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 151742, South Korea (e-mail: sgk{at}snu.ac.kr).
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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) and the aromatic hydrocarbon receptor (AhR) by polycyclic aromatic hydrocarbons (PAHs) leads to the induction of GSTA2 and quinone reductase via C/EBP
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
) plays an important role in hepatocyte-specific gene expression (19). The ratio of C/EBP
and C/EBP
is responsible for modulation of the transcriptional activities of the genes for cell proliferation and differentiation. For example, C/EBP
, 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
[-32P]dCTP (3000 mCi/mmol) and [
-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 transcriptionpolymerase chain reaction (RTPCR) 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 thiocyanatephenolchloroform 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 [-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
SDSpolyacrylamide 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 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 SDSPAGE 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 TrisHCl (pH 7.5), 1% Triton X-100, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM -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
, and C/EBP
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 [-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 TrisHCl [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
, C/EBP
, p300/CBP (N-terminal domains of C/EBP
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
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 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 immunoprecipitation-kinase assay kit (Upstate Biotechnology, Lake Placid, NY), according to the manufacturer's instructions. The reaction mixture contained 10 µCi of [
-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
-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 -glycerol phosphate, 5 mM EGTA (ethylene glycol bis [
-aminoethyl ether]-N,N,N',N'-tetraacetic acid), 1 mM sodium orthovanadate, 1 mM DTT, 25 mM MgCl2, 125 µM ATP, 10 µCi of [
-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
-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 NewmanKeuls 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Treatment of H4IIE cells with oltipraz (3100 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 1248 hours (Fig. 1, A). Immunoblot analysis confirmed that the GSTA2 protein was induced by oltipraz during the same time period (Fig. 1
, B).
|
|
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. 3, A). C/EBP binding activity increased 624 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. 3
, B). Competition experiments with antibodies directed against C/EBP
, C/EBP
, p300/CBP, and AhR indicated that oltipraz-induced C/EBP binding activity is specifically dependent on C/EBP
. Anti-C/EBP
antibody almost completely reduced the band intensity of the C/EBP binding complex (Fig. 3
, C).
|
Analysis of the C/EBP Response Element in the GSTA2 Promoter
A previous study (20) showed that activation of C/EBP 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
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. 4
, 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. 4
, B). Both
-naphthoflavone (
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. 4
, B).
|
To determine whether oltipraz induction of GSTA2 through the XRE involved C/EBP, 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. 4
, 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. 5, 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. 5
, 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. 5
, 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.
|
|
It has been proposed that ERK1/2 and p38 kinase might be associated with the induction of quinone reductase and -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. 7
, A). However, oltipraz did not alter phosphorylation of p38 kinase or JNK1/2 at any of the time points examined.
|
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. 8, 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. 8
, C). Hence, these results suggested that the induction of GSTA2 by oltipraz was not related to MKK1 inhibition.
|
To determine whether the chemical inhibition of MAP kinases or JNK1() transfection would block the oltipraz-induced nuclear translocation of C/EBP, we treated H4IIE cells with either PD98059 or SB203580. Neither inhibitor blocked the nuclear translocation of C/EBP
(Fig. 8
, E). In addition, C/EBP
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
by oltipraz.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 16 hours after treatment of cells with the pro-oxidant t-BHQ and that the GSTA2 mRNA level was elevated 624 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 timeat 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 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
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
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
share overlapping DNA binding sequences, and C/EBP
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 and other C/EBP proteins, such as C/EBP
. Active C/EBP
may compete with C/EBP
for the C/EBP binding site within the XRE. In the present study, we have demonstrated that C/EBP
is activated by oltipraz and that C/EBP
activation plays an important role in the induction of GSTA2. Our observation that oltipraz activates C/EBP
and induces its nuclear translocation has an important implication for the finding of C/EBP
(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 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
, which stimulates the expression of genes associated with liver cell proliferation (19). By contrast, dioxin, a PAH, suppresses the expression of C/EBP
and inhibits the growth fraction of cells (47). This difference in XRE activation between oltipraz and PAHs raises the possibility that activated C/EBP
and AhR may compete for the C/EBP binding site within the XRE in the GSTA2 promoter. In the present study, neither C/EBP
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
have been shown to interact with p300/CBP, the interaction of which is critical for C/EBP
transactivation (48). Thus, binding of activated C/EBP
to the C/EBP binding site within the XRE would induce the cooperative interaction of C/EBP
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 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
, 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 and in the induction of GSTA2. Whereas basal activity of PI3-kinase was sufficient for C/EBP
-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
may play an important role in cancer chemoprevention, as illustrated in Fig. 9
.
