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Transactivation of the PPAR-Responsive Enhancer Module in Chemopreventive Glutathione S-Transferase Gene by the Peroxisome Proliferator-Activated Receptor- and Retinoid X Receptor Heterodimer

National Research Laboratory, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea

	ABSTRACT

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Cancer chemopreventive agents transcriptionally induce glutathione S-transferase (GST), which can protect cells from chemical-induced carcinogenesis. Activation of either NF-E2-related factor-2 (Nrf2) or the CCAAT/enhancer binding protein-ß (C/EBPß) contributes to GST induction. Peroxisome proliferator-activated receptor- (PPAR) and the retinoic acid X receptor (RXR) play roles in regulating cell differentiation and chemoprevention. This study examined GSTA2 gene induction by the PPAR activator and 9-cis-retinoic acid (RA), a RXR ligand, and investigated the molecular basis of PPAR-RXR-mediated GSTA2 induction in the H4IIE hepatocytes. Either 15-deoxy- (12, 14)-prostaglandin J₂ (PGJ₂) or RA induced GSTA2 with Nrf2 and C/EBPß activation. When compared with PGJ₂ or RA alone, PGJ₂ + RA enhanced GSTA2 induction, with increases in Nrf2 and C/EBPß activation. PGJ₂ + RA increased the luciferase reporter gene activity in the cells transfected with the –1.65-kb flanking region of the GSTA2 gene. Thiazolidinedione PPAR agonists, troglitazone, rosiglitazone, and pioglitazone, in combination with RA, potentiated GSTA2 induction, confirming that the activation of the PPAR and RXR heterodimer contributed to GSTA2 expression. Deletion of the antioxidant response element- or C/EBP-binding sites or the overexpression of dominant-negative mutant of C/EBP abolished the reporter gene expression. PGJ2 + RA increased the binding of the PPAR – RXR heterodimer to the putative PPAR-response elements (PPREs) in the GSTA2 promoter. Specific mutations of these multiple PPRE sites resulted in the complete loss of its responsiveness to PGJ2 + RA, which suggests that these binding sites function as a PPRE-responsive enhancer module (PPREM). Transactivation of PPREM by the PPAR – RXR heterodimer was verified by the effective GSTA2 induction in the cells treated with PGJ2 + RA after transfecting them with the plasmids encoding PPAR1 and RXR. In conclusion, the PPAR – RXR heterodimer promotes GSTA2 induction by activating PPREM in the GSTA2 gene, as well as inducing Nrf2 and C/EBPß activation.

	INTRODUCTION

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Glutathione S-transferases (GSTs) are responsible for the cellular metabolism as well as detoxification of several xenobiotics and carcinogenic compounds primarily in the liver and, to a much lesser extent, in the extrahepatic organs. Glutathione conjugates formed in the liver can either be excreted intact in bile, or they can be converted to mercapturic acids in the kidney and excreted in the urine (1) . The loss of GST protection can increase the susceptibility of preneoplastic hepatocytes to chemical-induced genotoxicity during chemical carcinogenesis. Therefore, GST induction is not only for cell detoxification and survival but also cancer prevention.

The activation of the antioxidant response element (ARE) by reactive oxygen species from prooxidants contributes to the regulation of Phase II enzymes, including the GSTs (2 , 3) . The signals activated by oxidative stress stimulate the transduction of NF-E2-related factor (Nrf) activity and ARE activation (4 , 5) . The protein binding to the ARE consensus sequence involves the Nrf proteins, as well as Maf family members (6 , 7) . Oxidative stress induces GSTA2 via ARE activation, which involves the Nrf proteins (8 , 9) . Transcription factors of the CCAAT/enhancer binding protein (C/EBP) family play roles in cell differentiation and exert their function by regulating the expression of tissue-specific genes, as well as cell proliferation (10 , 11) . In response to growth stimuli, the members of the C/EBP family involved in the transcription of the tissue-specific genes include C/EBPß and C/EBP (12) . C/EBP constitutes an essential distinct pathway for GST induction. Previous studies have shown that C/EBPß activation and its binding to the C/EBP-response element plays a crucial role in the induction of the GSTA2 gene (13 , 14) .

The peroxisome proliferator-activated receptors (PPARs) are well-characterized transcription factors that are members of the nuclear hormone receptor superfamily (15) . There are three PPARs subtypes, including PPAR, PPARß, and PPAR, which have distinct distribution patterns. PPAR is mainly present in the liver, heart, and kidney, whereas PPARß is expressed ubiquitously (15) . PPAR is predominantly expressed in the adipose tissue and, to a lesser extent, in the liver (15) . PPAR activators up-regulate the expression of the genes involved in adipocyte differentiation (16) . Although the basal expression of the PPAR in the liver is relatively low, hepatic PPAR is pronouncedly up-regulated by PPAR activators and under certain pathophysiological conditions (e.g., obesity) for the activation of several PPAR-responsive genes (17 , 18) . PPAR is also an important target for the development of new drugs aimed at preventing and treating cancer. A deficiency in PPAR expression can be a significant risk factor for carcinogenesis (19) . Ligands for PPAR suppress carcinogenesis in experimental models and induce the differentiation of human tumorigenic cells (20, 21, 22, 23) . The activated PPAR forms a heterodimer with retinoid X receptor (RXR) and binds to specific PPAR response elements in the promoter region of their target genes (24) . Therefore, PPAR agonists affect cell survival, growth, and differentiation by binding to the peroxisomal proliferator-response element (PPRE).

Hepatic stellate cells in the liver contain 40–70% of the body stores of retinoids. Retinoic acids, which are natural metabolites of circulating vitamin A, are essential for maintaining the normal pathway of epithelial tissues differentiation (25) . Natural and synthetic retinoids are also effective in preventing a variety of cancers in animals and in reversing preneoplastic lesions in humans (26, 27, 28, 29) . A large-scale study of the cancer chemopreventive role of retinoic acids in human has shown that retinoids exhibit a high degree of specificity in cancer chemoprevention (30) . The major retinoids responsible for the transcriptional regulation of many processes in the development and differentiation are all-trans and 9-cis-retinoic acids (RAs), which affect the important biological processes via their interaction with the nuclear receptors (25) . In particular, RA was identified as an activating ligand that is relatively selective for RXR, which must heterodimerize with a permissive partner (24 , 31 , 32) . RXR is essential for the high-affinity DNA binding of PPAR and serves as an auxiliary DNA-binding factor.

