Activation of CCAAT/enhancer-binding protein ß by 2'-amino-3'-methoxyflavone (PD98059) leads to the induction of glutathione S-transferase A2

Keon Wook Kang, Eun Young Park and Sang Geon Kim1

National Research Laboratory (MDT), College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Korea


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The induction of glutathione S-transferases by flavonoids is associated with cancer chemopreventive effects. We reported that 2'-amino-3'-methoxyflavone (PD98059), an MKK1 inhibitor, induces glutathione S-transferase A2 (rGSTA2). This report comparatively examines the role of CCAAT/enhancer-binding protein (C/EBP) and Nrf2 in the induction of rGSTA2 by PD98059. We first assessed whether the MKK1/ERK1/2 pathway regulated rGSTA2 induction. Northern and western blot analyses showed that PD98059 at the concentrations effective for the inhibition of MKK1 increased the rGSTA2 protein and mRNA levels in H4IIE cells. PD98059 also induced rGSTA2 in cells stably transfected with dominant-negative mutant of MKK1(–), which provided evidence that the inhibition of MKK1/ERK1/2 by PD98059 was not responsible for rGSTA2 induction. Gel shift assay and immunoblot analysis of subcellular fractions revealed that PD98059 caused nuclear translocation of C/EBPß and increased C/EBP DNA binding, which was super-shifted with anti-C/EBPß antibody. Nrf2 was not activated by PD98059. PD98059 increased the luciferase reporter gene activity in cells transfected with the C/EBP-containing -1.65 kb flanking region of the rGSTA2 gene. Deletion of the C/EBP-binding site or over-expression of dominant-negative mutant of C/EBP abolished the reporter gene activity. Flavone, a backbone structure of PD98059, also induced nuclear translocation of C/EBPß and C/EBP-mediated rGSTA2 gene induction. Inhibition of phosphatidylinositol 3-kinase abolished C/EBPß-mediated rGSTA2 induction by PD98059. These results provide evidence that PD98059 and flavone induce nuclear translocation of C/EBPß and activate the C/EBP-binding site in the rGSTA2 gene, which constitutes the distinct pathway for the enzyme induction irrespective of the inhibition of MKK1/ERK activity.

Abbreviations: AhR, Ah receptor; ARE, antioxidant response element; C/EBP, CCAAT/enhancer-binding protein; ERK, extracellular signal-regulated kinase; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; MAP kinase, mitogen-activated protein kinase; MEM, minimal essential medium; PI3-kinase, phosphatidylinositol 3-kinase; SDS, sodium dodecylsulfate; SSC, standard saline citrate; XRE, xenobiotic response element


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The induction of glutathione S-transferases (GST) is associated with cancer chemopreventive and cytoprotective effects. Activation of antioxidant response element (ARE) by reactive oxygens from pro-oxidants plays an important role in the regulation of phase II enzymes including GST (1,2). Signals activated by oxidative stress stimulate transduction of NF-E2-related factor (Nrf) activity and activation of ARE (3,4). The proteins binding to the ARE consensus oligonucleotide involve Nrf proteins and Maf family members (57). Oxidative stress induces rGSTA2 through the activation of ARE, which involves Nrf proteins (6,8).

2'-Amino-3'-methoxyflavone (PD98059) has been extensively used as a selective inhibitor of the serine/threonine-specific protein kinase activity of MKK1 (MEK1). PD98059 served as an inhibitory agent for the pathway involving MKK1/ERK and was found to be useful to identify some of the physiological roles of the cellular signaling pathway (9,10). We previously used PD98059 as an MKK1 inhibitor as part of the studies on the signaling pathways for the induction of phase II detoxifying genes (8,11). In the study, we found that PD98059 per se induced glutathione S-transferase A2 (rGSTA2) (8). Yu et al. reported that PD98059 blocked sulforaphane-induced ERK activation and ARE-mediated reporter activity in cells transiently transfected with a plasmid construct containing an ARE enhancer and that the inhibition of ERK activation by PD98059 diminished the ARE reporter activity (12). Given the wide use of PD98059 as a selective MAP kinase inhibitor and the conflicting reports on the effects of PD98059 on the expression of phase II detoxifying enzymes, we attempted to study the mechanistic basis for the induction of rGSTA2 by PD98059. In the present study, we first assessed whether the MKK1/ERK1/2 pathway regulated the expression of rGSTA2. We observed that the inhibition of the MKK1/ERK1/2 by dominant-negative mutant of MKK1 failed to activate the expression of rGSTA2. Hence, the pathway involving MKK1/ERK, which is inhibited by PD98059, is not likely to be responsible for the induction of rGSTA2.

Transcription factors of CCAAT/enhancer-binding protein (C/EBP) family have roles in the differentiation of cells and exert their function by regulating expression of tissue-specific genes and cell proliferation (13,14). In response to growth stimuli, the members of C/EBP family involved in the transcription of genes include C/EBPß and C/EBP{delta} (15). Also, the truncated C/EBP isoform or the ratio of C/EBP isoforms modulates transcriptional activities. We recently found that activation of C/EBPß and its binding to the C/EBP-response element play a critical role in the induction of the rGSTA2 gene (16). C/EBP constitutes an essential distinct pathway for GST induction. Therefore, we sought to determine the role of C/EBP activation in the rGSTA2 gene expression by PD98059.