|
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, 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
-mediated transcriptional activation.
Given the finding that C/EBP 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
-glutamylcysteine synthetase, mouse quinone reductase, human GST
, 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
may serve as a common transcriptional factor for the induction of phase II enzymes and cancer chemoprevention.
![]() |
NOTES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Sporn MB, Dunlop NM, Newton DL, Smith JM. Prevention of chemical carcinogenesis by vitamin A and its synthetic analogs (retinoids). Fed Proc 1976;35:13328.[Medline]
2 Kensler TW. Chemoprevention by inducers of carcinogen detoxication enzymes. Environ Health Perspect 1997;105 Suppl 4:96570.[Medline]
3 Rao CV, Rivenson A, Katiwalla M, Kelloff GJ, Reddy BS. Chemopreventive effect of oltipraz during different stages of experimental colon carcinogenesis induced by azoxymethane in male F344 rats. Cancer Res 1993;53:25026.[Abstract]
4 Wang JS, Shen X, He X, Zhu YR, Zhang BC, Wang JB, et al. Protective alterations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People's Republic of China. J Natl Cancer Inst
1999;91:34754.
5 Jacobson LP, Zhang BC, Zhu YR, Wang JB, Wu Y, Zhang QN, et al. Oltipraz chemoprevention trial in Qidong, People's Republic of China: study design and clinical outcomes. Cancer Epidemiol Biomarkers Prev 1997;6:25765.[Abstract]
6 Roebuck BD, Liu YL, Rogers AE, Groopman JD, Kensler TW. Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short-term molecular dosimetry. Cancer Res 1991;51:55016.[Abstract]
7 Bolton MG, Munoz A, Jacobson LP, Groopman JD, Maxuitenko YY, Roebuck BD, et al. Transient intervention with oltipraz against aflatoxin-induced hepatic tumorigenesis. Cancer Res 1993;53:3499504.[Abstract]
8 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
1997;51:22533.
9 Bergelson S, Pinkus R, Daniel V. Induction of AP-1 (Fos/Jun) by chemical agents mediates activation of glutathione S-transferase and quinone reductase gene expression. Oncogene 1994;9:56571.[Medline]
10 Venugopal R, Jaiswal AK. Nrf2 and Nrf1 in association with Jun proteins regulate antioxidant response element-mediated expression and coordinated induction of genes encoding detoxifying enzymes. Oncogene 1998;17:314556.[CrossRef][Medline]
11 Huang HC, Nguyen T, Pickett CB. Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor 2. Proc Natl Acad Sci U S A
2000;97:1247580.
12 Moinova HR, Mulcahy RT. An electrophile responsive element (EpRE) regulates beta-naphthoflavone induction of the human gamma-glutamylcysteine synthetase regulatory subunit gene. Constitutive expression is mediated by an adjacent AP-1 site. J Biol Chem
1998;273:146839.
13 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
2000;58:101725.
14 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
2001;59:114756.
15 Kim W, Gates KS. Evidence for thiol-dependent production of oxygen radicals by 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (oltipraz) and 3H-1,2-dithiole-3-thione: possible relevance to the anticarcinogenic properties of 1,2-dithiole-3-thiones. Chem Res Toxicol 1997;10:296301.[CrossRef][Medline]
16 Kwak MK, Egner PA, Dolan PM, Ramos-Gomez M, Groopman JD, Itoh K, et al. Role of phase 2 enzyme induction in chemoprotection by dithiolethiones. Mutat Res 2001;480481:30515.