From these observations, it is possible that PPAR and RXR agonists activate the expression of the liver-specific genes at the transcriptional level. The liver-specific target genes may include GSTs because there is an extremely high (i.e., 10 mM) concentration of glutathione in the liver. The conjugation of xenobiotics with glutathione, which are catalyzed by all classes of GSTs, is fundamentally different from their conjugation with other amino acids, because the substrates of glutathione conjugation include an enormous range of electrophilic xenobiotics or xenobiotics, which are biotransformed to electrophiles (33) . The GSTA2 gene is a representative member of the class GSTs that contains a xenobiotic-response element, putative phenobarbital-responsive element, glucocorticoid-responsive element, and an ARE. However, no functionally active PPRE or PPRE-responsive enhancer module (PPREM) has yet been identified in the GSTA2 gene. In the present study, we initially found the putative multiple PPREs in the promoter region of the GSTA2 gene. We determined whether the PPAR and RXR activators induce GSTA2 via activating PPAR – RXR heterodimer binding to the putative PPREs in H4IIE hepatocytes and, if so, whether the putative PPREs function as a module where there needs to be multiple nuclear binding sites in close proximity. We used 15-deoxy- (12 , 14) -prostaglandin J₂ (PGJ₂) and thiazolidinedione PPAR ligands as the PPAR agonists. It was found that the combined treatment of the PPAR agonist and RA both activated and induced Nrf2 and C/EBPß, which are the essential transcription factors for GSTA2 expression. More importantly, the PPAR-binding site cluster in the GSTA2 gene was identified as a functionally active PPREM.

	MATERIALS AND METHODS

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Materials.
[-³²P]dCTP (3000 mCi/mmol) and [-³²P]ATP (3000 mCi/mmol) were purchased from New England Nuclear (Arlington Heights, IL). The random prime-labeling kit was supplied by Promega (Madison, WI). PGJ₂ and troglitazone were purchased from Biomol Research Labs (St. Louis, MO). Pioglitazone and rosiglitazone were kindly supplied from Dong-A Pharmaceutical Co. (Shingal, Korea). RA, DTT, and 3-[N-morpholino]propanesulfonic acid were provided from Sigma Chemical (St. Louis, MO). Anti-PPAR antibody and anti-RXR antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The plasmid pGTB-1.65 construct containing the GSTA2-promoter region (–1651 to +66 bp from the transcription start site) was kindly provided by Dr. C. B. Pickett (Schering-Plough Corp. Inst., Kenilworth, NJ), and the C/EBP dominant-negative expression (AC/EBP) plasmid was a gift from Dr. C. Vinson (NIH, Besthesda, MD; Ref. 34 ). AC/EBP prevents the "normal" C/EBP from binding to DNA because C/EBP acts as a dimer. The expression plasmid of mouse PPAR1 (i.e., pCMX-mPPAR1) was kindly supplied from Dr. C. K. Glass (University of California, San Diego, CA). The human RXR expression plasmid (PECE-RXR) was provided by Dr. M. O. Lee (Sejong University, Seoul, Korea).

Construction of Plasmids.
The pGL-1651 reporter gene construct containing the promoter region of the GSTA2 gene was generated according to methods described previously (14) . Briefly, the region containing –1651 to +66 bp of the GSTA2 gene was amplified by PCR using pGTB-1.65 as a template. The PCR product was cloned into pGEM-T EasyVector (Promega) and subcloned into the pGL3 luciferase reporter plasmid. The deletion mutants of GSTA2 promoter-luciferase plasmid, pGL-C/EBP and pGL-ARE, in which the C/EBP binding site and ARE were deleted, respectively, were constructed by PCR-based methods. The specific PPRE mutant constructs of the GSTA2 promoter-luciferase reporter gene, pGL-mPPRE1, pGL-mPPRE2, and pGL-mPPRE3, were prepared by replacing the respective direct repeat (DR)1 site with 5'-CTCGAGAGAATTC-3'. The mutant plasmids were generated by the overlapping PCR technique and then ligated into the pGL3 plasmid. To increase RXR expression, the coding region of RXR derived from PECE-RXR (HindIII and EcoRI digestion) was subcloned into pCDNA3.1 (Invitrogen, Carlsbad, CA; i.e., pCDNA-RXR). The DNA sequences of the constructs were verified by sequence analysis using an ABI7700 DNA cycle sequencer.

Cell Culture.
H4IIE, a rat hepatocyte-derived cell line, and 3T3-L1, a murine preadipocyte cell line, were obtained from American Type Culture Collection (Rockville, MD). Cells were maintained in DMEM containing 10% FCS, 50 units/ml penicillin, and 50 µg/ml streptomycin at 37°C in a humidified atmosphere with 5% CO₂. 3T3-L1 preadipocytes were differentiated to adipocytes by incubating in the medium containing 10% FCS, 1 µM dexamethasone, 1 µg/ml insulin, and 0.5 mM 3-isobutyl-1-methylxanthine, as described previously (35) . Hepatic stellate cells and primary hepatocytes were isolated from male Sprague Dawley rats according to the method published previously with slight modifications (36) .

PPAR Agonist and RA Treatment.
PGJ₂, troglitazone, pioglitazone, rosiglitazone, and RA, dissolved in DMSO, were added to H4IIE cells and incubated for the indicated time period for each experiment at 37°C. Cells were then washed twice with ice-cold PBS before sample preparation.

Preparation of a cDNA Probe for GSTA2.
A cDNA probe for the GSTA2 gene was amplified by reverse transcription-PCR using selective primers (37) and cloned in the pGEM-T vector (Promega).

Preparation of Nuclear Extracts.
Nuclear extracts were prepared according to methods published previously (14) . Briefly, H4IIE cells in dishes were washed twice with ice-cold PBS and then scraped from the dishes with PBS and transferred to microtubes. Cells were then centrifuged at 2,500 x g for 3 min 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% NP40, 1 mM DTT, and 0.5 mM phenylmethylsulfonylfluoride. The lysates were incubated for 10 min on ice and then centrifuged at 7,200 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 DTT, and 1 mM phenylmethylsulfonyl fluoride and then incubated for 30 min on ice. The samples were then centrifuged at 15,800 x g for 10 min to obtain supernatants containing nuclear fractions. Nuclear fractions were stored at –70°C until use.