The flavonoids are a class of chemicals naturally occurring in fruits and vegetables (17). The flavonoids with the structure of polyphenolic benzo-{gamma}-pyrone may exert common biological functions. Many of the flavonoid compounds have anti-inflammatory, anti-angiogenic and apoptotic effects (1820). Other flavonoids including PD98059, flavone, ß-naphthoflavone and 3'-methoxy-4'-nitroflavone induce a G1 arrest in cell cycle and increase Ah receptor (AhR)-mediated gene expression (21). Flavonoids have attracted attention as potential cancer chemopreventive agents. The possible cancer chemopreventive effects of flavonoids, some of which have proven effective in clinical trials (22), may also result from the modulation of phase II detoxifying enzyme activities as well as the inhibition of CYP1A1/2 (23,24).

This study uses PD98059 and flavone, a backbone flavonoid structure of PD98059, to comparatively evaluate the roles of Nrf2 and C/EBP transcription factors and of ARE and C/EBP DNA-binding regions in the rGSTA2 induction. We now report that the flavonoid compounds promote nuclear translocation of C/EBPß, and increase C/EBPß binding to the C/EBP-binding site, but fail to activate Nrf2 and Nrf2 binding to the ARE. Activation of C/EBPß by PD98059 leads to the induction of the GST gene irrespective of the inhibition of MKK1/ERK1/2 activity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
[{gamma}-32P]ATP (3000 mCi/mmol) was purchased from PerkinElmer Life Science (Arlington Heights, IL). Anti-GST{alpha} antibody was supplied from Detroit R & D (Detroit, MI). Horseradish peroxidase-conjugated rabbit anti-goat IgG was obtained from Pierce Biotechnology (Rockford, IL). Anti-Nrf2 and anti-C/EBP{alpha}/ß antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). These antibodies specifically recognized their respective transcription factors without any cross-reactivity. Anti-phospho ERK1/2 and ERK1/2 antibodies were supplied from New England Biolabs (Beverly, MA). Horseradish peroxidase- or fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG was purchased from Zymed Laboratories (San Francisco, CA). PD98059 and flavone were obtained from Calbiochem (San Diego, CA) and Aldrich chemicals (Milwaukee, WI), respectively. Acrylamide and other reagents in the molecular studies were supplied from Sigma Chemical (St Louis, MO). The plasmid, pGTB-1.65 construct containing rGSTA2-promoter region (-1651 to +66) was kindly provided from Dr C.B.Pickett (Schering-Plough, Kenilworth, NJ). C/EBP-specific dominant-negative expression (AC/EBP) plasmid was a gift from Dr C.Vinson (National Institutes of Health, Bethesda, MD) (25). MKK1 dominant-negative mutant was gifted from Dr N.G.Ahn (Howard Hughes Medical Institute, University of Colorado, Boulder, CO).

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

Preparation of nuclear extracts
Nuclear extracts were prepared essentially according to the previously published method (26). Briefly, the cells in dishes were washed with ice-cold PBS. Cells were then scraped, transferred to microtubes, and allowed to swell after the addition of 100 µl hypotonic buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet P-40, 1 mM dithiothreitol and 0.5 mM phenylmethylsulfonylfluoride. The lysates were incubated for 10 min in ice and centrifuged at 7 200 g for 5 min at 4°C. Pellets containing crude nuclei were resuspended in 50 µl of extraction buffer containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 10 mM dithiothreitol and 1 mM phenylmethylsulfonylfluoride and then incubated for 30 min in ice. The samples were centrifuged at 15 800 g for 10 min to obtain supernatants containing nuclear fractions. Nuclear fractions were stored at -70°C until use.

Immunoblot analysis
After washing the cells twice with sterile PBS, the cells were scraped and sonicated for disruption. Cytosolic fractions were prepared by differential centrifugation. The subcellular preparations were stored at –70°C until use. Sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis and immunoblot analyses were performed according to previously published procedures (27). Cytosolic proteins were separated by 12% gel electrophoresis and electrophoretically transferred to nitrocellulose paper. The nitrocellulose paper was incubated with anti-GST{alpha} antibody, followed by incubation with horseradish peroxidase-conjugated secondary antibody, and developed using ECL® chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, UK) (28). Cells were lysed in the buffer containing 20 mM Tris–Cl (pH 7.5), 1% Triton X-100, 137 mM sodium chloride, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM ß-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonylfluoride and 1 µg/ml leupeptin. Cell lysates were centrifuged at 10 000 g for 10 min to remove debris. Active phosphorylated forms of ERK1/2 were immunochemically assessed in the cell lysates by using the specific antibody. Nuclear Nrf2 and C/EBP{alpha} forms were immunoblotted with the respective specific antibodies.

