mediated transcriptional activation of the phase II enzyme. PD98059, a flavonoid compound, has been claimed to be a ligand for the AhR and to function as an AhR antagonist (42). The result of the present study is in line with the finding that PD98059 antagonizes AhR-mediated response (42).

In conclusion, we provide evidence that oltipraz suppresses 3-MC induction of the CYP1A1 gene expression and that activation of C/EBP{beta} by oltipraz contributes to suppression of PAH-inducible AhR-mediated CYP1A1 expression. We demonstrated that activation of C/EBP{beta} constitutes a distinct pathway that negatively regulates PAH-mediated AhR activation of the XRE for the CYP1A1 gene transcription. The observation that oltipraz suppresses the potent and efficacious induction of CYP1A1 by 3-MC is at least in part consistent with its cancer chemopreventive effect. The current study brings additional insights into the chemopreventive effects of oltipraz to modify CYP1A1 gene regulation involved in procarcinogen metabolism.


   FOOTNOTES
 
* This work was supported by National Research Laboratory Program (2001), KISTEP, The Ministry of Science and Technology, Republic of Korea. 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} To whom correspondence should be addressed: College of Pharmacy, Seoul National University, Sillim-dong, Kwanak-gu, Seoul 151-742, South Korea. Tel.: 822-880-7840; Fax: 822-872-1795; E-mail: sgk{at}snu.ac.kr.

1 The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; 3-MC, 3-methylcholanthrene; AC/EBP, dominant-negative mutant C/EBP; AhR, aryl hydrocarbon receptor; ARE, antioxidant response element; ARNT, aryl hydrocarcbon receptor nuclear translocator; C/EBP, CCAAT/enhancer-binding protein; CYP1A1, cytochrome P4501A1; MEM, minimal essential medium; XRE, xenobiotic response element; GST, glutathione S-transferase. Back


   ACKNOWLEDGMENTS
 
The kind donation of pCMV500 and AC/EBP plasmids from Dr. C. Vinson is gratefully thanked.



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 ABSTRACT
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 RESULTS
 DISCUSSION
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