Preparation of Cytosolic Fractions.
H4IIE cells were washed twice with sterile PBS, scraped from their dishes, and sonicated to disrupt the membranes. Cytosolic fractions were prepared by differential centrifugation at 15,000 x g for 15 min and stored at –70°C until use. Protein content was determined by the Bradford assay (Bio-Rad protein assay kit; Bio-Rad, Hercules, CA).

Northern Blot Analysis.
Total RNA was isolated from H4IIE cells using the single-step method of thiocyanate-phenol-chloroform RNA extraction, and Northern blot analysis was carried out according to procedures described previously (37) . Briefly, total RNA was resolved by electrophoresis in a 1% agarose gel containing 2.2 M formaldehyde and transferred to nitrocellulose paper. The nitrocellulose paper was baked in a vacuum oven at 80°C for 2 h. The filter was then incubated with hybridization buffer containing 50% deionized formamide, 5 x Denhardt’s solution [0.1% Ficoll, 0.1% polyvinylpyrrolidine, and 0.1% BSA (Pentex Fraction V)], 0.1% SDS, 200 µg/ml sonicated salmon sperm DNA, and 5 x saline-sodium phosphate-EDTA [1 x saline-sodium phosphate-EDTA = 0.15 M NaCl, 10 mM NaH₂PO₄, and 1 mM Na₂EDTA (pH 7.4)] at 42°C for 1 h without probe. Hybridization was performed at 42°C for 18 h with a heat-denatured cDNA probe for GSTA2 that was random prime-labeled with [-³²P]dCTP. Filters were washed twice in 2 x SSC (1 x SSC = 150 mM NaCl and 15 mM sodium citrate) and 0.1% SDS for 10 min at room temperature and twice in 0.1 x SSC and 0.1% SDS for 10 min at room temperature. Filters were then washed once in a solution containing 0.1 x SSC and 0.1% SDS for 1 h at 60°C. After quantitation of GSTA2 mRNA levels via scanning densitometry, the membranes were stripped and rehybridized with a ³²P-labeled cDNA probe for 18S rRNA to control for RNA loading onto the membranes. Four separate experiments were performed with different RNA samples.

Immunoblot Analysis.
SDS-PAGE and immunoblot analysis were performed according to procedures published previously (37) . Briefly, proteins were separated by 7.5 or 12% gel electrophoresis and electrophoretically transferred to nitrocellulose paper. The replicate SDS-PAGE gels were stained with Coomassie Blue for verification of equal loading of proteins before immunoblotting. The nitrocellulose paper was incubated with an anti-GST antibody (Detroit R&D, Detroit, MI), followed by incubation with a horseradish peroxidase-conjugated secondary antibody. Specificity of the antibodies to the GST subunit has been confirmed previously (8 , 37) . Immunoreactive protein was visualized through incubation with an enhanced chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, United Kingdom; Ref. 8 ). Equal loading of proteins was verified by actin immunoblotting with a goat antiactin antibody (Santa Cruz Biotechnology). Three or four separate experiments were performed with different cytosolic samples. Changes in the levels of GSTA2 protein in the cells treated with PPAR agonist + RA relative to that in untreated control were determined via scanning densitometry. Similarly, PPAR, Nrf2, and C/EBPß were immunochemically detected with their respective antibodies (Santa Cruz Biotechnology). At least three separate experiments were performed with different subcellular fractions (or lysates) to confirm changes in the protein levels.

Scanning Densitometry.
Scanning densitometry of the Northern and immunoblots was performed with Image Scan & Analysis System (Alpha-Innotech Corp., San Leandro, CA). The area of each lane was integrated using the software AlphaEase version 5.5, followed by background subtraction.

Gel Shift Assay.
A double-stranded DNA probe containing the GSTA2 gene ARE end-labeled with [-³²P]ATP, and T₄ polynucleotide kinase was used for gel shift analysis. The sequence of the ARE-containing oligonucleotide was 5'-GATCATGGCATTGCACTAGGTGACAAAGCA-3'. Similarly, C/EBP gel shift analysis was carried out with the radiolabeled oligonucleotide, 5'-TGCAGATTGCGCAATCTGCA-3', which contained the C/EBP consensus sequence. The consensus PPRE sequence was 5'-CCAAGGTCAAAGGTCATGT-3'. To study the binding of nuclear hormone receptors to the DNA-binding sites, double-stranded PPRE oligonucleotides derived from PPRE1, 5'-CCATCGGTGATGACCTTGT-3'; PPRE2, 5'-TTGGCAGGAAGGATCAGTA-3'; and PPRE3, 5'-AACAGGACAAAGATTAAGA-3' present in the promoter region of the GSTA2 gene were used as probes. The reaction mixture contained 4 µl of 5 x binding buffer [containing 50 mM Tris-Cl (pH 7.5), 20% glycerol, 5 mM MgCl₂, 250 mM NaCl, 2.5 mM EDTA, 2.5 mM DTT, and 0.25 mg/ml poly dI-dC], 10 µg of nuclear extract, and sterile water up to a total volume of 20 µl. The reaction mixture was preincubated without probe at room temperature for 10 min. The probe (1 µl, containing 10⁶ cpm) was then added, and DNA-binding reactions were carried out for 30 min at room temperature. In some analyses, specificity of binding was determined by competition experiments, which were carried out by adding a 10-fold molar excess of an unlabeled ARE, C/EBP, or PPRE oligonucleotide to the reaction mixture before the labeled probe was added. SP-1 oligonucleotide (5'-ATTCGATCGGGGCGGGGCGAGC-3') was used as a negative control for competition experiments. In other analyses, known as immuno-inhibition assays, antibodies directed to Nrf2, C/EBP, C/EBPß, PPAR, and RXR (2 µg each) were added to the reaction mixture 20 min after the labeled probe was added, and the reaction was then continued for 1 h at 25°C. Samples were separated on 4% polyacrylamide gels at 100 V. The gels were fixed with 40% methanol/10% acetic acid, dried, and subject to autoradiography.