Gel retardation assay
Double stranded DNA probes containing the rGSTA2 gene ARE and the C/EBP consensus oligonucleotides were used for gel shift analyses after end-labeling of each probe with [{gamma}-32P]ATP and T4 polynucleotide kinase. The sequences of ARE-containing and C/EBP consensus oligonucleotides were (5'-GATCATGGCATTGCACTAGGTGACAAAGCA-3') and (5'-TGCAGATTGCGCAATCTGCA-3'), respectively. The reaction mixtures included 4 ml of 5x binding buffer containing 20% glycerol, 5 mM MgCl2, 250 mM NaCl, 2.5 mM EDTA, 2.5 mM dithiothreitol, 0.25 mg/ml poly dI–dC and 50 mM Tris–Cl (pH 7.5), 10 µg of nuclear extracts and sterile water in a total volume of 20 µl. The reaction mixtures were pre-incubated for 10 min. DNA-binding reactions were carried out at room temperature for 30 min after the addition of 1 µl probe (106 c.p.m.). Specificity of binding was determined by competition experiments, which were carried out by adding a 20-fold excess of unlabeled ARE or C/EBP oligonucleotide to the reaction mixture before the DNA-binding reaction. For super-shift assay, the antibodies (2 µg each) were added to the reaction mixture, and additionally incubated for 1 h at 25°C. Samples were loaded onto 4% polyacrylamide gels at 100 V. The gels were removed, fixed and dried, followed by autoradiography.

Stable plasmid transfection
H4IIE cells were transfected using Transfectam® according to the manufacturer’s instruction (Promega, Madison, WI). Cells were replated 24 h before transfection at a density of 2x106 cells in a 10 cm2 plastic dish. For use in MKK1 dominant-negative mutant [MKK1(-)] vectors, 20 µl of Transfectam® was mixed with 10 µg of each plasmid in 2.5 ml of minimal essential medium (MEM). Cells were transfected by addition of MEM containing each plasmid and Transfectam®, and then incubated at 37°C in a humidified atmosphere of 5% CO2 for 6 h. After addition of 6.25 ml of MEM containing 10% FCS, cells were incubated for an additional 48 h at 37°C and 50 µg/ml of geneticin was added to select the resistant colonies.

Immunocytochemistry
H4IIE cells were grown on Lab-TEK chamber slides® (Nalge Nunc International, Rochester, NY) and incubated in serum-deprived medium for 24 h. Standard immunocytochemical method was used as described previously (29). For immunostaining, cells were fixed in 100% methanol for 20 min and washed three times with PBS. After blocking in 5% bovine serum albumin in PBS for 1 h at room temperature or overnight at 4°C, cells were incubated for 1 h with polyclonal rabbit anti-Nrf2 or anti-C/EBPß antibody in PBS containing 0.5% bovine serum albumin. Cells were incubated with 1:100 dilution of FITC-conjugated goat anti-rabbit IgG after serial washings with PBS. Counter-staining with propidium iodide (PI) verified the location and integrity of nuclei. Stained cells were washed and examined using a laser scanning confocal microscope (Leica TCS NT, Leica Microsystems, Wetzlar, Germany).

Construction of rGSTA2 promoter-luciferase constructs and luciferase assay
Firefly luciferase reporter gene construct pGL-1651 was generated by ligation of the -1.65 promoter region of the rGSTA2 gene. Briefly, the flanking region was generated by PCR amplification using pGTB-1.65 as a template, inserted into pGEM-T (Promega), and subcloned into the XhoI–BglII sites of the pGL3 reporter plasmid (Promega). Chimeric gene constructs pGL-797 and pGL-197 were also prepared using PCR-amplified-deleted rGSTA2 promoter regions. pCMV500 and pCMV-AC/EBP constructs were obtained from Dr C.Vinson. To determine the promoter activity, we used dual-luciferase reporter assay system (Promega). Briefly, cells (7x105 cells/well) were replated in 6-well plates overnight, serum-starved for 6 h, and transiently transfected with each rGSTA2 promoter-luciferase construct and pRL-SV plasmid (Renilla luciferase expression for normalization) (Promega) using Lipofectamine® reagent for 3 h (Life Technologies, Gaithersburg, MD). Transfected cells were incubated in the medium containing 1% FCS for 3 h, and then exposed to PD98059 (30 µM) or flavone (10 µM) for 18 h. Firefly and Renilla luciferase activities in cell lysates were measured using a luminometer (Luminoskan®, Thermo Labsystems, Helsinki, Finland). The relative luciferase activity was calculated by normalizing rGSTA2 promoter-driven firefly luciferase activity to that of Renilla luciferase.

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


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
rGSTA2 induction and ERK inhibition by PD98059 and flavone
PD98059 induced rGSTA2 with the increase in the level of its mRNA (8). Western blot analysis was performed to confirm the extent of rGSTA2 induction by PD98059 in H4IIE cells. A time course study showed that PD98059 (30 µM) induced rGSTA2 with the maximal induction being observed at 24 h. PD98059 increased the level of rGSTA2 protein in a concentration-dependent manner (3–30 µM, 24 h) (Figure 1AGo). Because PD98059 at 30 µM significantly induced rGSTA2, the concentration was chosen in subsequent experiments. We also determined whether flavone, a backbone structure of PD98059, was capable of inducing the enzyme. Flavone induced rGSTA2 with the maximal induction being noted at 3 µM (Figure 1BGo). Anti-rGSTA1/2 antibody preferentially recognized the induction of rGSTA2 because the protein subunit was inducible (28,30).