17 Maheo K, Antras-Ferry J, Morel F, Langouet S, Guillouzo A. Modulation of glutathione S-transferase subunits A2, M1, and P1 expression by interleukin-1 in rat hepatocytes in primary culture. J Biol Chem
1997;272:1612532.
18 Daulhac L, Kowalski-Chauvel A, Pradayrol L, Vaysse N, Seva C. Src-family tyrosine kinases in activation of ERK-1 and p85/p110-phosphatidylinositol 3-kinase by G/CCKB receptors. J Biol Chem
1999;274:2065763.
19 Diehl AM. Roles of CCAAT/enhancer-binding proteins in regulation of liver regenerative growth. J Biol Chem
1998;273:308436.
20 Pimental RA, Liang B, Yee GK, Wilhelmsson A, Poellinger L, Paulson KE. Dioxin receptor and C/EBP regulate the function of the glutathione S-transferase Ya gene xenobiotic response element. Mol Cell Biol 1993;13:436573.[Abstract]
21 Vasiliou V, Puga A, Chang CY, Tabor MW, Nebert DW. Interaction between the Ah receptor and proteins binding to the AP-1-like electrophile response element (EpRE) during murine phase II [Ah] battery gene expression. Biochem Pharmacol 1995;50:205768.[CrossRef][Medline]
22 Mischoulon D, Rana B, Bucher NL, Farmer SR. Growth-dependent inhibition of CCAAT enhancer-binding protein (C/EBP) gene expression during hepatocyte proliferation in the regenerating liver and in culture. Mol Cell Biol
1992;12:255360.[Abstract]
23 Amato SF, Swart JM, Berg M, Wanebo HJ, Mehta SR, Chiles TC. Transient stimulation of the c-Jun-NH2-terminal kinase/activator protein 1 pathway and inhibition of extracellular signal-regulated kinase are early effects in paclitaxel-mediated apoptosis in human B lymphoblasts. Cancer Res 1998;58:2417.[Abstract]
24 Fritz G, Kaina B. Activation of c-Jun N-terminal kinase 1 by UV irradiation is inhibited by wortmannin without affecting c-Jun expression. Mol Cell Biol
1999;19:176874.
25 Mansat-de Mas V, Bezombes C, Quillet-Mary A, Bettaieb A, D'orgeix AD, Laurent G, et al. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin. Mol Pharmacol
1999;56:86774.
26 Zipper LM, Mulcahy RT. Inhibition of ERK and p38 MAP kinases inhibits binding of Nrf2 and induction of GCS genes. Biochem Biophys Res Commun 2000;278:48492.[CrossRef][Medline]
27 Yu R, Lei W, Mandlekar S, Weber MJ, Der CJ, Wu J, et al. Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals. J Biol Chem
1999;274:2754552.
28 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
1998;18:96777.
29 Schreiber E, Harshman K, Kemler I, Malipiero U, Schaffner W, Fontana A. Astrocytes and glioblastoma cells express novel octamer-DNA binding proteins distinct from the ubiquitous Oct-1 and B cell type Oct-2 proteins. Nucleic Acids Res 1990;18:5495503.[Abstract]
30 Nancy V, Wolthuis RM, de Tand MF, Janoueix-Lerosey I, Bos JL, de Gunzburg J. Identification and characterization of potential effector molecules of the Ras-related GTPase Rap2. J Biol Chem
1999;274:873745.