Luciferase Reporter Gene Analysis.
We used the dual-luciferase reporter assay system (Promega). Briefly, H4IIE cells (7 x 10⁵ cells/well) were replated in six-well plates overnight, serum starved for 12 h, and transiently transfected with each GSTA2 promoter-luciferase construct and pRL-SV plasmid (a plasmid that encodes for Renilla luciferase and is used to normalize transfection efficacy) in the presence of LipofectAMINE Plus Reagent (Life Technologies, Inc. Gaithersburg, MD) for 3 h. Transfected cells were incubated in DMEM containing 1% FCS (Life Technologies) for 3 h and exposed to PGJ2 + RA (50 nM each) in medium containing 10% FCS for 18 h at 37°C. Firefly and Renilla luciferase activities in cell lysates were measured using a Luminoskan luminometer (Thermo Labsystems, Helsinki, Finland). The activity of firefly luciferase was measured by adding Luciferase Assay Reagent II (Promega) according to the manufacturer’s instruction, and, after quenching the reaction, the Renilla luciferase reaction was initiated by adding Stop & Glo reagent (Promega). The relative luciferase activity was calculated by normalizing firefly luciferase activity to that of Renilla luciferase.

Statistical Analysis.
Scanning densitometry was performed with Image Scan & Analysis System (Alpha-Innotech). One-way 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 <0.01. All statistical tests were two sided.

	RESULTS

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GSTA2 Induction by PGJ₂.
The PPAR1 expression level was higher than that of PPAR2 in most tissue types (15) . To examine the role of PPAR in GSTA2 expression, we first assessed the PPAR levels in H4IIE hepatocytes and other representative cells. Immunoblot analysis revealed that mainly PPAR1 was expressed in H4IIE cells (Fig. 1 , left panel). PPAR1 was also expressed in the primary cultured hepatocytes or stellate cells isolated from the rat liver (Fig. 1 , middle panel). It was reported that the PPAR expression level was high in adipocytes (1 is expressed to a greater extent than 2) but not in the preadipocytes,¹⁶ which was also confirmed by immunoblot analysis (Fig. 1 , right panel).

View larger version (13K): [in this window] [in a new window]   Fig. 1. Expression of peroxisome proliferator-activated receptor (PPAR) in H4IIE cells and other types of cells. Total cell lysates were prepared from H4IIE cells, primary cultured hepatocytes, hepatic stellate cells, 3T3-L1 preadipocytes, and 3T3-L1 adipocytes, as described in "Materials and Methods." Immunoblot analyses were conducted with the lysate preparations. Each lane contained 10, 20, or 30 µg of lysate proteins. Closed and open arrowheads, PPAR1 and PPAR2, respectively. Results were confirmed by repeated experiments.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Induction of GSTA2 by PGJ2. A, immunoblot analyses of the GSTA2 protein. Immunoblot analyses show the levels of GSTA2 protein in H4IIE cells treated with 100-1000 nM PGJ2 for 24 h (left) or 300 nM PGJ2 for 1–24 h. Each lane was loaded with 10 µg of cytosolic proteins. Immunoreactive protein was visualized through incubation with horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection kit. Equal loading of proteins was verified by probing the replicate blots for actin. Changes in GSTA2 protein levels relative to control were assessed by scanning densitometry. B, Northern blot analyses of GSTA2 mRNA. Northern blot analysis was performed with total RNA fractions (30 µg each) prepared from the cells incubated with 100-1000 nM PGJ2 for 24 h (left) or in the cells treated with 300 nM PGJ2 for 3–24 h (right). The equal loading of RNA in each lane was confirmed by rehybridization of the stripped membrane with a 32P-labeled probe for 18S rRNA. Changes in the GSTA2 mRNA expression relative to control were assessed by scanning densitometry and normalized by RNA loading. C, immunoblot analyses of NF-E2-related factor (Nrf)2 or CAAT/enhancer binding protein (C/EBP)ß and gel shift assays of Nrf2 or C/EBPß DNA binding. Nuclear fractions were prepared from H4IIE cells treated with 300 nM PGJ2 for 1–12 h. Gel shift assays were conducted with nuclear extracts prepared from H4IIE cells exposed to PGJ2 (300 nM) for 1–12 h and 5 ng of radiolabeled antioxidant response element (ARE) or C/EBP-binding oligonucleotide. Arrowheads, the DNA bound with proteins. Results were confirmed by three separate experiments, and a representative immunoblot is shown. Data represent the mean ± SD with three separate experiments. One-way ANOVA was used for comparisons of multiple group means followed by Newman-Keuls test (significant as compared with control, *, P < 0.05; **, P < 0.01; control level = 1).

GSTA2 Induction by RA.
Next, we determined whether RA could induce GSTA2. Treatment of the H4IIE cells with RA (10–300 nM), a ligand of RXR, for 24 h increased the GSTA2 protein level in a concentration-dependent manner (Fig. 3A) . RA at concentrations of 100 or 300 nM induced GSTA2 2–3-fold with the maximum induction being observed at 300 nM. A time course study showed that RA gradually induced the GSTA2 protein level from 6 h with the maximum increase being noted at 24 h. Northern blot analyses confirmed that the level of GSTA2 mRNA increased after exposure of cells to 300 nM RA (Fig. 3B) . The GSTA2 mRNA level peaked at 12 h.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Induction of glutathione S-transferase (GST)A2 by 9-cis-retinoic acid (RA). A, immunoblot analyses of the GSTA2 protein in the cells treated with 10–300 nM RA for 24 h or 300 nM RA for 1–24 h. Each lane was loaded with 10 µg of cytosolic proteins. B, Northern blot analyses of GSTA2 mRNA. Northern blot analysis was performed with total RNA fractions (30 µg each) prepared from the cells incubated with 10–300 nM RA for 24 h (left) or 300 nM RA for the indicated times (right). C, immunoblot analyses of NF-E2-related factor (Nrf)2 or CCAAT/enhancer binding protein (C/EBP)ß and gel shift assays of Nrf2 or C/EBPß DNA binding. Nuclear fractions were prepared from H4IIE cells treated with 300 nM RA for 1–12 h. Gel shift assays were conducted as described in the legend to Fig. 2 C. Immunoblot analyses of Nrf2 and C/EBPß in the nuclear fractions. Nrf2 and C/EBPß were immunoblotted in the nuclear fractions of the cells treated with 300 nM RA for 1–12 h. Results were confirmed by three separate experiments, and a representative immunoblot is shown. Data represent the mean ± SD with four separate experiments. One-way ANOVA was used for comparisons of multiple group means followed by Newman-Keuls test (*, P < 0.05, significant as compared with control; **, P < 0.01, control level = 1).