View larger version (53K):
[in this window]
[in a new window]
 
Fig. 1. The effects of PD98059 or flavone on the expression of rGSTA2 and phosphorylation of ERK1/2. (A) Immunoblot analysis of rGSTA2 in cells treated with PD98059. (B) Immunoblot analysis of rGSTA2 in cells treated with flavone. A representative immunoblot shows the level of rGSTA2 protein in H4IIE cells treated with PD98059 (3–30 µM) or flavone (1–10 µM) for 24 h. Each lane was loaded with 10 µg of cytosolic proteins. The relative rGSTA2 level was assessed by scanning densitometry. Data represent the mean ± SD with three separate experiments (significant as compared with control, *P < 0.05; **P < 0.01; control level = 1). (C) The effects of PD98059 (3–30 µM) or flavone (3–30 µM) on the phosphorylation of ERK1/2. The extents of ERK1/2 activation were assessed by immunoblotting of phosphorylated forms of ERK1/2. Closed and open arrowheads indicate the phosphorylated (p-ERK) and unphosphorylated (ERK) forms of ERK1/2. Data represent the mean ± SD with three separate experiments (significant as compared with control, *P < 0.05; **P < 0.01; phosphorylation of control cells = 100%).

 
PD98059 is frequently used as an inhibitor of MKK1. The inhibition of ERK1/2 phosphorylation by PD98059 in H4IIE cells was confirmed by immunoblot analysis. PD98059 at 30 µM significantly (70%) inhibited constitutive phosphorylation of ERK1/2 at the concentrations effective for the induction of rGSTA2 (Figure 1CGo). Flavone at 30 µM, which is a 10-fold greater concentration than that for rGSTA2 induction, weakly inhibited (20%) phosphorylation of ERK1/2. Thus, it was apparent that the induction of rGSTA2 by flavone was not related with MKK1 inhibition.

Lack of correlation between MKK1 inhibition and GST induction by PD98059
Because the inhibition of MKK1 by PD98059 paralleled rGSTA2 induction, we assessed the effect of ERK1/2 inhibition on rGSTA2 induction. We next determined the effect of PD98059 on the level of rGSTA2 mRNA in cells stably transfected with the vector of MKK1 dominant-negative mutant [MKK1(-)] and compared the extent of an increase in rGSTA2 mRNA with that by a known rGSTA2 inducer ß-naphthoflavone (ß-NF) (31). The level of rGSTA2 mRNA was not increased by MKK1(-) stable transfection alone (Figure 2AGo). Northern blot analysis showed that PD98059 increased the rGSTA2 mRNA level in MKK1(-) cells (12 h) (Figure 2AGo). ß-NF also increased the level of rGSTA2 mRNA in MKK1(-) cells. The ratio of PD98059-inducible to ß-NF-inducible rGSTA2 mRNA levels in MKK1(-) cells was identical to that in control cells. Immunoblot analysis confirmed that phosphorylation of ERK1/2 was notably inhibited in cells stably transfected with the MKK1(-) plasmid (Figure 2BGo). These experiments verified the notion that the inhibition of ERK1/2 activation by PD98059 was not responsible for the induction of rGSTA2.



View larger version (49K):
[in this window]
[in a new window]
 
Fig. 2. Increase in rGSTA2 mRNA by PD98059 in MKK1(-) cells. (A) Northern blot analysis of rGSTA2 mRNA was performed in H4IIE cells or cells stably transfected with MKK1(-). A representative blot shows the level of rGSTA2 mRNA in cells treated with PD98059 (30 µM, 12 h). ß-Naphthoflavone (ß-NF, 30 µM, 12 h) was used as a control. Each lane was loaded with 40 µg of total RNA. (B) The effect of MKK1(-) transfection on ERK1/2 phosphorylation. The extent of ERK phosphorylation was immunoblotted with the lysates (20 µg) of control cells or cells stably transfected with MKK1(-). Closed and open arrowheads indicate phosphorylated (p-ERK) and unphosphorylated (ERK) forms of ERK1/2, respectively.

 
No activation of Nrf2 by PD98059
A previous study from this laboratory showed that oxidative stress (e.g. tert-butylhydroquinone or sulfur amino acid deprivation) activates Nrf2 and increases Nrf2 binding to the ARE at early times (e.g. 3–6 h), which leads to rGSTA2 induction (8,28). We determined whether PD98059 activated Nrf2 binding to the ARE. Gel shift analysis showed that PD98059 failed to increase the band intensity of protein binding to the ARE consensus oligonucleotide (Figure 3AGo, left). Flavone was also inactive in the ARE activation. tert-Butylhydroquinone (30 µM, 6 h), which was used a positive control, increased the band intensity of protein binding to the ARE (Figure 3AGo, middle). Competition experiments using excess ARE or SP-1 oligonucleotide confirmed the specificity of protein binding to the ARE (Figure 3AGo, right). The specificity of Nrf2 binding to the ARE was also verified by immunocompetition analyses, and the gel mobility of the ARE oligonucleotide bound with Nrf2 was determined previously (8,28). Immunocytochemistry and immunoblot analysis of subcellular fractions confirmed that PD98059 failed to cause Nrf2 to translocate into the nucleus (Figure 3B and CGo). The data indicated that the induction of rGSTA2 by PD98059 was not mediated with ARE activation involving nuclear translocation of Nrf2.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 3. The effect of PD98059 or flavone on Nrf2 binding to the ARE. (A) Gel shift analysis of protein binding to the ARE. Nuclear extracts were prepared from H4IIE cells incubated with 30 µM PD98059 or 10 µM flavone for 3 or 6 h and subjected to gel shift analysis. All lanes contained 10 µg of nuclear extracts and 5 ng of radiolabeled ARE oligonucleotide. Arrowheads indicate DNA bound with Nrf2 protein. The band intensity of Nrf2 binding to the ARE was increased by tert-butylhydroquinone (t-BHQ, 30 µM, 6 h) that was used as a positive control. Competition experiments using excess ARE or SP-1 oligonucleotide confirmed the specificity of protein binding to the ARE. (B) Immunocytochemistry of Nrf2 in cells treated with PD98059 (30 µM, 6 h). The same fields were counter-stained with propidium iodide (PI). (C) The level of nuclear Nrf2 protein. Nrf2 was immunoblotted with anti-Nrf2 antibody in the nuclear fraction prepared from cells treated with 30 µM PD98059 or 10 µM flavone for 1–12 h. Results were confirmed by repeated experiments.

