ARTICLE

Essential Role of Phosphatidylinositol 3-Kinase-Dependent CCAAT/Enhancer Binding Protein {beta} Activation in the Induction of Glutathione S-Transferase by Oltipraz

Keon Wook Kang, Il Je Cho, Chang Ho Lee, Sang Geon Kim

Affiliations of authors: K. W. Kang, I. J. Cho, S. G. Kim, National Research Laboratory, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea; C. H. Lee, Department of Pharmacology and Institute of Biomedical Science, College of Medicine, Hanyang University, Seoul.

Correspondence to: Sang Geon Kim, Ph.D., College of Pharmacy, Seoul National University, Sillim-dong, Kwanak-gu, Seoul 151–742, South Korea (e-mail: sgk{at}snu.ac.kr).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Cancer chemopreventive agents transcriptionally induce genes whose protein products can protect cells from chemical-induced carcinogenesis. Oltipraz, a dithiolthione, exerts chemopreventive responses through glutathione S-transferase (GST) induction. We investigated the role of the CCAAT/enhancer binding protein (C/EBP) in the induction of the GSTA2 gene (alpha class) by oltipraz and identified the enhancer element(s) responsible for GSTA2 gene expression. Methods: H4IIE rat hepatocyte-derived cells were treated with oltipraz, and GSTA2 expression was determined by northern and immunoblot analyses. The activation of C/EBP{beta} and {alpha} forms and NF-E2-related factor 2 (Nrf2) was assessed by immunochemical assays. C/EBP{beta}-DNA binding activity was determined by subcellular fractionation and electrophoretic mobility shift assays. The role of the C/EBP binding site in the induction of the GSTA2 gene was assessed by luciferase reporter-gene activity. The role of phosphatidylinositol 3-kinase (PI3-kinase) and mitogen-activated protein (MAP) kinase signaling pathways in C/EBP-mediated GSTA2 induction was studied by using chemical inhibitors, overexpression vectors, and dominant-negative mutants. All statistical tests were two-sided. Results: Oltipraz induced GSTA2 mRNA and protein expression. In oltipraz-treated cells, C/EBP{beta} translocated to the nucleus and bound to the consensus sequence of C/EBP (TTGCGCAA). Oltipraz treatment increased luciferase reporter-gene activity in H4IIE cells transfected with the C/EBP-containing regulatory region of the GSTA2 gene. Deletion of the C/EBP binding site or overexpression of a dominant-negative mutant form of C/EBP (AC/EBP) abolished the reporter gene activity. PI3-kinase, but not MAP kinases, was required for C/EBP{beta}-dependent induction of GSTA2 by oltipraz. Conclusions: Oltipraz-induced GSTA2 gene expression is dependent upon PI3-kinase-mediated nuclear translocation and binding of C/EBP{beta} to the C/EBP response element in the GSTA2 gene promoter.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cancer chemoprevention can be defined as the use of naturally occurring or synthetic agents to prevent, inhibit, or reverse the process of carcinogenesis (1). A large body of experimental data has shown that this approach is feasible. Chemopreventive agents transcriptionally induce a battery of genes whose protein products can protect cells from chemical-induced carcinogenesis (2). The dithiolthione oltipraz (5-[2-pyrazinyl]-4-methyl-1,2-dithiol-3-thione) is being developed as a chemopreventive agent for malignancies, such as liver and colorectal cancer (2,3). A phase IIa randomized chemoprevention trial of oltipraz in residents of Qidong, China, showed that oltipraz might be active as a chemopreventive agent (4,5). In experimental cancer prevention studies, oltipraz was shown to substantially reduce tumor incidence and multiplicity (e.g., rodent colon and liver cancer) (3,6,7). Comprehensive mechanistic studies suggest that oltipraz exerts cancer chemopreventive effects through the induction of glutathione S-transferase (GST), a phase II detoxifying enzyme (2,7). GST induction also accounts for the cytoprotective effect of oltipraz against toxicant-induced injury and {gamma}-ray irradiation (6,8).

A cascade of molecular events initiates activation of transcription factors and stimulates induction of antioxidant genes in cells treated with xenobiotics. Activation of antioxidant response elements (AREs) by reactive oxygens (produced by quinoid chemicals via redox cycling) plays an important role in the regulation of phase II enzymes. AREs coordinately regulate the expression of antioxidant genes, including GST and quinone reductase (911). Proteins that bind to AREs include nuclear factor (NF)-E2-related factor 2 (Nrf2) proteins and musculoaponeurotic fibrosarcoma (Maf) family members (10,12,13). Molecular signals activated by oxidative stress stimulate translocation of Nrf2 to the nucleus, where it binds and activates AREs (1012). We have previously reported that tert-butylhydroquinone (t-BHQ), a representative pro-oxidant, induces GSTA2 (alpha form) through nuclear translocation of Nrf2, which binds to the ARE in the GSTA2 promoter in H4IIE rat hepatocyte-derived cells (14).

Oltipraz, in conjunction with thiols, such as glutathione and dithiothreitol, mediates the conversion of molecular oxygen to reactive oxygen radicals in vitro, which raises the possibility that oltipraz induces GST expression through the production of reactive oxygen species (15). Exposure of rodents to oltipraz triggers nuclear accumulation of Nrf2 and enhances Nrf2 binding activity to the ARE (16). On the basis of these findings, it was suggested (15) that oxygen species produced from oltipraz induce GSTA2 gene transcription via Nrf2 binding to the ARE in the GSTA2 promoter. Nevertheless, cellular activation of Nrf2 DNA binding by oltipraz seems to be not as strong as that of other pro-oxidants (e.g., t-BHQ) (14,16). The induction of GST by oltipraz was strong and persistent in both cells and animals (8,17). By contrast, the induction of GST by t-BHQ was transient and attenuated at later time periods (14). The potent induction of GSTA2 in response to oltipraz may result from the activation of other transcription factors besides Nrf2.

Previous studies in our laboratory have shown that oxidative stress, evoked by decreased glutathione or t-BHQ, activates both phosphatidylinositol 3-kinase (PI3-kinase) and Akt and that PI3-kinase plays an essential role in Nrf2/ARE-mediated GSTA2 induction (13,14). PI3-kinase has also been implicated in the activation of molecular signals involved in cell survival and proliferation in response to growth factors (18). The CCAAT/enhancer binding protein (C/EBP) family plays an important role in regulating the expression of hepatocyte-specific genes, particularly those associated with cell survival and cell proliferation (19). C/EBP proteins, of which there are at least four isoforms, form homodimers and heterodimers and bind to a consensus C/EBP binding DNA sequence. The activation of the C/EBP alpha isoform (C/EBP{alpha}) and the aromatic hydrocarbon receptor (AhR) by polycyclic aromatic hydrocarbons (PAHs) leads to the induction of GSTA2 and quinone reductase via C/EBP{alpha} binding to the C/EBP binding site within or close to the xenobiotic response element (XRE), which is present in the promoter regions of these genes (20,21). Among the C/EBP isoforms, C/EBP beta (C/EBP{beta}) plays an important role in hepatocyte-specific gene expression (19). The ratio of C/EBP{alpha} and C/EBP{beta} is responsible for modulation of the transcriptional activities of the genes for cell proliferation and differentiation. For example, C/EBP{alpha}, whose expression varies reciprocally with that of other C/EBPs, inhibits the proliferative activity of hepatocytes (22).

A number of cellular stresses activate mitogen-activated protein (MAP) kinases and concomitantly induce transactivation of the stress-activated target genes (2325). For example, p38 MAP kinase induces phase II enzymes in H4IIE and HepG2 cells (13,26), and it has been claimed that extracellular signal-regulated kinase (ERK; an MAP kinase) mediates the induction of quinone reductase by sulforaphane and t-BHQ (27). However, we found that the induction of GSTA2 by t-BHQ in H4IIE cells was not regulated by MAP kinases (14).