Synergistic Induction of GSTA2 by PGJ₂ + RA.
PPAR heterodimerizes with the RXR for activation, and the PPAR – RXR heterodimers are widely expressed in the major organs, including the liver (15) . We determined whether PGJ₂ + RA enhanced GSTA2 induction. GSTA2 was induced 2-fold by 100 nM RA but not 100 nM PGJ₂. In contrast to the weak induction of GSTA2 by each agent alone, PGJ₂ + RA (100 nM each) synergistically increased the GSTA2 protein level (Fig. 4A) . A preliminary study reported that PGJ₂ + RA induced GSTA2 to the greatest extent at the 1:1 molar ratio. To determine the dose-response effect, the GSTA2 level was further assessed in the cells treated with PGJ₂ + RA each at equal concentrations (i.e., 15, 50, or 100 nM) for 24 h (Fig. 4B) . The dose-response study revealed that GSTA2 induction was efficaciously increased by the PGJ₂ + RA treatment (i.e., 3–4-fold increases at concentrations of 15 or 50 nM). Therefore, 50 nM PGJ₂ and RA each were selected for the subsequent experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Induction of glutathione S-transferase (GST)A2 by prostaglandin J2 (PGJ2) + 9-cis-retinoic acid (RA). A, representative immunoblot analysis of the GSTA2 protein in the H4IIE cells incubated with PGJ2 with or without RA for 24 h. B, the relative levels of GSTA2 in the cells treated with PGJ2 + RA (1:1) for 24 h. GSTA2 protein levels were assessed by scanning densitometry of immunoblots. Each lane was loaded with 10 µg of cytosolic proteins. Immunoreactive protein was visualized through incubation with horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection kit. Data represent the mean ± SD with three separate experiments (significant as compared with control, **, P < 0.01; the level of GSTA2 in control cells = 1).

View larger version (25K): [in this window] [in a new window]   Fig. 5. Induction of glutathione S-transferase (GST)A2 by peroxisome proliferator-activated receptor- agonists in combination with 9-cis-retinoic acid (RA). A, immunoblot analyses of the GSTA2 in H4IIE cells treated with troglitazone and/or RA, rosiglitazone, and/or RA or pioglitazone and/or RA. Western blot analysis was performed in the cells treated with peroxisome proliferator-activated receptor- agonist (1–10 µM) in the absence or presence of RA (50 nM) for 24 h. Results were confirmed by repeated experiments, and representative blots are shown. B, immunoblot analyses of the GSTA2 in primary cultured hepatocytes treated with each peroxisome proliferator-activated receptor- agonist (1–3 µM) and/or RA (50 nM) for 48 h. Data represent the mean ± SD with three separate experiments (*, P < 0.05, significant as compared with untreated control; **, P < 0.01, the level of GSTA2 in control = 1).

Activation of Nrf2 and C/EBPß by PGJ₂ + RA.
To confirm that PGJ₂ + RA (50 nM each) activates Nrf2, immunoblot and gel shift assays were performed with the nuclear extracts of the cells treated with PGJ₂ + RA. PGJ₂ + RA increased the nuclear Nrf2 levels and its binding to a radiolabeled ARE consensus oligonucleotide at 6–12 h. Binding of the protein to the ARE was competed with an excess amount of unlabeled ARE (Fig. 6A) . Immunoblot and gel shift analyses showed that the PGJ₂ + RA treatment increased the C/EBPß levels in the nuclear fractions and C/EBPß binding to the C/EBP binding oligonucleotide compared with the control (Fig. 6B) . Competition experiments using excess quantities of unlabeled C/EBP or SP-1 oligonucleotides confirmed the specificity of C/EBP DNA binding. Supershift experiments indicated that the C/EBP-binding complex comprised of C/EBPß (Fig. 6B) . The band intensity of C/EBP DNA-binding complex was reduced by the anti-C/EBPß antibody but not the anti-PPAR or -RXR antibodies. Immunocytochemistry confirmed that Nrf2 and C/EBPß, which were located mainly in the cytoplasm of control cells, had perinuclear and nuclear localization at 6–12 h in the cells treated with PGJ₂ + RA, suggesting that these transcription factors translocate into the nucleus (data not shown).

View larger version (47K): [in this window] [in a new window]   Fig. 6. Activation of NF-E2-related factor (Nrf)2 and CCAAT/enhancer binding protein (C/EBP)ß by prostaglandin J2 (PGJ2) + 9-cis-retinoic acid (RA). A, immunoblot analysis of nuclear Nrf2 and gel shift analysis. Nrf2 was immunoblotted in the nuclear extracts prepared from H4IIE cells incubated with PGJ2 + RA (50 nM each) for 1–12 h. Gel shift analysis was performed with the nuclear fractions. Arrowheads, the antioxidant response element (ARE) binding complex. For competition assays, 10-fold molar excess of unlabelled ARE oligonucleotide or SP-1 oligonucleotide was added to the reaction mixture that included radiolabeled oligonucleotide (5 ng) and nuclear extracts prepared from H4IIE cells treated with PGJ2 + RA for 6 h. B, immunoblot analysis of C/EBPß and gel shift analysis of C/EBPß binding to the C/EBP-binding site. Nuclear extracts were prepared from H4IIE cells incubated with PGJ2 + RA (50 nM each) for 1–12 h. Arrowheads, the C/EBPß bound with DNA. Immunocompetition or unlabelled excess oligonucleotide assays of C/EBP binding to the C/EBP-binding site were performed with the nuclear extracts prepared from the cells treated with PGJ2 + RA (50 nM each) for 6 h. Results were confirmed by three separate experiments. Data represent the mean ± SD with three separate experiments (*, P < 0.05, significant as compared with control; **, P < 0.01, the band intensity in control cells = 1).

View larger version (19K): [in this window] [in a new window]   Fig. 7. Induction of NF-E2-related factor (Nrf)2 and CCAAT/enhancer binding protein (C/EBP)ß by prostaglandin J2 (PGJ2) + 9-cis-retinoic acid (RA). Immunoblot analyses of Nrf2 (A) and C/EBPß (B) in the cells treated with PGJ2 + RA. The levels of Nrf2 or C/EBPß were determined in the lysates of cells treated with PGJ2 + RA (50 nM each) for 1–12 h. Each lane was loaded with 15 µg of cell lysates. Data represent the mean ± SD with three separate experiments (significant as compared with control, **, P < 0.01; the level of Nrf2 or C/EBPß in control cells = 1).