 
C/EBP activation by PD98059
We showed previously that a dithiolthione chemopreventive agent activates C/EBPß and that C/EBPß binding to the C/EBP-binding site present in the promoter region of the rGSTA2 gene leads to the enzyme induction (16). We next tested whether PD98059 was capable of activating C/EBP protein. Gel shift assay revealed that C/EBP-binding activity to a radiolabeled C/EBP-binding oligonucleotide began to slightly increase 3 h after treatment of cells with PD98059 (Figure 4AGo, left). A maximal increase was observed at 6 h. C/EBP DNA binding gradually decreased from the maximum at 12 h. Competition experiments using excess amounts of unlabeled C/EBP or SP-1 oligonucleotides (10-fold) confirmed the specificity of C/EBP protein binding. Competition experiments with the highly specific antibodies directed against C/EBPß, C/EBP{alpha} and AhR nuclear translocator-1 (ARNT1) indicated that C/EBP-binding complex comprised of C/EBPß, but not C/EBP{alpha} or ARNT1 (Figure 4AGo, right). Anti-C/EBPß antibody almost completely reduced the band intensity and super-shifted the retarded band. Flavone also increased C/EBPß binding to the C/EBP-binding site (Figure 4AGo, right).



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 4. Activation of C/EBPß by PD98059 or flavone. (A) Gel shift analysis of C/EBP binding to the C/EBP-binding site. Nuclear extracts were prepared from cells treated with 30 µM PD98059 for 1–12 h. Competition experiments using excess C/EBP or SP-1-binding oligonucleotide confirmed the specificity of C/EBP binding. All lanes contained 15 µg of nuclear extracts and 5 ng of radiolabeled C/EBP consensus oligonucleotide. Immunodepletion experiments were carried out by incubating the nuclear extracts (PD98059, 30 µM, 6 h; flavone, 10 µM, 6 h) with the specific polyclonal antibody directed against C/EBPß, C/EBP{alpha} or ARNT1. The closed and open arrowheads indicate shifted and super-shifted (SS) DNA bound with C/EBP protein, respectively. Super-shift analysis was carried out by pre-incubating the nuclear extracts with the respective antibody for 1 h. (B) Immunocytochemistry of C/EBPß. Cells were treated with 30 µM PD98059 or 10 µM flavone for 6 h. Cellular localization of C/EBPß was immunochemically assessed by using anti-C/EBPß antibody. Either PD98059 or flavone induced nuclear translocation of cytoplasmic C/EBPß. The same fields were counter-stained with propidium iodide (PI). (C) Immunoblot analyses of nuclear C/EBPß. Nuclear fractions were obtained from cells treated with PD98059 or flavone for 1–12 h, and the levels of C/EBPß and {alpha} forms in each fraction were determined by immunoblotting with the respective antibodies. Results were confirmed by repeated experiments.

 
Increases in signals obtained in gel shift assays might occur as a result of the nuclear translocation of C/EBPß. Immunocytochemistry verified nuclear translocation of C/EBPß in cells treated with PD98059 or flavone (Figure 4BGo). Whereas C/EBPß was located predominantly in the cytoplasm of control cells, C/EBPß had nuclear localization at 6 h after treatment with PD98059 or flavone. This result indicated that C/EBPß moved into the nucleus. Western blot analysis was conducted to further confirm the translocation of C/EBPß. The levels of C/EBPß in the nuclear fractions were increased 6–12 h after treatment of cells with PD98059 or flavone in H4IIE cells (with a variable increase at 3 h) (Figure 4CGo). The level of nuclear C/EBP{alpha} was weakly changed at the time points. The data support the notion that C/EBPß, but not C/EBP{alpha}, was activated by PD98059 and served as a component actively binding to the C/EBP-binding site.