These findings raise a number of questions: 1) Does oltipraz regulate the activation of C/EBP and promote C/EBP-mediated expression of the GSTA2 gene? 2) If so, what is the role of PI3-kinase in the C/EBP-mediated induction of GSTA2 by oltipraz? and 3) What is the role of the MAP kinase signaling pathway in the induction of GSTA2 by oltipraz? Consequently, we investigated the role of the C/EBP signaling pathway in the induction of GSTA2 by oltipraz and aimed to identify the enhancer elements responsible for the induction of the GSTA2 gene.


    MATERIALS AND METHODS
 Top
 Notes
 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). 5-Bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium was obtained from Life Technologies (Gaithersburg, MD). The random prime-labeling kit was purchased from Promega (Madison, WI). PD98059 and LY294002 were obtained from Calbiochem (San Diego, CA). Wortmannin, SB203580, dithiothreitol (DTT), and 3-[N-morpholino]propanesulfonic acid (MOPS) were purchased from Sigma-Aldrich (St. Louis, MO). The plasmid pGTB-1.65 construct containing the GSTA2-promoter region (–1651 to +66) was provided by Dr. C. B. Pickett (Schering-Plough Research Institute, Kenilworth, NJ), and the C/EBP dominant-negative expression (AC/EBP) plasmid was a gift from Dr. C. Vinson (National Institutes of Health, Bethesda, MD) (28). AC/EBP prevents the "normal" C/EBP from binding to DNA because C/EBP acts as a heterodimer. PI3-kinase p110 and p85 overexpression vectors were provided by Dr. J. Downward (Imperial Cancer Research Fund, London, U.K.) and Dr. A. Toker (The Boston Biomedical Research Institute, Boston, MA), respectively. The MAP kinase kinase 1 (MKK1) dominant-negative mutant was a gift from Dr. N. G. Ahn (Howard Hughes Medical Institute, University of Colorado, Boulder, CO). The c-Jun N-terminal kinase 1 (JNK1) dominant-negative mutant (KmJNK1) was provided by Dr. N. Dhanasekaran (Fels Institute for Cancer Research and Molecular Biology, Department of Biochemistry, Temple University, Philadelphia, PA).

Cell Culture

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

Oltipraz Treatment

H4IIE cells were plated at a density of 1 x 106 per 10-cm2 dish, grown to 80%–90% confluency in medium containing 10% FCS and then incubated in serum-free medium for 24 hours. Oltipraz (Aventis Pharma France, Vitry-sur-Seine, France) (30 µM), dissolved in dimethyl sulfoxide (DMSO), was added to the H4IIE cells and incubated for the indicated time period for each experiment at 37 °C. Cells were then washed twice with ice-cold phosphate-buffered saline (PBS) before sample preparation.

Preparation of a Complementary DNA Probe for GSTA2

A complementary DNA (cDNA) probe for the GSTA2 gene was amplified by reverse transcription–polymerase chain reaction (RT–PCR) using selective primers (8) and was cloned into the pGEM+T vector (Promega).

Preparation of Nuclear Extracts

Nuclear extracts were prepared according to a previously published method (29). Briefly, H4IIE cells (1 x 107) in dishes were washed twice with ice-cold PBS and then scraped from the dishes with 1 mL of PBS and transferred to microtubes. Cells were then centrifuged at 2000g for 5 minutes. The supernatant was discarded, and the cell pellet was allowed to swell after the addition of 100 µL of hypotonic buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet P-40, 1 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The lysates were incubated for 10 minutes on ice and then centrifuged at 7200g for 5 minutes 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 PMSF and then incubated for 30 minutes on ice. The samples were then centrifuged at 15 800g for 10 minutes to obtain supernatants containing nuclear fractions. Nuclear fractions were stored at –70 °C until use.

Preparation of Cytosolic Fractions

H4IIE cells (1 x 107) were washed twice with PBS, scraped from their dishes (in 1 mL of PBS), and sonicated to disrupt the membranes. Cytosolic fractions were prepared by differential centrifugation at 15 000g for 15 minutes and stored at –70 °C until use. Protein content was determined by the Bradford assay (Bio-Rad Protein Assay Kit®, Bio-Rad Laboratories, Hercules, CA).

Northern Blot Analysis

Total RNA was isolated from H4IIE cells by using the single-step method of thiocyanate–phenol–chloroform RNA extraction, and northern blot analysis was carried out according to previously described procedures (8). Briefly, total RNA (30 µg) was resolved by electrophoresis through a 1% agarose gel containing 2.2 M formaldehyde and transferred to a nitrocellulose membrane. The nitrocellulose membrane was baked in a vacuum oven at 80 °C for 2 hours. The filter was then incubated with hybridization buffer containing 50% deionized formamide, 5x Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidine, and 0.1% bovine serum albumin [Pentex Fraction V; Sigma-Aldrich]), 0.1% sodium dodecyl sulfate (SDS), 200 µg/mL of sonicated salmon sperm DNA and 5x SSPE (1x SSPE = 0.15 M NaCl, 10 mM NaH2PO4, and 1 mM Na2EDTA, pH 7.4) at 42 °C for 1 hour without probe. Hybridization was performed at 42 °C for 18 hours with a heat-denatured cDNA probe for rGSTA2 that was random prime-labeled with [{alpha}-32P]dCTP. Filters were washed twice in 2x saline sodium citrate (SSC) (1x SSC = 150 mM NaCl and 15 mM sodium citrate) and 0.1% SDS for 10 minutes at room temperature and twice in 0.1x SSC and 0.1% SDS for 10 minutes at room temperature. Filters were then washed once in a solution containing 0.1x SSC and 0.1% SDS for 1 hour at 60 °C. Filters were then exposed to autoradiographic film at –70 °C. After quantitation of GSTA2 mRNA levels via scanning densitometry, the membranes were stripped and rehybridized with a 32P-labeled cDNA probe for 18S ribosomal RNA (rRNA) to control for RNA loading onto the membranes. Four separate experiments were performed with different RNA samples.

Immunoblot Analysis

SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis were performed according to a previously published procedure (8). Briefly, the cytosolic (1 or 10 µg) and nuclear (20 µg) fractions were separated by 7.5% and 12% gel electrophoresis, respectively, and were transferred to nitrocellulose membranes by electroblotting. The nitrocellulose membrane was incubated with the anti-rat GSTA1 and GSTA2 (GSTA1/2) antibody (Biotrin International, Dublin, Ireland) or anti-GST{alpha} antibody (1 : 1000) (Detroit R&D, Detroit, MI), followed by incubation with alkaline phosphatase or horseradish peroxidase-conjugated secondary antibody (Zymed Laboratories, San Francisco, CA). Specificity of the antibodies to the GST subunit has been confirmed previously (8,13). Immunoreactive proteins were detected after incubation with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium or by an enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences UK Ltd., Buckinghamshire, U.K.) (8,13). Equal loading of proteins was verified by actin immunoblotting with goat anti-actin antibody (1 : 2000) (Santa Cruz Biotechnology, Santa Cruz, CA). Four separate experiments were performed with different cytosolic samples. Changes in the levels of GSTA2 protein in oltipraz-treated cells relative to those in untreated cells were determined via scanning densitometry. The replicate SDS–PAGE gels were stained with Coomassie blue for verification of equal loading of proteins prior to immunoblotting.

Active phosphorylated forms of ERK, JNK, and p38 kinase were measured in cell lysates by immunoblotting. Cells (1 x 107) were lysed in buffer containing 20 mM Tris–HCl (pH 7.5), 1% Triton X-100, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM {beta}-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM PMSF, and 1 µg/mL leupeptin. Cell lysates were boiled for 5 minutes and then centrifuged at 15 000g for 15 minutes at 4 °C to remove debris. Phosphorylated ERK, JNK, and p38 kinase were immunochemically assessed by using specific antibodies to the phosphorylated forms (New England Biolabs, Beverly, MA), according to the manufacturer's recommended protocol. Similarly, nuclear Nrf2, C/EBP{alpha}, and C/EBP{beta} were immunochemically detected with their respective antibodies (Santa Cruz Biotechnology). Three separate experiments were performed with different lysates to assess changes in the protein levels.

Scanning Densitometry

Scanning densitometry of the northern blots and immunoblots was performed with an Image Scan & Analysis System (Alpha-Innotech, San Leandro, CA). The area of each lane was integrated using the software AlphaEaseTM version 5.5 (Alpha-Innotech) 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' that contained the C/EBP consensus sequence. The reaction mixture contained 4 µL of 5x binding buffer (containing 20% glycerol, 5 mM MgCl2, 250 mM NaCl, 2.5 mM EDTA, 2.5 mM DTT, 0.25 mg/mL poly dI-dC and 50 mM Tris–HCl [pH 7.5]), 10 µg of nuclear extract, and sterile water up to a total volume of 20 µL. The reaction mixture was pre-incubated without probe at room temperature for 10 minutes. The probe (1 µL, containing 106 cpm) was then added, and DNA-binding reactions were carried out for 30 minutes at room temperature. In some analyses, specificity of binding was determined by competition experiments, which were carried out by adding a 20-fold molar excess of an unlabeled ARE or C/EBP to the reaction mixture before the labeled probe was added. Specific protein-1 (SP-1) oligonucleotide (5'-ATTCGATCGGGGCGGGGCGAGC-3') was used as a negative control for competition experiments. In other analyses, known as immuno-inhibition assays, antibodies to C/EBP{alpha}, C/EBP{beta}, p300/CBP (N-terminal domains of C/EBP{beta} interact with p300/CBP) or AhR (2 µg each) were added to the reaction mixture 20 minutes after the labeled probe was added, and the reaction was then continued for another hour 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 subjected to autoradiography.

Immunocytochemistry of C/EBP{beta}

H4IIE cells were grown on Lab-TEK chamber slides (Nalge Nunc International, Rochester, NY) in a medium containing 10% FCS and further incubated in serum-free Dulbecco's modified Eagle medium for 6 hours at 37 °C. Standard immunocytochemical methods were used for immunostaining of C/EBP, as previously described (30). Briefly, cells were fixed in 100% methanol for 30 minutes, washed three times with PBS, and blocked in PBS containing 5% bovine serum albumin (BSA) for 1 hour at 37 °C. The cells were then incubated for 1 hour with polyclonal rabbit anti-C/EBP{beta} antibody (1 : 100) in PBS containing 0.5% BSA at 37 °C, washed several times with PBS, and incubated for 30 minutes with fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (1 : 100) (Zymed Laboratories) at 37 °C. Counterstaining with propidium iodide (2 µg/mL) was used to verify the location and integrity of nuclei. Stained cells were washed five times with PBS and examined by using a laser-scanning confocal microscope (Leica TCS NT; Leica Microsystems, Wetzlar, Germany).

Construction of GSTA2 Promoter-Luciferase Constructs and Luciferase Assay

The pGL-1651 reporter gene construct was generated by ligating the region 1.65 kb upstream of the transcription start site of the GSTA2 gene to the firefly luciferase reporter gene coding sequences. A series of chimeric gene constructs pGL-1128, pGL-797, and pGL-197 with promoter deletions were also created. pGL-1128 contains the XRE, which comprises the C/EBP binding sequence and the ARE. The pGL-797 gene construct, in which the XRE was deleted from the promoter, contains only the ARE and was used as a vector containing the minimal promoter region. The pCMV-AC/EBP construct, which contains a coding sequence for the expression of the dominant-negative mutant of C/EBP, and pCMV500 (an empty vector, which was used as a control) were obtained from Dr. C. Vinson (28).

To determine the promoter activity of the segments of the GSTA2 promoter in the pGL-1128, pGL-797, and pGL-197 constructs, 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 hours, and transiently transfected with each GSTA2 promoter-luciferase construct (1 µg) and pRL-SV plasmid (5 ng) (a plasmid that encodes for Renilla luciferase and is used to normalize transfection efficacy) in the presence of Lipofectamine Plus® Reagent (Life Technologies) for 3 hours. Transfected cells were incubated in Dulbecco's modified Eagle medium containing 1% FCS for 3 hours and exposed to 30 µM oltipraz in medium for 12 hours at 37 °C. The activity of firefly luciferase was measured by adding Luciferase Assay Reagent II (Promega) according to the manufacturer's recommended protocol and, after quenching the reaction, the Renilla luciferase reaction was initiated by adding Stop & Glo® reagent (Promega). Firefly and Renilla luciferase activities in cell lysates were measured by using a Luminoskan luminometer (Thermo Labsystems, Helsinki, Finland). The relative luciferase activity was calculated by normalizing firefly luciferase activity to that of Renilla luciferase.

Akt Activity

The Akt activity in cell lysates (500 µg) was assayed by using an Akt1/PKB{alpha} immunoprecipitation-kinase assay kit (Upstate Biotechnology, Lake Placid, NY), according to the manufacturer's instructions. The reaction mixture contained 10 µCi of [{gamma}-32P]ATP, 500 µg of cell lysate, and 100 µM of a peptide substrate (RPRAATF) derived from the phosphorylation site of glycogen synthase kinase-3 in a volume of 10 µL. The reaction was allowed to proceed for 10 minutes at 37 °C and was terminated by adding 20 µL of 40% trichloroacetic acid. An aliquot (25 µL) of the reaction mixture was spotted on P81 phosphocellulose paper, which was then washed three times with 0.75% phosphoric acid for 5 minutes each and then once with acetone for 5 minutes. The paper was transferred to 5 mL of scintillation cocktail, and the radioactivity (in cpm) of phosphorylated substrate was measured using a {beta}-scintillation counter (Wallac, PerkinElmer Life Sciences, Gaithersburg, MD). A sample assayed without cell lysates was used as a blank control.

MKK1 Activity

The MKK1 activity was assayed in vitro using an MEK1 assay kit (Upstate Biotechnology), according to the manufacturer's recommended protocol. Briefly, 0.2 U of active MEK1 was added to a mixture containing 20 mM MOPS (pH 7.2), 25 mM {beta}-glycerol phosphate, 5 mM EGTA (ethylene glycol bis [{beta}-aminoethyl ether]-N,N,N',N'-tetraacetic acid), 1 mM sodium orthovanadate, 1 mM DTT, 25 mM MgCl2, 125 µM ATP, 10 µCi of [{gamma}-32P]ATP, and 2 U of inactive MAP kinase 2, with or without oltipraz, and incubated for 30 minutes at 30 °C to activate the MAP kinase 2. The substrate (myelin basic protein, 20 µg) was then added to the reaction mixture, and the sample was incubated for an additional 10 minutes at 30 °C. An aliquot (25 µL) of the reaction mixture was spotted on P81 phosphocellulose paper, washed three times with 0.75% phosphoric acid for 5 minutes each time and then with acetone for 5 minutes, and the radioactivity of the paper was measured using a {beta}-scintillation counter. A sample assayed without MEK1 was used as a blank control.

Stable Plasmid Transfection

H4IIE cells were transfected with the plasmids KmJNK1(–), pcDNA3-CMV-PI3-kinase p85(+), pcDNA3-CMV-PI3-kinase p110(+), or pMCL-CMV-MKK1(–) using Transfectam according to the manufacturer's instructions (Promega). KmJNK1(–) codes for the dominant-negative mutant of JNK1, JNK1(–); pcDNA3-CMV-PI3-kinase p85(+) codes for the overexpression of the p85 subunit of PI3-kinase, p85(+); pcDNA3-CMV-PI3-kinase p110(+) codes for the overexpression of the p110 subunit of PI3-kinase, p110(+); and pMCL-CMV-MKK1(–) codes for dominant-negative mutant of MKK1, MKK1(–). Briefly, cells were replated 24 hours before transfection at a density of 2 x 106 cells in a 10-cm2 plastic dish. Cells were transfected by the addition of 2.5 mL of minimal essential medium (MEM) containing 10 µg of each plasmid and 20 µL of Transfectam and then incubated at 37 °C in a humidified atmosphere of 5% CO2 for 6 hours. After addition of 6.25 mL of MEM containing 10% FCS, cells were incubated for an additional 48 hours at 37 °C, and 50 µg/mL of geneticin was added to select the resistant colonies.

Statistical Analysis

One-way analysis of variance (ANOVA) was used to assess statistical significance of differences among treatment groups. For each statistically significant effect of treatment, the Newman–Keuls test was used for comparisons between multiple group means. The data were expressed as means ± 95% confidence intervals (CI). All statistical tests were two-sided.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of GSTA2

Treatment of H4IIE cells with oltipraz (3–100 µM) for 24 hours increased the level of GSTA2 protein in a concentration-dependent manner (data not shown). Oltipraz at a concentration of 3 µM induced expression of GSTA2 protein twofold relative to control, with maximal induction observed at a concentration of 30 µM (data not shown). An oltipraz concentration of 30 µM was therefore used in all subsequent experiments. Northern blot analysis showed that the level of GSTA2 mRNA began to increase 6 hours after exposure of cells to oltipraz and plateaued at 12–48 hours (Fig. 1Go, A). Immunoblot analysis confirmed that the GSTA2 protein was induced by oltipraz during the same time period (Fig. 1Go, B).



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Fig. 1. Induction of glutathione S-transferase A2 (GSTA2) by oltipraz. A) Northern blot analysis of GSTA2 mRNA in H4IIE cells. Northern blot analysis was performed with total RNA fractions (30 µg each) prepared from cells incubated with oltipraz (30 µM) for 3–48 hours. The equal loading of RNA in each lane was confirmed by rehybridization of the stripped membrane with a 32P-labeled probe for 18S ribosomal RNA (rRNA). Changes in the GSTA2 mRNA expression relative to control were assessed by scanning densitometry and normalized by RNA loading. Bars represent the mean of four experiments ±95% confidence intervals (CIs). mRNA levels at 6, 12, 24, and 48 hours were statistically significantly different from control (P = .024, P<.001, P<.001, and P = .002, respectively) (control level = 1). B) Immunoblot analysis of the GSTA2 protein in cytosolic extracts from H4IIE cells treated with oltipraz (30 µM) for 6–48 hours. Each lane was loaded with 10 µg of cytosolic proteins. Immunoreactive proteins bound with a polyclonal anti-rat GSTA2 antibody were detected with an alkaline phosphatase-conjugated secondary antibody and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium. Equal loading of proteins was verified by probing a replicate blot for actin. Changes in GSTA2 protein levels relative to control were assessed by scanning densitometry. Data represent the mean of four experiments ±95% CIs. Protein levels at 6, 12, 24, and 48 hours were statistically significantly different from control (P = .008, P = .001, P<.001, and P = .002, respectively) (control level = 1).

 
Nrf2 is essential for ARE-mediated induction of phase II detoxifying enzymes. To assess whether oltipraz increased the binding activity of Nrf2 to the ARE, nuclear extract, isolated from H4IIE cells treated with oltipraz, was probed with radiolabeled GSTA2 gene ARE. Gel shift analysis revealed that oltipraz marginally increased the band intensity of ARE binding at 3 and 6 hours (Fig. 2Go, A). By contrast, Nrf2 binding to ARE in cells treated with t-BHQ (14) was strong. Competition experiments using excess ARE or SP-1 oligonucleotide confirmed the specificity of protein binding to the ARE (Fig. 2Go, B). Subcellular fractionation and immunoblot analyses confirmed that oltipraz weakly increased the level of Nrf2 in the nuclear fraction relative to control, whereas t-BHQ strongly induced nuclear translocation of Nrf2 (Fig. 2Go, C). These data suggested that induction of GSTA2 by oltipraz might be mediated by the activation of other transcriptional factors in addition to Nrf2.



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Fig. 2. Effect of oltipraz on activation of nuclear factor (NF)-E2-related factor 2 (Nrf2) binding to the antioxidant response element (ARE). A) Gel shift analysis of the ARE transcription complex. Nuclear extracts were prepared from H4IIE cells treated with oltipraz (30 µM) for 1–12 hours. Five micrograms of nuclear extracts was then incubated for 30 minutes with 5 ng of radiolabeled oligonucleotide containing the ARE sequence and separated on a 4% polyacrylamide gel. Arrowhead indicates the ARE binding complex. B) Competition assays with 20-fold molar excess of unlabeled ARE oligonucleotide or specific protein-1 (SP-1) oligonucleotide added to the reaction mixture that included radiolabeled ARE-containing oligonucleotide (5 ng) and nuclear extracts prepared from H4IIE cells treated with oltipraz (30 µM) for 6 hours. C) Immunoblot analysis of the level of Nrf2 in the nuclear fraction of H4IIE cells treated with oltipraz (30 µM) for 1–12 hours or tert-butylhydroquinone (t-BHQ) (30 µM) for 6 hours. Immunoreactive protein bound with rabbit anti-Nrf2 antibody was visualized through incubation with horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection kit. Control lanes included nuclear extracts prepared from untreated H4IIE cells. Equal loading of proteins was verified by actin immunoblotting. Results were confirmed by three separate experiments, and a representative immunoblot is shown.

 
Activation of C/EBP{beta} by Oltipraz

Activation of GSTA2 gene expression by PAHs depends on the AhR response element and the portion of the XRE that is similar to the C/EBP binding site (20). To test whether the induction of GSTA2 gene expression by oltipraz was mediated by C/EBP, gel shift analysis of protein binding to the C/EBP binding site was performed with nuclear extracts of H4IIE cells using a radiolabeled C/EBP binding site. Treatment of the cells with oltipraz resulted in a time-dependent increase in C/EBP binding compared with nuclear extract from untreated cells (Fig. 3Go, A). C/EBP binding activity increased 6–24 hours after oltipraz treatment. Addition of a 20-fold excess of an unlabeled C/EBP binding oligonucleotide to the nuclear extract completely abolished the binding activity, whereas excess unlabeled SP-1 oligonucleotide did not inhibit binding, suggesting that the binding protein is C/EBP (Fig. 3Go, B). Competition experiments with antibodies directed against C/EBP{alpha}, C/EBP{beta}, p300/CBP, and AhR indicated that oltipraz-induced C/EBP binding activity is specifically dependent on C/EBP{beta}. Anti-C/EBP{beta} antibody almost completely reduced the band intensity of the C/EBP binding complex (Fig. 3Go, C).



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Fig. 3. Effect of oltipraz on activation of CCAAT/enhancer binding protein {beta} (C/EBP{beta}) binding to the C/EBP binding site. A) Gel shift analysis of protein binding to the C/EBP binding site. Nuclear extracts were prepared from H4IIE cells incubated with or without oltipraz (30 µM) for 1–24 hours. Ten micrograms of nuclear extracts was then incubated for 30 minutes with 5 ng of radiolabeled C/EBP binding site. Arrowhead indicates the DNA bound with C/EBP. B) Twentyfold molar excess of unlabeled C/EBP oligonucleotide or specific protein-1 (SP-1) oligonucleotide were added to the nuclear extracts from cells treated with oltipraz (30 µM) for 6 hours. DNA-binding reactions were performed by gel shift analysis. Arrowhead indicates the DNA bound with C/EBP. C) Immunocompetition assay of C/EBP binding to the C/EBP binding site. Nuclear extracts prepared from H4IIE cells treated with oltipraz (30 µM) for 6 hours were incubated with polyclonal antibodies against C/EBP{alpha}, C/EBP{beta}, p300/CBP, or Ah receptor (AhR) for 1 hour. The immunodepleted extracts were then mixed with labeled probe for the C/EBP binding site. Arrowhead indicates the DNA bound with C/EBP. D) Immunocytochemical analysis of C/EBP{beta}. H4IIE cells were treated with oltipraz (30 µM) for 6 or 24 hours. C/EBP{beta} was immunochemically localized using anti-C/EBP{beta} antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody. The same fields were counterstained with propidium iodide to verify the location and integrity of nuclei. E) Immunoblot analysis of C/EBP{beta} in the nuclear fraction. Nuclear fractions were prepared from H4IIE cells treated with oltipraz (30 µM) for 1–24 hours, and the C/EBP{beta} and C/EBP{alpha} forms in each fraction were immunoblotted with their respective antibodies. Arrowheads indicate nuclear C/EBP{beta} and C/EBP{alpha}. Equal loading of proteins was verified by actin immunoblotting. Results were confirmed by three separate experiments, and a representative blot is shown.