View larger version (25K): [in this window] [in a new window]   Fig. 8. The role of antioxidant response element (ARE) or CCAAT/enhancer binding protein (C/EBP)-response element in the induction of glutathione S-transferase (GST)A2 promoter-luciferase gene by prostaglandin J2 (PGJ2) + 9-cis-retinoic acid (RA). A, the deletion mutants of GSTA2 luciferase chimeric construct. B, the effects of deletion mutation of ARE or C/EBP-response element on the induction of luciferase activity by PGJ2 + RA. Dual luciferase reporter assays were performed on the lysates of H4IIE cells that had been cotransfected with the GSTA2-luciferase gene construct pGL-1651 (firefly luciferase) and pRL-SV (Renilla luciferase) at a ratio of 200:1 and subsequently exposed to PGJ2, RA, or PGJ2 + RA (50 nM each). Activation of the reporter gene was calculated as a change in the ratio of firefly luciferase activity to Renilla luciferase activity. PGJ2 + RA significantly induced luciferase activity in H4IIE cells transiently transfected with GSTA2 chimeric gene construct pGL-1651, which contains both the C/EBP response element and ARE of the GSTA2 promoter. Deletion mutation of the C/EBP response element or ARE completely abolished the luciferase induciblity by PGJ2 + RA. Inset, the inhibition of PGJ2 + RA-inducible pGL-1651 reporter gene activation by the dominant-negative mutant of C/EBP (AC/EBP). H4IIE cells were cotransfected with pGL-1651/pRL-SV (200:1) in combination with pCMV500 or pCMV-AC/EBP at a ratio of 1:1, and luciferase activity was measured at 18 h after transfection. The experimental value for luciferase activity was expressed as a relative luciferase unit of cell lysate and represented the mean ± SD with four separate experiments (significant as compared with control, PGJ2, or RA alone, **, P < 0.01; significant as compared with the respective treatment in cells transfected with pGL-1651, ##, P < 0.01; significant as compared with pCMV500-transfected cells treated with PGJ2 + RA, , P < 0.01).

Binding of the PPAR and RXR Heterodimer to the PPREs.
Studies have shown that PPAR heterodimerizes with RXR (probably RXR), which is activated by RA (38 , 39) . Searching the GSTA2 promoter region with the PPRE consensus sequence, AGGTCANAGGTCA, resulted in three putative response elements (i.e., PPRE1, PPRE2, and PPRE3), to which the activating PPAR – RXR heterodimer might bind. The putative PPRE1 sequence present at –792 bp from the transcription start site contained a reverse DR1 site, whereas the other DR1 sites named PPRE2 and PPRE3 were present at –746 and –549 bp, respectively (Fig. 9A) .

View larger version (56K): [in this window] [in a new window]   Fig. 9. Gel shift analyses of peroxisome proliferator-activated receptor (PPAR) and retinoic acid X receptor (RXR) binding to the putative PPAR-response elements (PPREs) in the glutathione S-transferase (GST)A2 promoter. A, sequence of the GSTA2 promoter region containing the three PPAR- and RXR-binding sites named PPRE1, PPRE2, and PPRE3. Gel shift analyses of protein binding to PPRE1 (B), PPRE2 (C), or PPRE3 (D). Nuclear extracts prepared from H4IIE cells exposed to prostaglandin J2 (PGJ2) + 9-cis-retinoic acid (RA; 50 nM each) for 6 h were incubated for 30 min with 5 ng of radiolabeled PPRE1, PPRE2, or PPRE3 oligonucleotide and separated on 4% polyacrylamide gel. Arrowheads, the PPREs bound with proteins. E, competition assays for protein binding to the PPRE3. For immunocompetition analyses, the nuclear extracts were incubated with an anti-PPAR antibody or -RXR antibody for 1 h and then mixed with labeled probe for the PPRE3-binding site. Competition assays with 10-fold molar excess of unlabelled PPRE3 or SP-1 oligonucleotide added to the reaction mixture that included radiolabeled PPRE3 oligonucleotide (5 ng) and the nuclear extracts. Results were confirmed by separate experiments. Data represent the mean ± SD with three separate experiments (*, P < 0.05, significant as compared with control; **, P < 0.01, the band intensity in control cells = 1).

Functional Analysis of the PPREs in the GSTA2 Promoter.
When the PPRE1 sequence located between the nucleotides –792 and –781 bp in pGL-1651 was mutated (Fig. 10A ; i.e., pGL-mPPRE1), the inducible luciferase activity by PGJ₂ + RA was completely reduced to that of the control (Fig. 10B) . For comparison, this study determined the effect of 3-methylcholanthrene, an AhR-xenobiotic-response element-mediating GSTA2 inducer, on the expression of pGL-mPPRE1 promoter reporter gene. Exposure of the H4IIE cells transfected with pGL-mPPRE1 to 3-methylcholanthrene increased the luciferase activity 8-fold compared with the untreated control (Fig. 10B) . The increase in luciferase activity was comparable with that in pGL-1651-transfected cells treated with 3-methylcholanthrene. These results indicated that the PPRE1 in the GSTA2 gene is selectively activated by PGJ₂ + RA. The role of the PPRE2 or PPRE3 site in responsiveness was also examined using mutation analyses (Fig. 10A) . Mutation of each of the PPRE sites almost completely abolished the inducible expression of the pGL-1651 luciferase chimeric gene by PGJ₂ + RA (Fig. 10C) . Either pGL-mPPRE2 or pGL-mPPRE3 also responded to 3-methylcholanthrene. These results demonstrate that PGJ₂ + RA directly activates the PPRE enhancer activities in the GSTA2 gene and that the multiple PPRE-binding sites are essential for the full ligand responsiveness.

View larger version (32K): [in this window] [in a new window]   Fig. 10. The role of putative peroxisome proliferator-activated receptor-response elements (PPREs) in the transcription of glutathione S-transferase A2 promoter-luciferase gene by prostaglandin J2 (PGJ2) + 9-cis-retinoic acid (RA). A, the PPRE mutant constructs of pGL-1651. B, the effects of PPRE mutation on the induction of luciferase activity by PGJ2 + RA. Dual luciferase reporter assays were performed in H4IIE cells transiently transfected with pGL-mPPRE1, in which the PPRE1 was mutated. C, inducible glutathione S-transferase A2-luciferase activities in the cells transfected with the PPRE mutant plasmids. The relative luciferase inducibility by PGJ2 + RA was obtained from the ratio of activity in pGL-mPPRE2- or pGL-mPPRE3-transfected cells to that in pGL-1651-transfected cells. The experimental value for luciferase activity represented the mean ± SD with four separate experiments (significant as compared with pGL-1651-transfected cells exposed to PGJ2 + RA, **, P < 0.01). 3-MC, 3-methylcholanthrene

Effect of PPAR1 – RXR Overexpression on the GSTA2 Transactivation.

View larger version (40K): [in this window] [in a new window]   Fig. 11. The functional role of peroxisome proliferator-activated receptor (PPAR)1 and retinoid X receptor (RXR) overexpression in gene transactivation. A, the effects of PPAR1 and RXR overexpression or ligand activation of PPAR and RXR on the expression of NF-E2-related factor (Nrf)2 and CCAAT/enhancer binding protein (C/EBP)ß. The levels of Nrf2 and C/EBPß were immunochemically measured in the cells transfected with the plasmids encoding PPAR1 and/or RXR. Cells were transfected with an empty vector or the PPAR1 and/or RXR plasmids (500 ng each) in the presence of LipofectAMINE for 3 h, exposed to the medium containing 1% FCS for 12 h, and then treated with prostaglandin J2 (PGJ2) + 9-cis-retinoic acid (RA; 50 nM each) for an additional 12-h time period. pCMX and pCDNA3.1 were included as empty vectors to transfect cells with the same amount of plasmids (a total of 1 µg of DNA). In another set of experiments, H4IIE cells were treated with PPAR-selective agonists (3 µM) with or without RA (50 nM) for 12 h. The levels of Nrf2 and C/EBPß were immunochemically determined in the cell lysates. Each lane contained 15 µg of proteins. Results were confirmed by repeated experiments. B, gel shift analysis of PPAR and RXR binding to the putative peroxisome proliferator-activated receptor-response element (PPRE). Nuclear extracts were prepared from the H4IIE cells transfected with the PPAR1 and/or RXR plasmids with or without PGJ2 + RA (50 nM each). Fifteen µg of nuclear extracts were incubated for 30 min with 5 ng of radiolabeled PPRE oligonucleotide and separated on 4% polyacrylamide gel. Arrowhead, the protein PPRE-binding complex. The nuclear extract was immunodepleted with an anti-PPAR antibody or -RXR antibody (2 µg). Excess of unlabelled PPRE oligonucleotide was added to the reaction mixture for a competition assay. Results were confirmed by repeated experiments. C, activation of the pGL-1651 glutathione S-transferase A2 gene by PPAR1 and RXR overexpression. H4IIE cells were transfected with the pGL-1651 luciferase reporter plasmid (1 µg) and plasmid of Renilla luciferase (5 ng) after transfection with the PPAR1 and/or RXR plasmid (500 ng each) for 3 h and incubated for 12 h in the medium with 1% FCS. The cells were treated with PGJ2 + RA (50 nM each) for 18 h, and the cell extracts were assayed for firefly and Renilla luciferase activities. The fold inductions of the normalized luciferase activity were compared with that in control cells transfected with the pGL-1651 (1 µg) and empty plasmids (500 ng each of pCMX and pCDNA3.1). Data represented the mean ± SE with three separate experiments (significant as compared with the cells transfected with empty plasmids, **, P < 0.01; significant as compared with the cells transfected the PPAR1 and RXR plasmids, ##, P < 0.01).

The extents of protein binding to the PPRE-binding sites in the GSTA2 gene were next determined. PPAR1 and RXR overexpression in the H4IIE cells resulted in an increase in the band intensity of protein binding to PPRE3, which was enhanced by the PGJ2 + RA treatment (Fig. 11B) . The addition of either the anti-PPAR or -RXR antibodies decreased the intensity of the shifted band, indicating that the binding proteins include PPAR and RXR.

To examine the effect of the PPAR and RXR heterodimer on transactivation of the GSTA2 gene, functional reporter gene analysis was performed on the cells transfected with pGL-1651. The functional role of the PPAR and RXR heterodimer in responsiveness was verified by an increase in the luciferase inducibility (Fig. 11C) . The luciferase activity in the cells transfected with the plasmids encoding PPAR1 and RXR was comparable with that of the control cells treated with PGJ₂ + RA. The luciferase reporter gene expression level was further increased after exposing the cells transfected with the PPAR1 and RXR plasmids to PGJ₂ + RA (Fig. 11C) . The results demonstrate that the PPAR and RXR heterodimer, which interacts with the multiple PPRE-binding sites, makes crucial contribution to the inducible expression of the GSTA2 gene.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The nuclear receptors are ligand-dependent transcription factors that regulate the gene networks involved in controlling cell growth, morphogenesis, differentiation, and homeostasis. Activation of the nuclear receptor homodimers and heterodimers in response to the ligands requires binding of the nuclear receptors to the response elements that contain two core recognition sequences (i.e., half-sites). PPARs, including PPAR, PPARß/, and PPAR, constitute a subfamily of the nuclear receptor superfamily activated by a variety of natural and chemical ligands. The PPAR genes are involved in regulating the lipid metabolism (40, 41, 42) . The RXRs, as members of the nuclear receptor superfamily, are the common heterodimeric partner for many receptors, including PPAR. The RXRs are modular proteins with a highly conserved central DNA-binding domain and less conserved ligand-binding domain (43) . RXR serves as a permissive partner of PPARs. Although the effects of the PPAR subfamily on the lipid metabolism have been extensively studied, a proper examination of the role of PPARs in the transcriptional regulation of the Phase II detoxification genes has not been reported.