31 Kim SG, Sung M, Kang KW, Kim SH, Son MH, Kim WB. DA-125, a novel anthracycline derivative showing high-affinity DNA binding and topoisomerase II inhibitory activities, exerts cytotoxicity via c-Jun N-terminal kinase pathway. Cancer Chemother Pharmacol 2001;47: 5118.[CrossRef][Medline]
32 Kensler TW, Egner PA, Dolan PM, Groopman JD, Roebuck BD. Mechanism of protection against aflatoxin tumorigenicity in rats fed 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) and related 1,2-dithiol-3-thiones and 1,2-dithiol-3-ones. Cancer Res 1987;47:42717.[Abstract]
33 Mehta RG, Steele V, Kelloff GJ, Moon RC. Influence of thiols and inhibitors of prostaglandin biosynthesis on the carcinogen-induced development of mammary lesions in vitro. Anticancer Res 1991;11:58791.[Medline]
34 Wattenberg LW, Bueding E. Inhibitory effects of 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) on carcinogenesis induced by benzo [a]pyrene, diethylnitrosamine and uracil mustard. Carcinogenesis 1986;7:137981.[Abstract]
35 Kensler TW, Groopman JD, Eaton DL, Curphey TJ, Roebuck BD. Potent inhibition of aflatoxin-induced hepatic tumorigenesis by the monofunctional enzyme inducer 1,2-dithiole-3-thione. Carcinogenesis 1992;13:95100.[Abstract]
36 Buetler TM, Bammler TK, Hayes JD, Eaton DL. Oltipraz-mediated changes in aflatoxin B1 biotransformation in rat liver: implications for human chemointervention. Cancer Res 1996;56:230613.[Abstract]
37 Prochaska HJ, De Long MJ, Talalay P. On the mechanisms of induction of cancer-protective enzymes: a unifying proposal. Proc Natl Acad Sci U S A 1985;82:82326.[Abstract]
38 Talalay P, De Long MJ, Prochaska HJ. Identification of a common chemical signal regulating the induction of enzymes that protect against chemical carcinogenesis. Proc Natl Acad Sci U S A 1988;85:82615.[Abstract]
39 Wattenberg LW. Chemoprevention of cancer. Cancer Res 1985;45:18.[Medline]
40 McMahon M, Itoh K, Yamamoto M, Chanas SA, Henderson CJ, McLellan LI, et al. The Cap`n'Collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes. Cancer Res
2001;61:3299307.
41 Enomoto A, Itoh K, Nagayoshi E, Haruta J, Kimura T, O'Connor T, 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
2001;59:16977.
42 Ramos-Gomez M, Kwak MK, Dolan PM, Itoh K, Yamamoto M, Talalay P, 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 U S A
2001;98:34105.
43 Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, et al. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem
2000;275:160239.
44 Kountouras J, Boura P, Lygidakis NJ. Liver regeneration after hepatectomy. Hepatogastroenterology 2001;48:55662.[Medline]
45 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 2000;164:14.[CrossRef][Medline]
46 Glusker JP, Zacharias DE, Carrell HL. Molecular structures of the chemical carcinogens 7-chloromethylbenz(a)anthracene and 7-chloromethyl-12-methylbenz(a)anthracene. Cancer Res 1976;36:242835.[Abstract]
47 Bauman JW, Goldsworthy TL, Dunn CS, Fox TR. Inhibitory effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on rat hepatocyte proliferation induced by 2/3 partial hepatectomy. Cell Prolif 1995;28:43751.[Medline]
48 Mink S, Haenig B, Klempnauer KH. Interaction and functional collaboration of p300 and C/EBP. Mol Cell Biol
1997;17:660917.[Abstract]
49 Ushio-Fukai M, Alexander RW, Akers M, Yin Q, Fujio Y, Walsh K, et al. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem
1999;274:22699704.
50 Eberhardt W, Huwiler A, Beck KF, Walpen S, Pfeilschifter J. Amplification of IL-1-induced matrix metalloproteinase-9 expression by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-
B and activating protein-1 and involves activation of the mitogen-activated protein kinase pathways. J Immunol
2000;165:578897.
51 Khan KM, Falcone DJ, Kraemer R. Nerve growth factor activation of Erk-1 and Erk-2 induces matrix metalloproteinase-9 expression in vascular smooth muscle cells. J Biol Chem
2002;277:23539.
52 Zanke BW, Boudreau K, Rubie E, Winnett E, Tibbles LA, Zon L, et al. The stress-activated protein kinase pathway mediates cell death following injury induced by cis-platinum, UV irradiation, or heat. Curr Biol 1996;6:60613.[Medline]
Manuscript received March 12, 2002; revised October 12, 2002; accepted November 6, 2002.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |