Cancer Research Donn Young Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, E. Y.
Right arrow Articles by Kim, S. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, E. Y.
Right arrow Articles by Kim, S. G.
[Cancer Research 64, 3701-3713, May 15, 2004]
© 2004 American Association for Cancer Research

Epidemiology and Prevention

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

Eun Young Park, Il Je Cho and Sang Geon Kim

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


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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-{gamma} (PPAR{gamma}) and the retinoic acid X receptor (RXR) play roles in regulating cell differentiation and chemoprevention. This study examined GSTA2 gene induction by the PPAR{gamma} 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-{delta} (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{gamma} agonists, troglitazone, rosiglitazone, and pioglitazone, in combination with RA, potentiated GSTA2 induction, confirming that the activation of the PPAR{gamma} 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{gamma} – 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{gamma} – RXR heterodimer was verified by the effective GSTA2 induction in the cells treated with PGJ2 + RA after transfecting them with the plasmids encoding PPAR{gamma}1 and RXR{alpha}. In conclusion, the PPAR{gamma} – RXR heterodimer promotes GSTA2 induction by activating PPREM in the GSTA2 gene, as well as inducing Nrf2 and C/EBPß activation.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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{delta} (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{alpha}, PPARß, and PPAR{gamma}, which have distinct distribution patterns. PPAR{alpha} is mainly present in the liver, heart, and kidney, whereas PPARß is expressed ubiquitously (15) . PPAR{gamma} is predominantly expressed in the adipose tissue and, to a lesser extent, in the liver (15) . PPAR{gamma} activators up-regulate the expression of the genes involved in adipocyte differentiation (16) . Although the basal expression of the PPAR{gamma} in the liver is relatively low, hepatic PPAR{gamma} is pronouncedly up-regulated by PPAR{gamma} activators and under certain pathophysiological conditions (e.g., obesity) for the activation of several PPAR{gamma}-responsive genes (17 , 18) . PPAR{gamma} is also an important target for the development of new drugs aimed at preventing and treating cancer. A deficiency in PPAR{gamma} expression can be a significant risk factor for carcinogenesis (19) . Ligands for PPAR{gamma} suppress carcinogenesis in experimental models and induce the differentiation of human tumorigenic cells (20, 21, 22, 23) . The activated PPAR{gamma} forms a heterodimer with retinoid X receptor (RXR){alpha} and binds to specific PPAR response elements in the promoter region of their target genes (24) . Therefore, PPAR{gamma} 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{alpha}, which must heterodimerize with a permissive partner (24 , 31 , 32) . RXR{alpha} 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{gamma} and RXR{alpha} 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 {alpha} 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{gamma} and RXR activators induce GSTA2 via activating PPAR{gamma} – 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-{delta} (12 , 14) -prostaglandin J2 (PGJ2) and thiazolidinedione PPAR{gamma} ligands as the PPAR{gamma} agonists. It was found that the combined treatment of the PPAR{gamma} 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
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
[{alpha}-32P]dCTP (3000 mCi/mmol) and [{gamma}-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{gamma} antibody and anti-RXR{alpha} 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 PPAR{gamma}1 (i.e., pCMX-mPPAR{gamma}1) was kindly supplied from Dr. C. K. Glass (University of California, San Diego, CA). The human RXR{alpha} expression plasmid (PECE-RXR{alpha}) 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-{Delta}C/EBP and pGL-{Delta}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{alpha} expression, the coding region of RXR{alpha} derived from PECE-RXR{alpha} (HindIII and EcoRI digestion) was subcloned into pCDNA3.1 (Invitrogen, Carlsbad, CA; i.e., pCDNA-RXR{alpha}). 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{gamma} 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 [{alpha}-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{alpha} 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{gamma} agonist + RA relative to that in untreated control were determined via scanning densitometry. Similarly, PPAR{gamma}, 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 [{gamma}-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{alpha}, C/EBPß, PPAR{gamma}, and RXR{alpha} (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
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GSTA2 Induction by PGJ2.
The PPAR{gamma}1 expression level was higher than that of PPAR{gamma}2 in most tissue types (15) . To examine the role of PPAR{gamma} in GSTA2 expression, we first assessed the PPAR{gamma} levels in H4IIE hepatocytes and other representative cells. Immunoblot analysis revealed that mainly PPAR{gamma}1 was expressed in H4IIE cells (Fig. 1Citation , left panel). PPAR{gamma}1 was also expressed in the primary cultured hepatocytes or stellate cells isolated from the rat liver (Fig. 1Citation , middle panel). It was reported that the PPAR{gamma} expression level was high in adipocytes ({gamma}1 is expressed to a greater extent than {gamma}2) but not in the preadipocytes,16 which was also confirmed by immunoblot analysis (Fig. 1Citation , right panel).



View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Expression of peroxisome proliferator-activated receptor (PPAR){gamma} 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, PPAR{gamma}1 and PPAR{gamma}2, respectively. Results were confirmed by repeated experiments.

 
We then determined whether or not PGJ2, an activator of PPAR{gamma}, induced GSTA2 in H4IIE cells. PGJ2, at concentrations of 300 or 1000 nM, increased the GSTA2 protein levels (Fig. 2A)Citation . A time course study showed that PGJ2 (300 nM) induced GSTA2 2-fold at 6–24 h. The GSTA2 mRNA levels were also increased by PGJ2 (Fig. 2B)Citation . Apparently, PGJ2 at relatively high concentrations weakly induced GSTA2.



View larger version (37K):
[in this window]
[in a new window]
[Download PPT slide]
  		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).

 
To determine whether PGJ2 increases the binding activity of Nrf2 to the ARE, the nuclear extract isolated from the H4IIE cells treated with 300 nM PGJ2 was probed with the radiolabeled GSTA2 gene ARE. Subcellular fractionation and immunoblot analyses showed that PGJ2 weakly increased the Nrf2 level in the nuclear fraction (Fig. 2CCitation , left panel). Immunoblot assay of the nuclear fractions and gel shift analysis confirmed that PGJ2 weakly enhanced the band intensity of Nrf2 ARE binding at 6 h, whereas that of C/EBPß was minimally affected by PGJ2 (Fig. 2CCitation , right panel). Gel shift analysis of protein binding to the C/EBP-binding site was performed with the nuclear extracts using a radiolabeled C/EBP-binding oligonucleotide. PGJ2 minimally changed C/EBP DNA binding, which varied during the 6–12-h time period (Fig. 2CCitation , right panel).

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)Citation . 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)Citation . The GSTA2 mRNA level peaked at 12 h.



View larger version (44K):
[in this window]
[in a new window]
[Download PPT slide]
  		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. 2Citation 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).

 
This study next examined whether or not RA activated Nrf2 and C/EBPß. RA at 300 nM weakly induced the nuclear translocation of Nrf2 and C/EBPß (Fig. 3C)Citation , as evidenced by the subcellular fractionation and immunoblot analysis. Gel shift analyses confirmed that RA at 300 nM weakly increased Nrf2 and C/EBPß binding to their respective consensus DNA oligonucleotides (Fig. 3C)Citation . Immuno-inhibition experiments using specific antibodies directed against Nrf2, PPAR{gamma}, and RXR{alpha} indicated that an increase in the Nrf2-ARE DNA-binding activity by RA was dependent on Nrf2 (Fig. 3C)Citation . Correspondingly, the shift in the C/EBP DNA-binding complex was the result of C/EBPß.

Synergistic Induction of GSTA2 by PGJ2 + RA.
PPAR{gamma} heterodimerizes with the RXR for activation, and the PPAR{gamma} – 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)Citation . 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)Citation . 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]
[Download PPT slide]
  		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).

 
Synergistic Induction of GSTA2 by PPAR Agonists in Combination with RA.
The studies were extended to verify the effects of the thiazolidinedione PPAR{gamma} agonists in combination with RA on the GSTA2 expression. Immunoblot analysis revealed that troglitazone (1–10 µM) + RA (50 nM) synergistically induced GSTA2, whereas either troglitazone (1–10 µM) or RA alone only weakly increased the GSTA2 protein level (Fig. 5A)Citation . Rosiglitazone at 1–10 µM moderately induced GSTA2, which was also enhanced by a combination treatment with RA. Similarly, pioglitazone + RA markedly increased the GSTA2 expression level when compared with the individual agent alone (Fig. 5A)Citation . These results strongly support the conclusion that the activation of the PPAR{gamma} and RXR heterodimer leads to the induction of GSTA2.



View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Induction of glutathione S-transferase (GST)A2 by peroxisome proliferator-activated receptor-{gamma} 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-{gamma} 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-{gamma} 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).

 
Additional experiments were performed in the primary cultured hepatocytes to confirm that the activation of the PPAR{gamma} and RXR heterodimer induces GSTA2 in normal cells. Because the enhancement effect for GSTA2 expression by the PPAR{gamma} agonist was saturated at 10 µM in H4IIE cells, 1 or 3 µM the PPAR{gamma} agonist were chosen for this experiment. Troglitazone, rosiglitazone, or pioglitazone in combination with RA induced GSTA2 to a greater extent when compared with the PPAR{gamma} agonist alone at the concentrations examined (Fig. 5B)Citation . Hence, the PPAR{gamma} and RXR heterodimer enhance GSTA2 expression in hepatocytes.

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)Citation . 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)Citation . 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)Citation . The band intensity of C/EBP DNA-binding complex was reduced by the anti-C/EBPß antibody but not the anti-PPAR{gamma} or -RXR{alpha} 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]
[Download PPT slide]
  		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).

 
Increases in the Nrf2 and C/EBPß Expression Levels by PGJ2 + RA.
Nrf2 and C/EBPß expression was next examined to determine whether it was affected by PGJ2 + RA. Immunoblot analysis revealed that PGJ2 + RA notably increased the Nrf2 levels in the total cell lysates at 6–12 h (Fig. 7A)Citation . C/EBPß expression also began to increase 3 h after the PGJ2 + RA treatment. C/EBPß was induced 2–3-fold after 6–12 h in the cells exposed to PGJ2 + RA (Fig. 7B)Citation . This indicates that PGJ2 + RA increased the Nrf2 and C/EBPß expression level, suggesting that induction of Nrf2 and C/EBPß might also contribute to Nrf2- and C/EBPß-mediated GSTA2 induction.



View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		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).

 
Analysis of ARE and C/EBP-Response Element in the GSTA2 Promoter.
The functional role of ARE and C/EBP-response element in transactivation of the GSTA2 gene by PGJ2 + RA was next examined. The reporter gene assays were performed using the H4IIE cells transfected with pGL-1651, which contained the luciferase structural gene downstream of the –1.65-kb GSTA2 promoter region (Fig. 8A)Citation .



View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		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, {dagger}{dagger}, P < 0.01).

 
Exposure of the pGL-1651-transfected cells to PGJ2 + RA resulted in a 6-fold increase in the luciferase activity (Fig. 8B)Citation . Either PGJ2 or RA alone (50 nM each) was minimally active. To precisely define the role of the ARE sequence for the induction of GSTA2 by PGJ2 + RA, this study prepared pGL-{Delta}ARE, which is a chimeric gene construct with the ARE deleted (Fig. 8A)Citation . The relative luciferase-inducible activity by PGJ2 + RA was markedly decreased in the H4IIE cells transfected with pGL-{Delta}ARE, compared with that in the cells transfected with pGL-1651 (Fig. 8B)Citation . C/EBPß was next examined to determine whether it plays a role in GSTA2 induction by PGJ2 + RA. The luciferase inducibility was completely abolished in the cells transfected with pGL-{Delta}C/EBP (Fig. 8B)Citation , which is a chimeric gene construct where the C/EBP response element was deleted (Fig. 8A)Citation . To further confirm the functional role of C/EBPß in GSTA2 transcription, the constitutively active dominant-negative mutant of C/EBP (AC/EBP) was expressed in combination with the pGL-1651 luciferase reporter in H4IIE cells. The expression of AC/EBP substantially inhibited the ability of PGJ2 + RA to stimulate reporter gene expression from the pGL-1651 plasmid (Fig. 8BCitation , inset). The ability of PGJ2 + RA to stimulate reporter gene expression from pGL-1651 was not inhibited by transfecting the cells with pCMV500, a control plasmid. These results provide evidence that the ARE and C/EBP-response elements are both necessary for GSTA2 induction by PGJ2 + RA.

Binding of the PPAR{gamma} and RXR Heterodimer to the PPREs.
Studies have shown that PPAR{gamma} heterodimerizes with RXR (probably RXR{alpha}), 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{gamma} – 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)Citation .



View larger version (56K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 9. Gel shift analyses of peroxisome proliferator-activated receptor (PPAR){gamma} and retinoic acid X receptor (RXR){alpha} 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{gamma}- and RXR{alpha}-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{gamma} antibody or -RXR{alpha} 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).

 
As part of the efforts to examine how PGJ2 + RA enhances the GSTA2 gene induction, the activities of protein binding to the PPREs were determined. Gel shift analyses revealed that PGJ2 + RA increased the band intensities of protein binding to either the PPRE1 or PPRE2 oligonucleotide at 3–12 h (Fig. 9, B and C)Citation . Similarly, PGJ2 + RA treatment resulted in increases in protein binding to the PPRE3 at 3–6 h (Fig. 9D)Citation . The protein–PPRE3-binding complex was immunodepleted with either an anti-PPAR{gamma} antibody or anti-RXR{alpha} antibody (Fig. 9E)Citation . Competition experiments using excess amounts of unlabeled SP-1 or the PPRE3 oligonucleotide confirmed the specificity of protein DNA binding (Fig. 9E)Citation . The specificity of PPAR{gamma} – RXR heterodimer binding to the other PPREs was also confirmed by immunodepletion and competition analyses with the unlabeled oligonucleotides (data not shown).

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. 10ACitation ; i.e., pGL-mPPRE1), the inducible luciferase activity by PGJ2 + RA was completely reduced to that of the control (Fig. 10B)Citation . 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)Citation . 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)Citation . 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)Citation . 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]
[Download PPT slide]
  		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 PPAR{gamma}1 – RXR{alpha} Overexpression on the GSTA2 Transactivation.
This study examined the effects of PPAR{gamma}1 and RXR{alpha} overexpression with or without ligands on the Nrf2, C/EBPß, and GSTA2 gene expression. The H4IIE cells were transfected with the plasmids encoding PPAR{gamma}1 and RXR{alpha}, which were then exposed to PGJ2 + RA. Either PPAR{gamma}1 or RXR{alpha} alone elicited a modest increase in the Nrf2 expression level. The concomitant expression of PPAR{gamma}1 and RXR{alpha} to the larger extent enhanced the level of Nrf2 level in the total cell lysates (Fig. 11ACitation , top panel). Furthermore, PGJ2 + RA potentiated Nrf2 induction in the cells transfected with the PPAR{gamma}1 and RXR{alpha} plasmids. Similarly, PPAR{gamma}1 and RXR{alpha} overexpression caused an increase in the C/EBPß level in the total cell lysates. C/EBPß was further induced by PGJ2 + RA in the cells transfected with the plasmids (Fig. 11ACitation , top panel).



View larger version (40K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 11. The functional role of peroxisome proliferator-activated receptor (PPAR){gamma}1 and retinoid X receptor (RXR){alpha} overexpression in gene transactivation. A, the effects of PPAR{gamma}1 and RXR{alpha} overexpression or ligand activation of PPAR{gamma} 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 PPAR{gamma}1 and/or RXR{alpha}. Cells were transfected with an empty vector or the PPAR{gamma}1 and/or RXR{alpha} 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{gamma}-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{gamma} and RXR{alpha} binding to the putative peroxisome proliferator-activated receptor-response element (PPRE). Nuclear extracts were prepared from the H4IIE cells transfected with the PPAR{gamma}1 and/or RXR{alpha} 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{gamma} antibody or -RXR{alpha} 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 PPAR{gamma}1 and RXR{alpha} 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 PPAR{gamma}1 and/or RXR{alpha} 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 PPAR{gamma}1 and RXR{alpha} plasmids, ##, P < 0.01).

 
Additional experiments were performed using PPAR{gamma} selective agonists with or without RA to confirm that the ligand activation of PPAR{gamma} and RXR led to Nrf2 or C/EBPß. RA (50 nM), pioglitazone, or rosiglitazone alone (3 µM) failed to induce Nrf2 in the cell lysates. As expected, an increased Nrf2 expression level was noted in the cells treated with pioglitazone + RA or rosiglitazone + RA (Fig. 11ACitation , bottom panel). However, troglitazone (3 µM) with or without RA induced Nrf2, which suggested that troglitazone might elicit oxidative stress. Pioglitazone, rosiglitazone, or troglitazone in conjunction with RA induced C/EBPß to the greater extent, compared with their respective treatment alone (Fig. 11ACitation , bottom panel).

The extents of protein binding to the PPRE-binding sites in the GSTA2 gene were next determined. PPAR{gamma}1 and RXR{alpha} 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)Citation . The addition of either the anti-PPAR{gamma} or -RXR{alpha} antibodies decreased the intensity of the shifted band, indicating that the binding proteins include PPAR{gamma} and RXR{alpha}.

To examine the effect of the PPAR{gamma} 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{gamma} and RXR heterodimer in responsiveness was verified by an increase in the luciferase inducibility (Fig. 11C)Citation . The luciferase activity in the cells transfected with the plasmids encoding PPAR{gamma}1 and RXR{alpha} 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 PPAR{gamma}1 and RXR{alpha} plasmids to PGJ2 + RA (Fig. 11C)Citation . The results demonstrate that the PPAR{gamma} 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{alpha}, PPARß/{delta}, and PPAR{gamma}, 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 PPAR{gamma}1 is expressed in the adipocytes and hepatic stellate cells with PPAR{gamma}2 being less frequently expressed (15 , 16 , 44) . In the current study, it was shown that PPAR{gamma}1 was sufficiently expressed in the H4IIE and primary-cultured hepatocytes. It was revealed that either PPAR{gamma} or RXR agonist results in the induction of GSTA2 with increases in the mRNA and that treatment of the cells with the PPAR{gamma} 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{gamma}. Enhanced GSTA2 induction by PGJ2 + RA, compared with that by each agent alone, demonstrates that PPAR{gamma} and RXR heterodimer activation may contribute to the enzyme induction. This was further supported by the observation that the PPAR{gamma} 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{gamma} agonist (1–10 µM) for GSTA2 induction may have resulted from the difference in their equilibrium dissociation rate constants for PPAR{gamma}. This is reflected by the high-affinity displacement of the binding of a radiolabeled thiazolidinedione to recombinant PPAR{gamma} by PGJ2 in a cell lysate system (45) . Therefore, it is likely that PPAR{gamma} activation contributes to GSTA2 induction. Despite the weak induction of GSTA2 by RA alone, RA potentiated the enzyme induction by PPAR{gamma} agonist. These results suggested that ligand activation of RXR{alpha} is needed for the full responsiveness in GSTA2 induction by the PPAR{gamma} 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{gamma} – RXR heterodimer. Therefore, both the presence of the binding sites for PPAR{gamma} – 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{gamma} 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{gamma} and RXR{alpha} 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{gamma} and RXR ligands, as evidenced by the binding site-deleted promoter–luciferase assay. The complete blockage of the PPAR{gamma} and RXR{alpha}-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. 12ACitation ; 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]
[Download PPT slide]
  		Fig. 12. Schematic diagrams illustrating the induction of glutathione S-transferase (GST)A2 by the peroxisome proliferator-activated receptor (PPAR){gamma} – 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{gamma} – 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{gamma} – RXR heterodimer induces C/EBPß and Nrf2.

 
The retinoic acid receptor and RXR{alpha} heterodimer activate transcription in response to the retinoic acid receptor-specific ligands on the DR5 elements but constitutively repress transcription on the DR1 elements (51) . The PPAR{gamma} and PPAR{gamma} – RXR{alpha} heterodimers have different DNA-binding specificities with preferential binding of the latter to DR1. The present finding of PPAR{gamma} and RXR heterodimer binding to the DR1 and functional role of the heterodimer in transactivating the GSTA2 gene excludes the possibility that the retinoic acid receptor-RXR heterodimer is involved in the transactivation of the GSTA2 gene. This is in parallel with the suppression of GSTP1 expression by RA in the presence of retinoic acid receptor as a result of the decreased transcription from its promoter (52) . PGJ2 alone (at nanomolar concentrations) is a weak inducer of GSTA2, because the PPAR{gamma} – RXR heterodimer requires the binding of an RXR ligand. The role of the PPAR{gamma} and RXR heterodimer in GSTA2 gene transactivation was confirmed by the comparable induction of GSTA2 by the thiazolidinedione PPAR{gamma} activator and RA. SRC-1 is a coactivator of PPAR{gamma} (53) . Binding of the ligand-activated PPAR{gamma} – RXR{alpha} heterodimer to its DNA-binding sites stimulates the interaction between PPAR{gamma} – RXR{alpha} and p160/SRC-1 (53) .

The observation that the ligand-activated PPAR{gamma} – 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{gamma} – RXR heterodimer to all of the PPRE sites is crucial for the inducible gene activation (Fig. 12B)Citation . 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{gamma} – 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{gamma} and RXR{alpha} to their corresponding ligands. This study on the regulation of GSTA2 gene expression by the PPAR{gamma} – 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 transactivation. Now, we report that the activating PPAR{gamma} and RXR heterodimer functionally regulates PPREM activity via the proximal promoter of the GSTA2 gene and confers full responsiveness to the PPAR{gamma} and RXR ligands in association with the activating Nrf2 and C/EBPß (Fig. 12B)Citation . Both Nrf2 and C/EBPß are up-regulated in a PPAR{gamma}- and RXR-dependent manner. However, the induction of Nrf2 and C/EBPß resulted in little or no activation of the GSTA2 promoter in the cells transfected with a GSTA2 promoter–reporter construct containing the sequentially mutated PPAR response elements. These results suggest that the PPAR response module, which was activated by the ligand-bound PPAR{gamma} and RXR heterodimer, may be essential for the formation of the transactivation complexes comprising Nrf2 and C/EBPß. The essential role of PPAR{gamma} binding to its target DNA element for enhancesome-mediated gene activation is further supported by the findings that activating transcription factors, such as PPAR{gamma}, Nrf2, and C/EBPß, form complexes with coregulators (e.g., CBP/p300; Ref. 53 ) and that PPAR{gamma} binds to Nrf2 (54) . An additional study revealed that PPAR{gamma} and RXR{alpha} agonists induced other forms of GST, including GSTA3 in H4IIE cells (data not shown). A search of the GenBank database showed that the putative PPRE containing DR1 element is located in the promoter region of the rat GSTA3 gene (GenBank no. AF067442). Therefore, the PPAR{gamma} – RXR heterodimer may serve as a common transcriptional factor complex for inducing the GSTs in the liver.

In conclusion: (a) PPAR{gamma} agonist + RA both activate and induce Nrf2 and C/EBPß; (b) activating Nrf2 and C/EBPß binding to the antioxidant- and C/EBP-response elements stimulates the expression of the GSTA2 gene in response to the PPAR{gamma} and RXR agonists; (c) the promoter region of the GSTA2 gene contains the multiple PPRE cluster, which interacts with the PPAR{gamma} – RXR heterodimer; and (d) the PPRE-binding sites present in the GSTA2 promoter might function as a PPREM for transactivation the GSTA2 gene. The fact that PPAR{gamma} – RXR activation plays a key role in GST induction via direct gene transactivation, as well as the coordinate induction of the activating Nrf2 and C/EBPß, provides implication that dietary components, including fatty acids and retinoids, strongly affect GST regulation, whose transcription confers cancer chemoprevention, as well as the detoxification of xenobiotics and toxicants.


   ACKNOWLEDGMENTS
 
We thank Drs. C. Vinson and C. B. Pickett for the kind donations of pCMV500 and AC/EBP plasmids and the constructs containing rGSTA2-promoter region, respectively.


   FOOTNOTES
 
Grant support: National Research Laboratory Program (2001), 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.

Requests for reprints: Sang Geon Kim, College of Pharmacy, Seoul National University, Sillim-dong, Kwanak-gu, Seoul 151-742, South Korea. Phone: 822-880-7840; Fax: 822-872-1795; E-mail: sgk{at}snu.ac.kr

Received 12/16/03. Revised 2/24/04. Accepted 3/ 4/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Salinas AE, Wong MG. Glutathione S-transferases-A review. Curr Med Chem, 6: 279-309, 1999.[Medline]
  2. Nguyen T, Pickett CB. Regulation of rat glutathione S-transferase Ya subunit gene expression. DNA-protein interaction at the antioxidant responsive element. J Biol Chem, 267: 13535-9, 1992.[Abstract/Free Full Text]
  3. Liu S, Pickett CB. The rat liver glutathione S-transferase Ya subunit gene: characterization of the binding properties of a nuclear protein from HepG2 cells that has high affinity for the antioxidant response element. Biochemistry, 35: 11517-21, 1996.[CrossRef][Medline]
  4. Itoh K, Chiba T, Takahashi S, et al An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun, 236: 313-22, 1997.[CrossRef][Medline]
  5. Ishii T, Itoh K, Takahashi S, et al Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem, 275: 16023-9, 2000.[Abstract/Free Full Text]
  6. Wasserman WW, Fahl WE. Functional antioxidant responsive elements. Proc Natl Acad Sci USA, 94: 5361-6, 1997.[Abstract/Free Full Text]
  7. Itoh K, Ishii T, Wakabayashi N, Yamamoto M. Regulatory mechanisms of cellular response to oxidative stress. Free Radic Res, 31: 319-24, 1999.[Medline]
  8. Kang KW, Ryu JH, Kim SG. The essential role of phosphatidylinositol 3-kinase and of p38 mitogen activated protein kinase activation in the antioxidant response element-mediated rGSTA2 induction by decreased glutathione in H4IIE hepatoma cells. Mol Pharmacol, 58: 1017-25, 2000.[Abstract/Free Full Text]
  9. Kang KW, Cho MK, Lee CH, Kim SG. Activation of phosphatidylinositol 3-kinase and Akt by tert-butylhydroquinone (t-BHQ) is responsible for antioxidant response element-mediated rGSTA2 induction in H4IIE cells. Mol Pharmacol, 59: 1147-56, 2001.[Abstract/Free Full Text]
  10. Rastegar M, Lemaigre FP, Rousseau GG. Control of gene expression by growth hormone in liver: key role of a network of transcription factors. Mol Cell Endocrinol, 164: 1-4, 2000.[CrossRef][Medline]
  11. Kountouras J, Boura P, Lygidakis NJ. Liver regeneration after hepatectomy. Hepatogastroenterology, 48: 556-62, 2001.[Medline]
  12. Liao J, Piwien-Pilipuk G, Ross SE, et al CCAAT/enhancer-binding protein ß (C/EBPß) and C/EBP{delta} contribute to growth hormone-regulated transcription of c-fos. J Biol Chem, 274: 31597-604, 1999.[Abstract/Free Full Text]
  13. Kang KW, Park EY, Kim SG. Activation of CCAAT/enhancer-binding protein ß by 2'-amino-3' methoxyflavone (PD98059) leads to the induction of glutathione S-transferase A2. Carcinogenesis, 24: 475-82, 2003.[Abstract/Free Full Text]
  14. Kang KW, Cho IJ, Lee CH, Kim SG. Essential role of phosphatidylinositol 3-kinase-dependent CCAAT/enhancer binding protein ß activation in the induction of glutathione S-transferase by oltipraz. J Natl Cancer Inst (Bethesda), 95: 53-66, 2003.[Abstract/Free Full Text]
  15. Dubuquoy L, Dharancy S, Nutten S, Pettersson S, Auwerx J, Desreumaux P. Role of peroxisome proliferator-activated receptor {gamma} and retinoid X receptor heterodimer in hepatogastroenterological diseases. Lancet, 360: 1410-8, 2002.[CrossRef][Medline]
  16. Chawla A, Schwarz EJ, Dimaculangan DD, Lazar MA. Peroxisome proliferator-activated receptor (PPAR) {gamma}: adipose-predominant expression and induction early in adipocyte differentiation. Endocrinology, 135: 798-800, 1994.[Abstract]
  17. Jiang JG, Johnson C, Zarnegar R. Peroxisome proliferator-activated receptor {gamma}-mediated transcriptional up-regulation of the hepatocyte growth factor gene promoter via a novel composite cis-acting element. J Biol Chem, 276: 25049-56, 2001.[Abstract/Free Full Text]
  18. Han J, Hajjar DP, Zhou X, Gotto AM, Jr, Nicholson AC. Regulation of peroxisome proliferator-activated receptor-{gamma}-mediated gene expression: a new mechanism of action for high density lipoprotein. J Biol Chem, 227: 23582-6, 2002.
  19. Sporn MB, Suh N, Mangelsdorf DJ. Prospects for prevention and treatment of cancer with selective PPAR{gamma} modulators (SPARMs). Trends Mol Med, 7: 395-400, 2001.[CrossRef][Medline]
  20. Mueller E, Sarraf P, Tontonoz P, et al Terminal differentiation of human breast cancer through PPAR{gamma}. Mol Cell, 1: 465-70, 1998.[CrossRef][Medline]
  21. Sarraf P, Mueller E, Jones D, et al Differentiation and reversal of malignant changes in colon cancer through PPAR{gamma}. Nat Med, 4: 1046-52, 1998.[CrossRef][Medline]
  22. Demetri GD, Fletcher CD, Mueller E, et al Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor-{gamma} ligand troglitazone in patients with liposarcoma. Proc Natl Acad Sci USA, 96: 3951-6, 1999.[Abstract/Free Full Text]
  23. Sarraf P, Mueller E, Smith WM, et al Loss-of-function mutations in PPAR{gamma} associated with human colon cancer. Mol Cell, 3: 799-804, 1999.[CrossRef][Medline]
  24. Kliewer SA, Umesono K, Noonan DJ, Heyman RA, Evans RM. Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature, 358: 771-4, 1992.[CrossRef][Medline]
  25. Ahuja HS, Szanto A, Nagy L, Davies PJ. The retinoid X receptor and its ligands: versatile regulators of metabolic function, cell differentiation and cell death. J Biol Regul Homeost Agents, 17: 29-45, 2003.[Medline]
  26. Lotan R. Retinoid and apoptosis: implication for cancer chemoprevention and therapy. J Natl Cancer Inst (Bethesda), 87: 1655-7, 1995.[Free Full Text]
  27. Lotan R. Retinoids in cancer chemoprevention. FASEB J, 10: 1031-9, 1996.[Abstract]
  28. Nagy L, Thomazy VA, Heyman RA, Davis PJ. Retinoid-induced apoptosis in normal and neoplastic tissues. Cell Death Differ, 5: 11-9, 1998.[CrossRef][Medline]
  29. Hansen LA, Sigman CC, Andreola F, Ross SA, Kelloff GJ, De Luca LM. Retinoids in chemoprevention and differentiation therapy. Carcinogenesis, 21: 1271-9, 2000.[Abstract/Free Full Text]
  30. Sun SY, Lotan R. Retinoids and their receptors in cancer development and chemoprevention. Crit Rev Oncol Hematol, 41: 41-55, 2002.[Medline]
  31. Zhang XK, Hoffmann B, Tran PB, Graupner G, Pfahl M. Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors. Nature, 355: 441-6, 1992.[CrossRef][Medline]
  32. Mukherjee R, Jow L, Croston GE, Paterniti JR, Jr. Identification, characterization, and tissue distribution of human peroxisome proliferator-activated receptor (PPAR) isoforms PPAR{gamma}2 versus PPAR{gamma}1 and activation with retinoid X receptor agonists and antagonists. J Biol Chem, 272: 8071-6, 1997.[Abstract/Free Full Text]
  33. Montserrat M, Cederbaum AI. Induction of catalase, {alpha}, and microsomal glutathione S-transferase in CYP2E1 overexpressing HepG2 cells and protection against short-term oxidative stress. Hepatology, 33: 652-61, 2001.[CrossRef][Medline]
  34. Ahn S, Olive M, Aggarwal S, Krylov D, Ginty DD, Vinson C. A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus-dependent transcription of c-fos. Mol Cell Biol, 18: 967-77, 1998.[Abstract/Free Full Text]
  35. Bernlohr DA, Angus CW, Lane MD, Bolanowski MA, Kelly TJ, Jr. Expression of specific mRNAs during adipose differentiation: identification of an mRNA encoding a homologue of myelin P2 protein. Proc Natl Acad Sci USA, 81: 5468-72, 1984.[Abstract/Free Full Text]
  36. Kang KW, Kim YG, Cho MK, et al Oltipraz regenerates cirrhotic liver through CCAAT/enhancer binding protein-mediated stellate cell inactivation. FASEB J, 16: 1988-90, 2002.[Abstract/Free Full Text]
  37. Kim SG, Nam SY, Kim JH, Cho CK, Yoo SY. Enhancement of radiation-inducible hepatic glutathione S-transferase Ya1, Yb1, Yb2, Yc1, and Yc2 expression by oltipraz: possible role in radioprotection. Mol Pharmacol, 51: 225-33, 1997.[Abstract/Free Full Text]
  38. Kim HI, Kim JW, Kim SH, Cha JY, Kim KS, Ahn YH. Identification and functional characterization of the peroxisomal proliferator response element in rat GLUT2 promoter. Diabetes, 49: 1517-24, 2000.[Abstract]
  39. Boulanger A, McLemore P, Copeland NG, et al Identification of ß-carotene 15, 15'-monooxygenase as a peroxisome proliferator-activated receptor target gene. FASEB J, 17: 1304-6, 2003.[Abstract/Free Full Text]
  40. Spiegelman BM, Flier JS. Adipogenesis and obesity: rounding out the big picture. Cell, 87: 377-89, 1996.[CrossRef][Medline]
  41. Desvergne B, Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev, 20: 649-88, 1999.[Abstract/Free Full Text]
  42. Ferre P. The biology of peroxisome proliferator-activated receptors: relationship with lipid metabolism and insulin sensitivity. Diabetes, 53: S43-50, 2004.[Abstract/Free Full Text]
  43. Holmbeck SM, Foster MP, Casimiro DR, Sem DS, Dyson HJ, Wright PE. High-resolution solution structure of the retinoid X receptor DNA-binding domain. J Mol Biol, 281: 271-84, 1998.[CrossRef][Medline]
  44. Galli A, Crabb D, Price D, et al Peroxisome proliferator-activated receptor gamma transcriptional regulation is involved in platelet-derived growth factor-induced proliferation of human hepatic stellate cells. Hepatology, 31: 101-8, 2000.[CrossRef][Medline]
  45. Young PW, Buckle DR, Cantello BC, et al Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor {gamma}. J Pharmacol Exp Ther, 284: 751-9, 1998.[Abstract/Free Full Text]
  46. Enomoto A, Itoh K, Nagayoshi E, et al High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes. Toxicol Sci, 59: 169-77, 2001.[Abstract/Free Full Text]
  47. Ramos-Gomez M, Kwak MK, Dolan PM, et al Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in Nrf2 transcription factor-deficient mice. Proc Natl Acad Sci USA, 98: 3410-5, 2001.[Abstract/Free Full Text]
  48. Chanas SA, Jiang Q, McMahon M, et al Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice. Biochem J, 365: 405-16, 2002.[CrossRef][Medline]
  49. Kang KW, Lee SJ, Park JW, Kim SG. Phosphatidylinositol 3-kinase regulates nuclear translocation of NF-E2-related factor 2 through actin rearrangement in response to oxidative stress. Mol Pharmacol, 62: 1001-10, 2002.[Abstract/Free Full Text]
  50. Mink S, Haenig B, Klempnauer KH. Interaction and functional collaboration of p300 and C/EBPß. Mol Cell Biol, 17: 6609-17, 1997.[Abstract]
  51. Xiao JH, Durand B, Chambon P, Voorhees JJ. Endogenous retinoic acid receptor (RAR)-retinoid X receptor (RXR) heterodimers are the major functional forms regulating retinoid-responsive elements in adult human keratinocytes. Binding of ligands to RAR only is sufficient for RAR-RXR heterodimers to confer ligand-dependent activation of hRAR ß 2/RARE (DR5). J Biol Chem, 270: 3001-11, 1995.[Abstract/Free Full Text]
  52. Xia C, Hu J, Ketterer B, Taylor JB. The organization of the human GSTP1–1 gene promoter and its response to retinoic acid and cellular redox status. Biochem J, 313: 155-61, 1996.
  53. Kodera Y, Takeyama K, Murayama A, Suzawa M, Masuhiro Y, Kato S. Ligand type-specific interactions of peroxisome proliferator-activated receptor {gamma} with transcriptional coactivators. J Biol Chem, 275: 33201-4, 2000.[Abstract/Free Full Text]
  54. Ikeda Y, Sugawara A, Taniyama Y, et al Suppression of rat thromboxane synthase gene transcription by peroxisome proliferator-activated receptor {gamma} in macrophages via an interaction with Nrf2. J Biol Chem, 275: 33142-50, 2000.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
H. Gong, J. He, J. H. Lee, E. Mallick, X. Gao, S. Li, G. E. Homanics, and W. Xie
Activation of the Liver X Receptor Prevents Lipopolysaccharide-induced Lung Injury
J. Biol. Chem., October 30, 2009; 284(44): 30113 - 30121.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
X. Zhao, R. Strong, J. Zhang, G. Sun, J. Z. Tsien, Z. Cui, J. C. Grotta, and J. Aronowski
Neuronal PPAR{gamma} Deficiency Increases Susceptibility to Brain Damage after Cerebral Ischemia
J. Neurosci., May 13, 2009; 29(19): 6186 - 6195.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
Q. T. Tran, L. Xu, V. Phan, S. B. Goodwin, M. Rahman, V. X. Jin, C. H. Sutter, B. D. Roebuck, T. W. Kensler, E.O. George, et al.
Chemical genomics of cancer chemopreventive dithiolethiones
Carcinogenesis, March 1, 2009; 30(3): 480 - 486.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
F. Ondrey
Peroxisome Proliferator-Activated Receptor {gamma} Pathway Targeting in Carcinogenesis: Implications for Chemoprevention
Clin. Cancer Res., January 1, 2009; 15(1): 2 - 8.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
M. Katzenellenbogen, L. Mizrahi, O. Pappo, N. Klopstock, D. Olam, J. Jacob-Hirsch, N. Amariglio, G. Rechavi, E. Domany, E. Galun, et al.
Molecular Mechanisms of Liver Carcinogenesis in the Mdr2-Knockout Mice
Mol. Cancer Res., November 1, 2007; 5(11): 1159 - 1170.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
G. Kronke, A. Kadl, E. Ikonomu, S. Bluml, A. Furnkranz, I. J. Sarembock, V. N. Bochkov, M. Exner, B. R. Binder, and N. Leitinger
Expression of Heme Oxygenase-1 in Human Vascular Cells Is Regulated by Peroxisome Proliferator-Activated Receptors
Arterioscler Thromb Vasc Biol, June 1, 2007; 27(6): 1276 - 1282.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
M.-S. Kim, S. M. Lee, W. D. Kim, S. H. Ki, A. Moon, C. H. Lee, and S. G. Kim
G{alpha}12/13 Basally Regulates p53 through Mdm4 Expression
Mol. Cancer Res., May 1, 2007; 5(5): 473 - 484.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
S. G. Kim and S. J. Lee
PI3K, RSK, and mTOR Signal Networks for the GST Gene Regulation
Toxicol. Sci., April 1, 2007; 96(2): 206 - 213.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
M. S. Ko, S. J. Lee, J. W. Kim, J. W. Lim, and S. G. Kim
DIFFERENTIAL EFFECTS OF THE OXIDIZED METABOLITES OF OLTIPRAZ ON THE ACTIVATION OF CCAAT/ENHANCER BINDING PROTEIN-{beta} AND NF-E2-RELATED FACTOR-2 FOR GSTA2 GENE INDUCTION
Drug Metab. Dispos., August 1, 2006; 34(8): 1353 - 1360.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S. J. Lee, E. K. Yang, and S. G. Kim
Peroxisome Proliferator-Activated Receptor-{gamma} and Retinoic Acid X Receptor {alpha} Represses the TGFbeta1 Gene via PTEN-Mediated p70 Ribosomal S6 Kinase-1 Inhibition: Role for Zf9 Dephosphorylation
Mol. Pharmacol., July 1, 2006; 70(1): 415 - 425.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Katzenellenbogen, O. Pappo, H. Barash, N. Klopstock, L. Mizrahi, D. Olam, J. Jacob-Hirsch, N. Amariglio, G. Rechavi, L. A. Mitchell, et al.
Multiple adaptive mechanisms to chronic liver disease revealed at early stages of liver carcinogenesis in the mdr2-knockout mice.
Cancer Res., April 15, 2006; 66(8): 4001 - 4010.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
H. Gong, S. V. Singh, S. P. Singh, Y. Mu, J. H. Lee, S. P. S. Saini, D. Toma, S. Ren, V. E. Kagan, B. W. Day, et al.
Orphan Nuclear Receptor Pregnane X Receptor Sensitizes Oxidative Stress Responses in Transgenic Mice and Cancerous Cells
Mol. Endocrinol., February 1, 2006; 20(2): 279 - 290.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S. J. Lee and S. G. Kim
Role of p90 Ribosomal S6-Kinase-1 in Oltipraz-Induced Specific Phosphorylation of CCAAT/Enhancer Binding Protein-beta for GSTA2 Gene Transactivation
Mol. Pharmacol., January 1, 2006; 69(1): 385 - 396.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
G. Dai, N. Chou, L. He, M. A. Gyamfi, A. J. Mendy, A. L. Slitt, C. D. Klaassen, and Y.-J. Y. Wan
Retinoid X Receptor {alpha} Regulates the Expression of Glutathione S-transferase Genes and Modulates Acetaminophen-Glutathione Conjugation in Mouse Liver
Mol. Pharmacol., December 1, 2005; 68(6): 1590 - 1596.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
E. J. Bae and S. G. Kim
Enhanced CCAAT/Enhancer-Binding Protein {beta}-Liver-Enriched Inhibitory Protein Production by Oltipraz, Which Accompanies CUG Repeat-Binding Protein-1 (CUGBP1) RNA-Binding Protein Activation, Leads to Inhibition of Preadipocyte Differentiation
Mol. Pharmacol., September 1, 2005; 68(3): 660 - 669.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S. H. Ki, I. J. Cho, D. W. Choi, and S. G. Kim
Glucocorticoid Receptor (GR)-Associated SMRT Binding to C/EBP{beta} TAD and Nrf2 Neh4/5: Role of SMRT Recruited to GR in GSTA2 Gene Repression
Mol. Cell. Biol., May 15, 2005; 25(10): 4150 - 4165.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
I. Avis, A. Martinez, J. Tauler, E. Zudaire, A. Mayburd, R. Abu-Ghazaleh, F. Ondrey, and J. L. Mulshine
Inhibitors of the Arachidonic Acid Pathway and Peroxisome Proliferator-Activated Receptor Ligands Have Superadditive Effects on Lung Cancer Growth Inhibition
Cancer Res., May 15, 2005; 65(10): 4181 - 4190.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
I-N. Park, I. J. Cho, and S. G. Kim
CERAMIDE, AN APOPTOTIC RHEOSTAT, INHIBITS CCAAT/ENHANCER BINDING PROTEIN-{beta} AND NF-E2-RELATED FACTOR-2 ACTIVATION: THE ROLE IN GLUTATHIONE S-TRANSFERASE A2 GENE REPRESSION
Drug Metab. Dispos., September 1, 2004; 32(9): 893 - 897.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, E. Y.
Right arrow Articles by Kim, S. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, E. Y.
Right arrow Articles by Kim, S. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online