There are reports showing that PPAR1 is expressed in the adipocytes and hepatic stellate cells with PPAR2 being less frequently expressed (15 , 16 , 44) . In the current study, it was shown that PPAR1 was sufficiently expressed in the H4IIE and primary-cultured hepatocytes. It was revealed that either PPAR or RXR agonist results in the induction of GSTA2 with increases in the mRNA and that treatment of the cells with the PPAR and RXR agonists synergistically induces GSTA2. This was also observed in the cells treated with nanomolar concentrations of PGJ₂ and RA. PGJ₂ at nanomolar concentrations served as a ligand of PPAR. Enhanced GSTA2 induction by PGJ₂ + RA, compared with that by each agent alone, demonstrates that PPAR and RXR heterodimer activation may contribute to the enzyme induction. This was further supported by the observation that the PPAR ligand, troglitazone, rosiglitazone, or pioglitazone, in combination with RA, induced GSTA2 to a much greater extent. A discrepancy in the effective concentration range between PGJ₂ (50 nM) and thiazolidinedione PPAR agonist (1–10 µM) for GSTA2 induction may have resulted from the difference in their equilibrium dissociation rate constants for PPAR. This is reflected by the high-affinity displacement of the binding of a radiolabeled thiazolidinedione to recombinant PPAR by PGJ₂ in a cell lysate system (45) . Therefore, it is likely that PPAR activation contributes to GSTA2 induction. Despite the weak induction of GSTA2 by RA alone, RA potentiated the enzyme induction by PPAR agonist. These results suggested that ligand activation of RXR is needed for the full responsiveness in GSTA2 induction by the PPAR activator.

The expression of the Phase II detoxifying enzyme is transcriptionally activated partly through the activation of Nrf2 via the Nrf2 binding to the ARE in the promoter regions of the target genes (2 , 3) . Nrf2 activation plays an important role in the induction of many Phase II detoxifying enzymes, and the lack of Nrf2/ARE activation increases the sensitivity to xenobiotics, including carcinogens (46, 47, 48) . We reported previously that tert-butylhydroquinone, a representative prooxidant, activated Nrf2 for GSTA2 induction (9 , 49) . In the current study, it was shown that either PGJ₂ or RA alone at relatively high concentrations elicited Nrf2 translocation and its DNA binding in the H4IIE cells, whereas PGJ₂ or RA at nanomolar concentrations required each other for Nrf2 activation. In addition, PGJ₂ + RA induced Nrf2. We found that the promoter region of the Nrf2 gene contains the putative PPRE site (GenBank no. U70474, mouse Nrf2 promoter), which potentially interacts with the activating PPAR – RXR heterodimer. Therefore, both the presence of the binding sites for PPAR – RXR in the promoter region of the Nrf2 gene and the efficacious GSTA2 induction by nanomolar concentrations of PGJ₂ and RA indicate that the PPAR and RXR heterodimer activates the Nrf2 gene induction, which contributes to the GSTA2 induction.

Previous studies showed that cancer chemopreventive agents, including oltipraz and flavonoids, activate C/EBPß and stimulate C/EBPß binding to the C/EBP-response element in the GSTA2 gene (13 , 14) . The essential role of the C/EBP-binding site in GSTA2 induction was further supported by an experiment using a dominant-negative mutant C/EBP (AC/EBP) in the reporter gene assay, which holds significant implication for the finding of C/EBPß as an important transcription factor for GSTA2 induction. The NH₂-terminal transactivation domains of C/EBPß interact with CBP/p300 coactivator after binding to the C/EBP site, which is essential for C/EBPß-mediated gene transactivation (50) . In the present study, it was observed that a combined treatment of the cells with either PGJ₂ and RA induced the nuclear translocation of C/EBPß and activating C/EBPß binding to the C/EBP response element. In addition, PGJ2 + RA increased the C/EBPß expression level. The C/EBPß gene contains PPRE sites (GenBank no. AY056052). It is likely that C/EBPß induction via PPAR and RXR heterodimer binding to the PPREs in the promoter region of the C/EBPß gene is also responsible for inducing GSTA2.

This study provides evidence that both the ARE and C/EBP-binding site have essential roles in the transactivation of the GSTA2 gene by PPAR and RXR ligands, as evidenced by the binding site-deleted promoter–luciferase assay. The complete blockage of the PPAR and RXR-mediated induction of the GSTA2 gene as a result of a deletion mutation of either the ARE or C/EBP-binding site confirms the previous observation that the binding of Nrf2 and C/EBPß to their response elements is simultaneously necessary for the full gene transactivation (Fig. 12A ; Refs. 13 and 14 ). It is inferred that the formation of the transcriptional protein complexes on the two essential binding sites is required for the full responsiveness to chemical inducers.

View larger version (25K): [in this window] [in a new window]   Fig. 12. Schematic diagrams illustrating the induction of glutathione S-transferase (GST)A2 by the peroxisome proliferator-activated receptor (PPAR) – retinoid X receptor (RXR) heterodimer. A, the expression of GSTA2 requires activation of CCAAT/enhancer binding protein (C/EBP)ß and NF-E2-related factor (Nrf)2, which involves nuclear translocation of C/EBPß and Nrf2 binding to their DNA-binding sites. B, the ligand-bound PPAR – RXR heterodimer promotes GSTA2 induction by activation of multiple putative PPAR-response element (PPRE)-binding sites present in the PPRE-responsive enhancer module of the GSTA2 gene, as well as by induction of activating C/EBPß and Nrf2. The PPAR – RXR heterodimer induces C/EBPß and Nrf2.

The observation that the ligand-activated PPAR – RXR heterodimer binds the regulatory PPREs independent of their activation and the induction of Nrf2 and C/EBPß led us to examine the functional role of each of these PPRE-binding sites containing the DRs related to the hexamer AGGTCA in the GSTA2 gene. Specific mutations of these nuclear binding sites in the GSTA2 promoter, which are present as a three PPRE cluster, resulted in the complete loss of its responsiveness to PGJ₂ + RA. We showed that all of the putative PPRE sites comprising DR1 are functionally active. Therefore, the binding of the activating PPAR – RXR heterodimer to all of the PPRE sites is crucial for the inducible gene activation (Fig. 12B) . Blockage of the ligand-dependent transcriptional response as a result of a mutation of the respective binding site suggests that the multiple putative PPREs function as a module in which the nuclear binding sites in close proximity to each other are essential for full ligand responsiveness. Therefore, it is highly likely that the PPAR-binding site cluster is the functionally active PPREM in the GSTA2 gene. Consistent with this, the essential role of the PPAR – RXR heterodimer and PPRE-binding elements in activation, the GSTA2 gene was further supported by the reporter gene induction experiments. It was observed that transcription of the GSTA2 gene was further enhanced by exposing the cells transfected with the plasmids encoding PPAR and RXR to their corresponding ligands. This study on the regulation of GSTA2 gene expression by the PPAR – RXR heterodimer at the promoter containing DR1 elements brings additional insights into the transcriptional control mechanism of the Phase II enzyme involved in carcinogen detoxification.

Nrf2 and C/EBPß play essential roles in gene transacti