 
To determine whether increases in band intensities obtained in gel shift assays occurred as a result of the nuclear translocation of C/EBP{beta}, the subcellular localization of C/EBP{beta} was determined by immunocytochemical analysis (Fig. 3Go, D). C/EBP{beta} was located predominantly in the cytoplasm of untreated H4IIE cells. However, when cells were treated with oltipraz, C/EBP{beta} showed nuclear localization at 6 hours. At 24 hours of oltipraz treatment, C/EBP{beta} was found in both the cytoplasm and the nucleus (Fig. 3Go, D). To verify these results, immunoblot experiments were conducted with nuclear fractions (i.e., nuclear C/EBP{beta} and nuclear C/EBP{alpha}). The level of nuclear C/EBP{beta} increased 6–24 hours after treatment of cells with oltipraz, whereas the level of nuclear C/EBP{alpha} remained relatively consistent (Fig. 3Go, E). These data provided evidence that C/EBP{beta}, but not C/EBP{alpha}, was activated by oltipraz in H4IIE cells and that C/EBP{beta} served as an active component in the C/EBP binding complex.

Analysis of the C/EBP Response Element in the GSTA2 Promoter

A previous study (20) showed that activation of C/EBP{alpha} and AhR is involved in the induction of GSTA2 expression via the XRE. Given this role of C/EBP in GSTA2 expression and the activation of C/EBP{beta} by oltipraz, we examined whether oltipraz might transcriptionally activate the GSTA2 gene via the C/EBP binding site within the XRE. Reporter gene assays were performed using H4IIE cells transfected with the mammalian cell expression vector pGL-1651, which contained the luciferase structural gene downstream of the –1.65-kb GSTA2 promoter region (Fig. 4Go, A). Exposure of transiently transfected cells to oltipraz resulted in a 7.4-fold (95% CI = 6.4-fold to 8.3-fold) increase in luciferase activity (Fig. 4Go, B). Both {beta}-naphthoflavone ({beta}NF), which interacts with the AhR, and t-BHQ, which activates the ARE, also transcriptionally activated the GSTA2 gene 4.0-fold (95% CI = 2.9-fold to 5.1-fold) and 2.3-fold (95% CI = 1.8-fold to 2.9-fold), respectively, with t-BHQ inducing the luciferase activity, presumably through the ARE sequence (Fig. 4Go, B).



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Fig. 4. Analysis of the glutathione S-transferase A2 (GSTA2) promoter region in promoter-luciferase reporter gene constructs. A) The size of the flanking insert in each GSTA2 chimeric gene construct. B) Induction of luciferase activity by oltipraz in H4IIE cells transiently transfected with GSTA2 chimeric gene construct pGL-1651, which contains both the xenobiotic response element (XRE) and the antioxidant response element (ARE) of the GSTA2 promoter. Lysates from H4IIE cells that had been co-transfected with the GSTA2-luciferase gene construct pGL-1651 (firefly luciferase) and pRL-SV (Renilla luciferase) at a ratio of 200 : 1 and subsequently treated with oltipraz (30 µM), {beta}-naphthoflavone ({beta}NF, 5 µM), or tert-butylhydroquinone (t-BHQ, 30 µM) for 12 hours were used for dual luciferase reporter assays. Activation of the reporter gene was calculated as a change in the ratio of firefly luciferase activity to Renilla luciferase activity. Data represent the mean of three experiments ±95% confidence intervals (CIs). Luciferase units in cells treated with oltipraz, {beta}NF, or t-BHQ were statistically significantly different from control (P<.001) (control level = 1). C) Basal and oltipraz-inducible luciferase activity of 5' deletion mutants of the GSTA2 gene. The chimeric gene constructs pGL-1651 and pGL-1128 contained both XRE and ARE elements, whereas pGL-797 had only the ARE element. pGL-197 was used as a vector containing the minimal promoter region. The relative luciferase inducibility was obtained from the ratio of activity in H4IIE cells treated with oltipraz (30 µM) to that in untreated H4IIE cells transfected with each chimeric construct. Data represented the mean of three experiments ±95% CIs. Change in luciferase units in cells transfected with pGL-797 and pGL-197 were statistically significantly different from cells transfected with pGL-1651 (P = .008 and P = .001, respectively). D) Inhibition of oltipraz-inducible pGL-1651 reporter gene activation by the dominant-negative mutant of C/EBP (AC/EBP). H4IIE cells were co-transfected with pGL-1651/pRL-SV (200 : 1) in combination with pCMV-AC/EBP at a ratio of 1 : 1. Transfected cells were incubated in medium containing 1% fetal calf serum for 12 hours and then exposed to oltipraz for 12 hours. Luciferase activity was measured at 24 hours after transfection. Transfected cells were incubated for 24 hours to ensure sufficient expression of AC/EBP. Luciferase activity was expressed as firefly luciferase units of cell lysate relative to that of lysed cells transfected with pCMV500 and is represented as the mean of three experiments ±95% CIs. Luciferase units in pCMV-A/CEBP-transfected cells treated with oltipraz were statistically significantly different from pCMV500-transfected cells treated with oltipraz (P<.001).

 
To precisely define the DNA sequence required for the induction of GSTA2 by oltipraz, a series of chimeric gene constructs with promoter deletions was prepared (pGL-1128, pGL-797, and pGL-197; Fig. 4Go, A). The relative luciferase-inducible activity by oltipraz in H4IIE cells transfected with pGL-1128, which contained both the XRE and the ARE, was comparable to that obtained in cells transfected with pGL-1651 (Fig. 4Go, C). Exposure of cells transfected with pGL-797 to oltipraz resulted in only 25% of the inducible luciferase activity that was observed with pGL-1651 or pGL-1128 (Fig. 4Go, C). In addition, the pGL-197 gene construct, which lacked nucleotides –1651 to –198 (i.e., lacked both the XRE and the ARE) of the GSTA2 gene flanking region, showed no luciferase inducibility. The promoter deletion experiments demonstrated that the position of the GSTA2 regulatory region that confers oltipraz responsiveness included XRE.

To determine whether oltipraz induction of GSTA2 through the XRE involved C/EBP{beta}, the constitutively active dominant-negative mutant of C/EBP (AC/EBP) was expressed in combination with the pGL-1651 luciferase reporter in H4IIE cells. Expression of AC/EBP almost completely inhibited the ability of oltipraz to stimulate reporter gene expression from the pGL-1651 plasmid (Fig. 4Go, D). Transfection of the cells with pCMV500, which was used as a control, did not inhibit the ability of oltipraz to stimulate reporter gene expression from pGL-1651. These data indicate that the C/EBP binding sequence within the XRE was likely to be responsible for GSTA2 induction.

Role of PI3-kinase in GSTA2 Induction by Oltipraz

Because pro-oxidants increase PI3-kinase activity (14) and activate one of its downstream mediators, Akt, we determined the effect of oltipraz on the PI3-kinase pathway by assaying Akt activity in H4IIE cells. Oltipraz treatment, at the time points examined (10 minutes through 6 hours), did not increase PI3-kinase/Akt activity (Fig. 5Go, A), whereas Akt activity was increased with t-BHQ, which was used a positive control. Therefore, we assessed whether the constitutive activity of PI3-kinase could modulate the oltipraz-inducible expression of GSTA2. H4IIE cells were pre-incubated with the PI3-kinase inhibitors wortmannin (500 nM) or LY294002 (30 µM) for 1 hour before the addition of oltipraz (30 µM). Northern and immunoblot analyses revealed that the PI3-kinase inhibitors completely blocked oltipraz-inducible GSTA2 mRNA and protein expression (Fig. 5Go, B, and 5, C, respectively). To confirm the role of PI3-kinase in the induction of GSTA2 by oltipraz, the effects of oltipraz on the level of GSTA2 protein were monitored in cells overexpressing either the p85 regulatory subunit or the p110 catalytic subunit of PI3-kinase (Fig. 5Go, D). Immunoblot analysis revealed that overexpression of the p85 subunit completely inhibited oltipraz-inducible increases in GSTA2 protein expression, whereas overexpression of the p110 subunit did not alter oltipraz-inducible increases in GSTA2 protein expression.



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Fig. 5. Effect of oltipraz on phosphatidylinositol 3-kinase (PI3-kinase)-dependent glutathione S-transferase A2 (GSTA2) induction. A) The effect of oltipraz on the activity of Akt. Akt activity was measured in H4IIE cells treated with oltipraz (30 µM) for 10 minutes to 6 hours by phosphorylation of a peptide substrate derived from the phosphorylation site of glycogen synthase kinase-3. Akt activity was measured in cells treated with tert-butylhydroquinone (t-BHQ; 30 µM, 10 minutes), as a positive control. Data represent the mean of three experiments ±95% confidence intervals (CIs). Akt activity at 10 minutes after t-BHQ treatment was statistically significantly different from control (P = .017; Akt activity in control cells = 1). B) The effects of PI3-kinase inhibitors on oltipraz-induced GSTA2 mRNA expression. The GSTA2 mRNA level was determined by northern blot analysis in H4IIE cells treated with oltipraz (30 µM) in the presence or absence of wortmannin (WO) (500 nM) or LY294002 (LY) (30 µM) for 12 hours. The equal loading of RNA in each lane was confirmed by rehybridization of the stripped membrane with a 32P-labeled probe for 18S ribosomal RNA (rRNA). The relative changes in GSTA2 mRNA level were assessed by scanning densitometry. Data represent the mean of four experiments ±95% CIs. mRNA levels in cells treated with oltipraz + WO or oltipraz + LY were statistically significantly different from cell treated with oltipraz alone (P<.001). C) The effects of PI3-kinase inhibitors on the induction of GSTA2 protein in H4IIE cells treated with oltipraz (30 µM). Immunoblot analyses were performed as described in Fig. 1Go, B. Each lane was loaded with 10 µg of cytosolic proteins. Immunoreactive proteins bound with polyclonal anti-rat GSTA2 antibody were detected with an alkaline phosphatase-conjugated secondary antibody and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium. Equal loading of proteins was verified by probing the replicate blot for actin. Data represent the mean of four experiments ±95% CIs. Protein levels in cells treated with oltipraz + WO or oltipraz + LY were statistically significantly different from cells treated with oltipraz alone (P<.001). D) The effect of oltipraz on the expression of GSTA2 in H4IIE cells stably transfected with an overexpression vector of the p85 regulatory subunit [p85(+)] or the p110 catalytic subunit [p110(+)] of PI3-kinase. H4IIE cells were incubated with oltipraz (30 µM) for 12 hours. The expression of GSTA2 was immunochemically measured by immmunoblot analysis. Each lane was loaded with 1 µg of cytosolic proteins. Immunoreactive protein bound with anti-GSTA2 antibody was visualized through incubation with horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence (ECL) detection kit. Equal loading of proteins was verified by actin immunoblot. Results were confirmed by four separate experiments.

 
To identify the possible relationship between the PI3-kinase pathway and the activation of C/EBP{beta} as a transcription factor required for oltipraz-inducible GSTA2 expression, the level of nuclear C/EBP{beta} was monitored in H4IIE cells treated with oltipraz in the presence or absence of PI3-kinase inhibitors. Both immunocytochemistry and subcellular fractionation analyses revealed that pretreatment of cells with wortmannin or LY294002 for 1 hour inhibited the activation of C/EBP by oltipraz at 6 hours (Fig. 6Go, A and B). The slight increase in the level of nuclear Nrf2 by oltipraz was also decreased by the PI3-kinase inhibitors (Fig. 6Go, B). These data suggest that the PI3-kinase pathway plays an essential role in the activation of C/EBP{beta} and in C/EBP{beta}-dependent GSTA2 gene expression.



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Fig. 6. Phosphatidylinositol 3-kinase (PI3-kinase)-dependence of oltipraz-induced nuclear localization of CCAAT/enhancer binding protein {beta} (C/EBP{beta}). A) Immunocytochemistry of C/EBP{beta}. H4IIE cells were pretreated with the PI3 kinase inhibitors wortmannin (WO) (500 nM) or LY294002 (LY) (30 µM) for 1 hour and then incubated with oltipraz (30 µM) for 6 hours. C/EBP{beta} was immunochemically detected using rabbit anti-C/EBP{beta} antibody. The cells were counterstained with propidium iodide to verify the location and integrity of nuclei. B) Immunoblot analyses of nuclear C/EBP{beta} and nuclear factor (NF)-E2-related factor 2 (Nrf2) in nuclear fractions prepared from H4IIE cells treated with oltipraz (30 µM) in the presence or absence of WO (500 nM) or LY294002 (30 µM) for 6 hours. Equal loading of proteins was verified by probing a replicate blot for actin with goat anti-actin antibody. Results were confirmed by three separate experiments, and a representative blot is shown.

 
Effects of Oltipraz on the Phosphorylation of MAP Kinases

It has been proposed that ERK1/2 and p38 kinase might be associated with the induction of quinone reductase and {gamma}-glutamylcysteine synthetase, respectively (26,27). Thus, we determined the effects of oltipraz on the activation of ERK1/2, p38 kinase, and JNK (another MAP kinase) in H4IIE cells. All three MAP kinases were activated by t-BHQ (14); by contrast, oltipraz inhibited constitutive phosphorylation of ERK1/2 at 1 hour and thereafter (Fig. 7Go, A). However, oltipraz did not alter phosphorylation of p38 kinase or JNK1/2 at any of the time points examined.



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Fig. 7. Effects of oltipraz on mitogen-activated protein (MAP) kinases and MAP kinase kinase 1 (MKK1). A) The effects of oltipraz on MAP kinase phosphorylation. H4IIE cells were treated with oltipraz (30 µM) for 10 minutes through 6 hours. Activation of ERK1/2, p38 kinase, and c-Jun N-terminal kinase (JNK) was assessed by immunoblot analysis of the phosphorylated forms (p–) of the MAP kinases in whole cell lysates. Results were confirmed by three separate experiments. Solid and open arrowheads indicate the phosphorylated and unphosphorylated forms of MAP kinases, respectively. B) Inhibition of MKK1 activity by oltipraz. The inhibitory effect of oltipraz on MKK1 activity was assessed in vitro, as described in the "Materials and Methods" section. MKK1 activity was assessed by MAP kinase 2 activity toward the substrate myelin basic protein. The extent of MKK1 inhibition by oltipraz (1–100 µM) was compared with that by PD98059 (10 µM), an MKK1 inhibitor. Data represent the mean of three experiments ±95% confidence intervals (CIs). Percent inhibition of MKK1 activity in the presence of 30 µM oltipraz, 100 µM oltipraz, or 10 µM PD98059 was statistically significantly different from control (P = .024, P = .001, and P<.001, respectively).

 
To assess whether the inhibition of ERK1/2 by oltipraz resulted from the inhibition of MKK1, a kinase that acts upstream of ERK1/2, we measured MKK1 activity in vitro. Oltipraz inhibited the activity of MKK1, with an IC50 (concentration that causes 50% inhibition of growth) of {approx}40 µM (Fig. 7Go, B). MKK1 activity was also inhibited 66.9% (95% CI = 63.5% to 70.3%; P<.001) by PD98059 (10 µM), an MKK1 inhibitor. These results suggested that the observed inhibition of ERK1/2 phosphorylation by oltipraz was consistent with inhibition of MKK1.

Contribution of the MAP Kinase Pathways to GSTA2 Induction

Previously, we showed that PD98059 treatment induced GSTA2 by increasing mRNA expression (14). To test whether the MKK1/ERK pathway is involved in the induction of GSTA2 by oltipraz, we first assessed the effect of PD98059 on the induction of GSTA2 by oltipraz. PD98059 did not alter GSTA2 expression in H4IIE cells treated with oltipraz (Fig. 8Go, A and B). We next monitored the expression of GSTA2 mRNA in H4IIE cells or cells stably transfected with a dominant-negative mutant of MKK1 [MKK1(–)] to establish whether the inhibition of MKK1 by oltipraz is associated with GSTA2 induction. GSTA2 mRNA was not increased by stable transfection with MKK1(–), and oltipraz was capable of increasing GSTA2 mRNA expression in MKK1(–) transfectants (Fig. 8Go, C). Hence, these results suggested that the induction of GSTA2 by oltipraz was not related to MKK1 inhibition.



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Fig. 8. Effects of mitogen-activated protein (MAP) kinase inhibition on oltipraz-inducible glutathione S-transferase A2 (GSTA2) expression. A) The effects of extracellular signal-regulated kinase (ERK1/2) and p38 kinase inhibitors on the increase in GSTA2 mRNA. H4IIE cells were treated with oltipraz (30 µM) for 12 hours in the presence of PD98059 (PD) (50 µM) or SB203580 (SB) (10 µM). Total RNA fractions (30 µg each) prepared from the cells were subjected to northern blot analysis. Data represent the mean of three experiments ±95% confidence intervals (CIs). mRNA levels in cells treated with oltipraz, oltipraz + PD, or oltipraz + SB were statistically significantly different from control (P<.001). B) The effects of ERK1/2 and p38 kinase inhibitors on GSTA2 expression. Immunoblot analyses of the level of GSTA2 proteins prepared from H4IIE cells that were treated with oltipraz (30 µM) for 24 hours in the presence or absence of PD or SB. Each lane was loaded with 10 µg of cytosolic proteins. Immunoreactive protein was visualized through incubation with alkaline phosphatase-conjugated secondary antibody and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium. Equal loading of proteins was verified by probing the replicate blot for actin. Data represent the mean of three experiments ±95% CI. Protein levels in cells treated with oltipraz, oltipraz + PD, or oltipraz + SB were statistically significantly different from control (P<.001). C) GSTA2 mRNA levels in H4IIE cells or in cells stably transfected with the dominant-negative mutant of MAP kinase kinase 1 [MKK1(–)]. The cells were treated with oltipraz (30 µM) for 12 hours. The equal loading of RNA in each lane was confirmed by rehybridization of the stripped membrane with a 32P-labeled probe for 18S ribosomal RNA (rRNA). D) GSTA2 protein levels in H4IIE cells or in cells stably transfected with the dominant-negative mutant of c-Jun N-terminal kinase 1 [JNK(–)], which were treated with or without oltipraz (30 µM) for 24 hours. Each lane was loaded with 1 µg of cytosolic proteins. Immunoreactive protein was visualized through incubation with horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence (ECL) detection kit. Equal loading of proteins was verified by actin immunoblot. E) Immunoblot analysis of nuclear CCAAT/enhancer binding protein {beta} (C/EBP{beta}). Nuclear fractions of C/EBP{beta} were prepared from H4IIE cells that were treated with oltipraz (30 µM) for 6 hours in the presence or absence of PD (50 µM) or SB (10 µM). C/EBP{beta} protein levels in H4IIE cells or in cells stably transfected with JNK1(–) were treated with or without oltipraz (30 µM) for 6 hours. Equal loading of proteins was verified by probing the replicate blot for actin. Results were confirmed by three separate experiments.

 
To determine the possible role of p38 kinase in the induction of GSTA2 by oltipraz, we used SB203580, a chemical inhibitor of p38 kinase. SB203580 (10 µM) did not inhibit the oltipraz-inducible increases in GSTA2 mRNA and protein expression (Fig. 8Go, A and B). To assess the role of the JNK cascade in the induction of GSTA2 by oltipraz, H4IIE cells were stably transfected with the dominant-negative mutant of JNK1 [JNK1(–)], in which JNK activity has been previously shown to be decreased in the JNK1(–) transfectants (14,31). Stable transfection of JNK1(–) did not change the expression of GSTA2, whereas oltipraz (30 µM, for 24 hours) increased the expression of GSTA2 in JNK1(–) cells (Fig. 8Go, D). These data suggest that none of the MAP kinase pathways examined here were responsible for the induction of GSTA2 by oltipraz.

To determine whether the chemical inhibition of MAP kinases or JNK1(–) transfection would block the oltipraz-induced nuclear translocation of C/EBP{beta}, we treated H4IIE cells with either PD98059 or SB203580. Neither inhibitor blocked the nuclear translocation of C/EBP{beta} (Fig. 8Go, E). In addition, C/EBP{beta} in JNK1(–) cells was responsive to oltipraz. Thus, none of the MAP kinase pathways examined in this study were involved in regulating the nuclear translocation of C/EBP{beta} by oltipraz.


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Oltipraz and the substituted 1,2-dithiole-3-thiones are excellent candidates for cancer chemoprevention because they are potent inducers of enzymes such as GST, which detoxify many carcinogens, including aflatoxin, dimethylbenzanthracene, azoxymethane, diethylnitrosamine, uracil mustard, benzo[a]pyrene, and N-butyl-N-(4-hydroxybutyl)nitrosamine (2,3234). In this study, we report for the first time that oltipraz activates C/EBP{beta}, whose nuclear translocation activates the C/EBP binding site present in the GSTA2 gene and that the PI3-kinase pathway regulates this nuclear translocation of C/EBP{beta}.

Oltipraz, which was initially considered to be a monofunctional inducer (35), activates both phase I and phase II enzymes (36). Considerable attention has been focused on the mechanistic basis for the induction of phase II enzymes by monofunctional and bifunctional inducers. Monofunctional inducers transcriptionally activate the expression of GSTA2 and quinone reductase genes through the ARE, whereas bifunctional inducers act through both the XRE and the ARE (3739). Nrf2 binding to ARE plays a critical role in the induction of phase II detoxifying enzymes, such as GST (13,14).

Evidence of the potential chemopreventive role of inducers of phase II enzymes comes from animal studies. For example, lack of Nrf2 binding to the ARE increases sensitivity of animals to xenobiotic-induced injury (40,41). Nrf2 knockout(-/-) mice develop liver and lung damage in response to toxicants (41), which has been attributed to diminished expression of phase II enzyme genes. In another study (42), oltipraz did not protect against benzo[a]pyrene-initiated cancer of the forestomach in Nrf2(-/-) mice compared with wild-type mice, and the Nrf2(-/-) mice developed a greater number of tumors than did the wild-type mice. Hence, activation of Nrf2, which controls constitutive and inducible expression of phase II detoxifying genes, may be one of the protective mechanisms against xenobiotics.

Cytotoxic pro-oxidants also induce phase II enzymes through the activation of Nrf2 (43). Oxidative stress from a pro-oxidant stimulates Nrf2 activation to compensate for an altered cellular oxidation state, and Nrf2 activation transcriptionally activates phase II enzymes (1014). In our previous study (14), we showed that nuclear Nrf2 binding to the ARE is activated 1–6 hours after treatment of cells with the pro-oxidant t-BHQ and that the GSTA2 mRNA level was elevated 6–24 hours after treatment. The GSTA2 mRNA level was highest 12 hours after treatment and gradually returned toward that of pretreatment levels by 24 hours. The time course of GSTA2 induction by oltipraz differed substantially from that induced by t-BHQ; that is, GSTA2 mRNA levels in cells treated with oltipraz remained high for a longer period of time—at least up to 48 hours. By contrast with the strong binding of Nrf2 to the ARE by t-BHQ, oltipraz only weakly stimulated Nrf2 binding activity. This finding may imply that a mechanism(s) other than that involving Nrf2 binding to the ARE is involved in the induction of GSTA2 by oltipraz.

Transcription factors of the C/EBP family have roles in the differentiation of cells and in the regulation of the expression of both tissue-specific genes and genes involved in cell proliferation (44,45). In this study, we found that activation of C/EBP{beta} by oltipraz preceded the persistent elevation of GSTA2 mRNA levels. This finding, together with the results of luciferase reporter-genes assays, supports the role of the C/EBP response element within the XRE sequence to which C/EBP binds in the induction of GSTA2. The crucial role of C/EBP{beta} binding to the C/EBP binding site was evidenced by the almost complete inability of oltipraz to stimulate luciferase reporter gene activity in the experiments using the dominant-negative mutant of C/EBP. C/EBP proteins are involved in the expression of liver-specific genes (19). In untreated cells, C/EBP{alpha} is likely to be part of the proteins that directly bind to the C/EBP binding site within the XRE (20). AhR and C/EBP{alpha} share overlapping DNA binding sequences, and C/EBP{alpha} enhances xenobiotic induction mediated by AhR through cooperative interactions with AhR (20).

The biologic effect evoked by C/EBP on the transcription of liver-specific genes might depend on the relative activities of C/EBP{alpha} and other C/EBP proteins, such as C/EBP{beta}. Active C/EBP{beta} may compete with C/EBP{alpha} for the C/EBP binding site within the XRE. In the present study, we have demonstrated that C/EBP{beta} is activated by oltipraz and that C/EBP{beta} activation plays an important role in the induction of GSTA2. Our observation that oltipraz activates C/EBP{beta} and induces its nuclear translocation has an important implication for the finding of C/EBP{beta} (mediated by GST induction) as a molecular target of cancer chemoprevention.

In general, AhR and the AhR nuclear translocator are often essential to XRE activation caused by carcinogenic planar aromatic compounds (46). In this study, however, we found that AhR was not a component of the protein complex (activated by oltipraz) binding to the C/EBP consensus sequence, although the binding sequences for C/EBP and ligand-activated AhR within the XRE did closely overlap. Lack of AhR as a binding protein for protein complex binding to the C/EBP site within the XRE may explain an important difference between the activation of XRE by oltipraz and that by PAHs. Competition of C/EBP{beta} for the C/EBP binding site within or closely proximal to the XRE may be associated with altered gene expression and cell proliferation by PAHs that activate AhR. Oltipraz activates C/EBP{beta}, which stimulates the expression of genes associated with liver cell proliferation (19). By contrast, dioxin, a PAH, suppresses the expression of C/EBP{beta} and inhibits the growth fraction of cells (47). This difference in XRE activation between oltipraz and PAHs raises the possibility that activated C/EBP{beta} and AhR may compete for the C/EBP binding site within the XRE in the GSTA2 promoter. In the present study, neither C/EBP{alpha} nor the p300/CBP coactivator was involved in C/EBP binding to the C/EBP binding site within the XRE. N-terminal transactivation domains of C/EBP{beta} have been shown to interact with p300/CBP, the interaction of which is critical for C/EBP{beta} transactivation (48). Thus, binding of activated C/EBP{beta} to the C/EBP binding site within the XRE would induce the cooperative interaction of C/EBP{beta} with p300/CBP for transactivation of the GSTA2 gene.

The fact that expression of GSTA2 markedly decreased in Nrf2(-/-) mice (41,42) implies that the activation of C/EBP{beta} and its binding to the C/EBP binding site within the XRE (induced by oltipraz) may require the constitutive binding of Nrf2 to the ARE for transcriptional activation of the GSTA2 gene. Thus, formation of a large complex comprising C/EBP{beta}, p300/CBP, and Nrf2 binding to two or more DNA sites may be required for induction of the GSTA2 gene.

Interestingly, we have previously shown that the activation of the PI3-kinase pathway is also involved in Nrf2/ARE-mediated GSTA2 induction and that PI3-kinase and Akt are activated in response to oxidative stress (13,14). PI3-kinase functions in cell growth, survival, and transformation, and its activity is regulated in a redox-sensitive manner (49). The present study shows that oltipraz treatment did not increase the activity of PI3-kinase/Akt, which raises the possibility that the production of reactive oxygen species induced by oltipraz in H4IIE cells is minimal. Although PI3-kinase was not activated by oltipraz, we found that the PI3-kinase signaling pathway plays an essential role in the activation of C/EBP{beta} and in the induction of GSTA2. Whereas basal activity of PI3-kinase was sufficient for C/EBP{beta}-dependent GSTA2 induction by oltipraz, overexpression of the constitutively active p110 catalytic subunit of PI3-kinase failed to alter constitutive or oltipraz-inducible GSTA2 expression in H4IIE cells, thus indicating that the increase in PI3-kinase activity alone was not sufficient to stimulate C/EBP-dependent GSTA2 expression. This is consistent with the hypothesis that the PI3-kinase-dependent mechanism that controls the activation of C/EBP{beta} may play an important role in cancer chemoprevention, as illustrated in Fig. 9Go.



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Fig. 9. Schematic diagram illustrating the proposed mechanism by which oltipraz induces glutathione S-transferase (GST). The phosphatidylinositol 3-kinase (PI3-kinase)-mediated nuclear translocation of CCAAT/enhanced protein {beta} (C/EBP{beta}) promoted by oltipraz leads to the induction of GST via the activation of the C/EBP response element within the xenobiotic response element (XRE) together with nuclear factor (NF)-E2-related factor 2 (Nrf2) binding to antioxidant response element (ARE). Active C/EBP{beta} may compete with C/EBP{alpha} for the C/EBP binding site.

 
Growth stimuli, a number of cellular stresses, and lethal insults (e.g., cytotoxic chemicals) engage the MAP kinases and concomitantly induce transactivation of the genes for cell proliferation, inflammation, or apoptosis (50,51). In the present study, oltipraz inhibited MKK1 activity in vitro and also suppressed phosphorylation of ERK1/2. PD98059, an ERK inhibitor, did not affect oltipraz-inducible GSTA2 expression. Hence, the ERK pathway is unlikely to be responsible for the induction of GSTA2 by oltipraz. This finding is further supported by the result of the MKK1(–) stable transfection experiment, in which the induction of GSTA2 by oltipraz was not associated with the inhibition of MKK1 activity. Hence, the pathway involving MKK1/ERK, which has been proposed to play a role in the transcriptional activation of the ARE enhancer-linked reporter gene or the induction of quinone reductase (27), is not likely to be responsible for GSTA2 induction.

Activation of p38 kinase or JNK is an early response of cells on exposure to a variety of stressful signals, such as heat, UV irradiation, and DNA-damaging agents (23,52). In this study, oltipraz did not alter phosphorylation of either p38 kinase or JNK. By contrast to our previous observation of p38 kinase-mediated GSTA2 induction by glutathione-depleted oxidative stress (13), p38 kinase was not responsible for the induction of GSTA2 by oltipraz. In addition, the finding that H4IIE cells stably transfected with JNK1(–) only slightly increased expression of the GSTA2 protein excludes the possibility that the JNK pathway is involved in the induction of GSTA2 by oltipraz. These results, together with the observation that the MAP kinases are not responsible for the oltipraz-induced nuclear translocation of C/EBP{beta}, suggest that none of the MAP kinase pathways examined in this study play a role in the induction of GSTA2, at least through the mechanism involving C/EBP{beta}-mediated transcriptional activation.

Given the finding that C/EBP{beta} might be a molecular target for GSTA2 induction, we searched the GenBank database for the C/EBP response elements in the regulatory regions of other phase II enzymes. The promoter sequences of representative phase II enzymes from the GenBank database were compared with the consensus sequence to which C/EBP binds. The genes that contained C/EBP as a core sequence that overlaps with or is closely proximal to the XRE include human {gamma}-glutamylcysteine synthetase, mouse quinone reductase, human GST{alpha}, and human heme oxygenase-1 (a C/EBP or C/EBP-like sequence is located at –1272 bp, –246 bp, –847 bp, and –1882 bp from the transcription start site, respectively). Therefore, C/EBP{beta} may serve as a common transcriptional factor for the induction of phase II enzymes and cancer chemoprevention.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Supported by the National Research Laboratory Program (2001), Korea Institute of Science and Engineering Evaluation and Planning, The Ministry of Science and Technology, Republic of Korea.

The kind donations of pCMV500 and pCMV-AC/EBP plasmids from Dr. Charles Vinson and pGTB-1.65 containing the GSTA2-promoter region from Dr. Cecil B. Pickett are gratefully acknowledged.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received March 12, 2002; revised October 12, 2002; accepted November 6, 2002.


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