Analysis of C/EBP-response element in the rGSTA2 promoter
Given the nuclear translocation of C/EBPß and C/EBPß binding to the C/EBP-binding site by PD98059, we then examined whether PD98059 transcriptionally activated the rGSTA2 gene via the C/EBP-response element. Reporter gene assays were performed using H4IIE cells transfected with a mammalian cell expression vector pGL-1651, which contained the luciferase structural gene and -1.65 kb rGSTA2 promoter (Figure 5AGo). Exposure of H4IIE cells, transiently transfected with the plasmid, to PD98059 (30 µM) resulted in a 7.2-fold increase in luciferase activity (Figure 5BGo). Flavone (10 µM) also transactivated the rGSTA2 gene (a 9.3-fold increase) (Figure 5BGo).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5. Analysis of the C/EBP-response element in the rGSTA2 promoter. (A) The size of the flanking insert in each rGSTA2 chimeric gene construct. (B) Induction of luciferase activity by PD98059 or flavone in H4IIE cells transiently transfected with rGSTA2 chimeric gene construct pGL-1651, which contained both C/EBP-response element and ARE. Dual luciferase reporter assay was performed with the lysates obtained from cells co-transfected with the rGSTA2-luciferase gene construct pGL-1651 (firefly luciferase) and pRL-SV (Renilla luciferase) (a ratio of 200:1) after exposure to PD98059 (30 µM) or flavone (10 µM) for 18 h. Activation of the reporter gene was calculated as a relative change to Renilla luciferase activity. Data represented the mean ± SD with three separate experiments (significant as compared with control, **P < 0.01; control level = 1). (C) PD98059- or flavone-inducible luciferase activity of 5' deletion mutants of the rGSTA2 gene. The chimeric gene construct pGL-1651 contained both C/EBP-response element and ARE, whereas pGL-797 had only the ARE. pGL-197 was used as a vector containing the minimal promoter region. The relative luciferase inducibility was obtained from the ratio of PD98059- or flavone-inducible activity to that in untreated cells transfected with each chimeric construct. Data represent the mean ± SD with three separate experiments (significant as compared with pGL-1651, **P < 0.01). (D) Inhibition of PD98059- or flavone-inducible pGL-1651 reporter gene activation by dominant-negative mutant of C/EBP (AC/EBP). Cells were co-transfected with pGL-1651/pRL-SV (200:1) in combination with pCMV-AC/EBP at a ratio of 1:1 and then the luciferase activity was measured 24 h after transfection. In this experiment, transfected cells were incubated for 24 h to ensure sufficient expression of AC/EBP. The experimental value for luciferase activity was expressed as the relative luciferase unit of cell lysates and represented the mean ± SD with three separate experiments (significant as compared with pCMV500 control, **P < 0.01; significant as compared with pCMV500-transfected cells treated with either PD98059 or flavone, {dagger}{dagger}P < 0.01).

 
To precisely define the sequence required for the inducible activity by PD98059, promoter deletion analyses were performed. In pGL-797, the C/EBP sequence was deleted from the promoter sequence (Figure 5AGo). The luciferase reporter gene inducibility to PD98059 or flavone was markedly decreased in cells transfected with pGL-797 (Figure 5CGo). Also, pGL-197, which lacked nucleotides -1651 to -198 of the rGSTA2 gene-flanking region (Figure 5AGo), showed no inducibility in response to PD98059 or flavone (Figure 5CGo). Promoter deletion experiments demonstrated that the regulatory region of the rGSTA2 gene, which responded to PD98059 or flavone, included the C/EBP-response element.

To determine whether C/EBP protein modulated the activation of C/EBP-response element present in the rGSTA2 gene by PD98059, constitutively active C/EBP-specific dominant-negative mutant (AC/EBP) was expressed in combination with pGL-1651 luciferase reporter in H4IIE cells. Expression of dominant-negative mutant C/EBP almost completely inhibited the ability of PD98059 or flavone to stimulate the reporter gene expression (Figure 5DGo). Transfection with pCMV500, which was used as a control vector, allowed pGL-1651 to respond to PD98059 or flavone. The data indicated that dominant-negative mutant of C/EBP restrained activation of C/EBPß and specifically interfered with the induction of rGSTA2 by the flavonoid compounds. Thus, the C/EBP-binding site present in the rGSTA2 gene was indeed responsible for the enzyme induction by PD98059.

Role of phosphatidylinositol 3-kinase (PI3-kinase) in rGSTA2 induction by PD98059
Previously, we reported that PI3-kinase was responsible for nuclear translocation of C/EBPß and subsequent rGSTA2 induction by a dithiolthione chemopreventive agent (16). We next determined whether PI3-kinase also regulated the expression of rGSTA2 by PD98059 (30 µM). Pre-treatment of cells with wortmannin (500 nM) or LY294002 (30 µM) chemical inhibitors of PI3-kinase significantly reduced PD98059-inducible rGSTA2 expression (24 h) (Figure 6AGo). Wortmannin or LY294002 alone did not affect the expression of rGSTA2 (Figure 6AGo). To confirm the role of PI3-kinase in the induction of rGSTA2 by PD98059, the effect of PD98059 on the level of rGSTA2 protein was monitored in cells over-expressing either the p110 catalytic subunit or the p85 regulatory subunit of PI3-kinase (Figure 6BGo). Over-expression of the p85 subunit notably inhibited the PD98059-inducible increase in rGSTA2 protein expression, as compared with that in cells over-expressing the p110 subunit. To further verify the relationship between the PI3-kinase cascade and the activation of C/EBPß as a transcription factor, the level of nuclear C/EBPß was monitored in cells exposed to PD98059 in the presence or absence of PI3-kinase inhibitor. Subcellular fractionation and immunoblot analyses showed that wortmannin or LY294002 inhibited the nuclear translocation of C/EBPß by PD98059 (6 h) (Figure 6CGo). The data provided evidence that the pathway of PI3-kinase controlled the activation of C/EBPß and C/EBP-mediated rGSTA2 gene expression.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 6. The role of PI3-kinase in the C/EBP-mediated rGSTA2 induction. (A) The effects of PI3-kinase inhibitors on the induction of rGSTA2. Expression of rGSTA2 subunit was measured in cells treated with PD98059 (30 µM) in the presence or absence of wortmannin (WO, 500 nM) or LY294002 (LY, 30 µM) for 24 h. Western blot analyses were performed, as described in Figure 1AGo. The levels of rGSTA2 were also determined in cells treated with wortmannin or LY294002 alone for 24 h. Relative rGSTA2 protein levels were assessed by scanning densitometry. Data represent the mean ± SD with three separate experiments (significant as compared with control, **P < 0.01; significant as compared with PD98059, {dagger}{dagger}P < 0.01; control level = 1). (B) The effect of PD98059 on the expression of rGSTA2 in H4IIE cells stably transfected with an over-expression vector of the p110 catalytic subunit [p110(+)] or the p85 regulatory subunit [p85(+)] of PI3-kinase. The cells were incubated with PD98059 for 24 h. The expression of rGSTA2 was measured by immmunoblot analysis. (C) PI3-kinase-mediated nuclear translocation of C/EBPß by PD98059. Nuclear fractions obtained from cells treated with PD98059 in the presence or absence of wortmannin or LY294002 for 6 h were subjected to immunoblot analysis of C/EBPß. The levels of nuclear C/EBPß were also determined in cells treated with wortmannin or LY294002 alone for 6 h. Results were confirmed by repeated experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
PD98059, which binds to MKK1 and prevents activation of downstream kinases (32,33), has been used as an inhibitory agent for the pathway involving MKK1 and ERK. The compound has selectivity for MKK1, but has no or minimal inhibitory activities against ERK1/2. No activity has been noted for the inhibition of the related pathways of p38 kinase and c-Jun N-terminal kinase (33). Thus, PD98059 has been used as a tool to identify some of the physiological roles of the cellular signaling pathway in the expression of drug metabolizing enzymes. Flavonoids, including PD98059, have other biological activities. Many flavonoids are ligands of the AhR. Thus, treatment of cells with PD98059 prior to the addition of 2,3,7,8-tetrachlorodibenzo-p-dioxin suppressed the accumulation of CYP1A1, CYP1B1 and NQO1 mRNAs (34). Some flavonoids cause cell cycle arrest in AhR-independent manner (35).

PD98059 has been used as an MKK1 inhibitor in dissecting the signaling pathways for the expression of phase II enzymes including NADPH:quinone oxidoreductase, GST, {gamma}-glutamyl cysteine synthetase and heme oxygenase-1. Our previous (8) and present studies showed that PD98059 induced rGSTA2 with the increase in its mRNA. Because rGSTA2 was also induced in cells stably transfected with dominant-negative mutant of MKK1 [MKK1(-)], the inhibitory effect of PD98059 on the activity of MKK1 appeared not to be responsible for the enzyme induction. In addition, flavone a backbone structure of PD98059 induced rGSTA2 at a concentration of 3–10 µM. At the concentrations effective for the induction of rGSTA2, flavone weakly inhibited phosphorylation of ERK1/2. This also supports the notion that PD98059 and flavone induce rGSTA2 irrespective of the inhibition of MKK1/ERK1/2 activity.

Binding of Nrf2 protein to the ARE plays a critical role in the induction of phase II detoxifying enzymes, and lack of Nrf2 expression increases sensitivity to xenobiotics (36,37). We recently reported that nuclear translocation of Nrf2 was mediated with actin rearrangement in response to oxidative stress (38). In contrast to the nuclear translocation of Nrf2 and Nrf2 binding to the ARE by proxidants (i.e. tert-butylhydroquinone), PD98059 failed to stimulate Nrf2 translocation and subsequently to activate the ARE present in the upstream region of the rGSTA2 gene. This implies that other mechanism(s) besides Nrf2 binding to the ARE are involved in the induction of rGSTA2 by PD98059. In the present study, we demonstrate that PD98059 induces nuclear translocation of C/EBPß, but not C/EBP{alpha}, and stimulates C/EBPß binding to the C/EBP-response element in the rGSTA2 gene. Activation of C/EBPß by PD98059 preceded the induction of rGSTA2, which in combination with the results of luciferase reporter gene and promoter deletion analyses support the essential role of C/EBPß translocation and C/EBPß binding to the C/EBP-binding site in the induction of rGSTA2. The crucial role of the C/EBP-response element in the induction of rGSTA2 by PD98059 was further evidenced by the experiment using the dominant-negative mutant of C/EBP in the reporter gene assay.

Our observation that PD98059 can stimulate nuclear translocation of C/EBPß and increase C/EBPß-binding activity to the C/EBP consensus oligonucleotide holds a significant implication for the finding of C/EBPß as an important transcription factor for rGSTA2 induction by PD98059. N-terminal transactivation domains of C/EBPß interact with CBP/p300 co-activator, which may be critical for C/EBPß transactivation (39). Thus, activating C/EBPß binding to the C/EBP-binding site would induce its cooperative interaction with CBP/p300 co-activator for transactivation of the rGSTA2 gene.

Previously, we and another group showed that PI3-kinase was responsible for nuclear translocation of Nrf2 (8,28,40). In particular, oxidative stress (e.g. glutathione depletion, tert-butylhydroquinone) increases the activities of PI3-kinase and Akt and that the pathway of PI3-kinase regulated translocation of Nrf2 to the nucleus (8,28). The PI3-kinase signaling pathway regulates rearrangement of actin microfilaments in response to oxidative stress and depolymerization of actin causes a complex of Nrf2 bound with actin to translocate into the nucleus (38). The PI3-kinase pathway also controls nuclear translocation of C/EBPß in response to a dithiolthione chemopreventive agent (16). In the present study, we further revealed that the signaling pathway of PI3-kinase regulated C/EBPß-mediated rGSTA2 induction by PD98059. The PI3-kinase-dependent C/EBPß-mediated induction of rGSTA2 by flavonoids has an implication for the finding of the distinct pathway of rGSTA2 induction, which differs from that of Nrf2-mediated enzyme induction by pro-oxidants. The potential cancer chemoprevention through dietary flavonoids may result from C/EBPß-mediated transcriptional activation of the phase II enzyme.

Chemical inhibition of PI3-kinase or transfection of the p85 subunit of PI3-kinase did not completely inhibit the induction of rGSTA2 by PD98059, suggesting that activation of other transcription factor(s), which is(are) regulated by other signaling pathways besides the PI3-kinase pathway, may also be involved in the enzyme induction. It has been shown that PD98059 or other flavonoids serve as agonists or antagonists of AhR (34). The C/EBP-binding sites in the phase II enzyme genes including the rGSTA2 gene are present within or closely proximal to the XRE. Thus, we cannot exclude the possibility that binding of PD98059 to AhR and subsequent interaction of the ligand-bound AhR or other proteins with the XRE be involved in the expression of rGSTA2.

In summary, PD98059 induces nuclear translocation of C/EBPß and activates the C/EBP-response element in the rGSTA2 gene, which constitutes the distinct pathway for the enzyme induction irrespective of their inhibition of MKK1/ERK activity. Also, the fact that both PD98059 and flavone activate C/EBP brings us to the attention that the pharmacological intervention of the MAP kinase pathway by PD98059 and other flavonoid inhibitors may skew our view of dissecting signaling pathway. PD98059 may not be pertinent as an MKK1 inhibitor in the situations in which the activation of C/EBP is influenced.


    Notes
 
1 To whom correspondence should be addressed Email: sgk{at}snu.ac.kr Back


    Acknowledgments
 
The kind donation of pCMV500 and AC/EBP plasmids from Dr C.Vinson and the constructs containing rGSTA2-promoter region from Dr C.B.Pickett are gratefully acknowledged. This work was supported by National Research Laboratory Program (2001), KISTEP, The Ministry of Science and Technology, Republic of Korea.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Nguyen,T. and Pickett,C.B. (1992) Regulation of rat glutathione S-transferase Ya subunit gene expression. DNA-protein interaction at the antioxidant responsive element. J. Biol. Chem., 267, 13535–13539.[Abstract/Free Full Text]
  2. Liu,S. and Pickett,C.B. (1996) The rat liver glutathione S-transferase Ya subunit gene: Characterization of the binding properties of a nuclear protein from HepG2 cells that has high affinity for the antioxidant response element. Biochemistry, 35, 11517–11521.[CrossRef][ISI][Medline]
  3. Itoh,K., Ishii,T., Wakabayashi,N. and Yamamoto,M. (1999) Regulatory mechanisms of cellular response to oxidative stress. Free. Radic. Res., 31, 319–324.[ISI][Medline]
  4. Ishii,T., Itoh,K., Takahashi,S., Sato,H., Yanagawa,T., Katoh,Y., Bannai,S. and Yamamoto,M. (2000) Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J. Biol. Chem., 275, 16023–16029.[Abstract/Free Full Text]
  5. Bergelson,S., Pinkus,R. and Daniel,V. (1994) Induction of AP-1 (Fos/Jun) by chemical agents mediates activation of glutathione S-transferase and quinone reductase gene expression. Oncogene, 9, 565–571.[ISI][Medline]
  6. Wasserman,W.W. and Fahl,W.E. (1997) Functional antioxidant responsive elements. Proc. Natl Acad. Sci. USA, 94, 5361–5366.[Abstract/Free Full Text]
  7. Itoh,K., Chiba,T., Takahashi,S. et al. (1997) An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem. Biophys. Res. Commun., 236, 313–322.[CrossRef][ISI][Medline]
  8. Kang,K.W., Cho,M.K., Lee,C.H. and Kim,S.G. (2001) Activation of phosphatidylinositol 3-kinase and Akt by tert-butylhydroquinone (t-BHQ) is responsible for antioxidant response element-mediated rGSTA2 induction in H4IIE cells. Mol. Pharmacol., 59, 1147–1156.[Abstract/Free Full Text]
  9. Park,H.J., Kim,B.C., Kim,S.J. and Choi,K.S. (2002) Role of MAP kinases and their cross-talk in TGFß1-induced apoptosis in FaO rat hepatoma cell line. Hepatology, 35, 1360–1371.[ISI][Medline]
  10. Lee,S.E., Chung,W.J., Kwak,H.B., Chung,C.H., Kwack,K.B., Lee,Z.H. and Kim,H.H. (2001) Tumor necrosis factor-{alpha} supports the survival of osteoclasts through the activation of Akt and ERK. J. Biol. Chem., 276, 49343–49349.[Abstract/Free Full Text]
  11. Kang,K.W., Novak,R.F., Lee,C.H. and Kim,S.G. (2002) Induction of microsomal epoxide hydrolase by sulfur amino acid deprivation via the pathway of c-Jun N-terminal kinase and its extracellular exposure during cell death. Free Radic. Biol. Med., 32, 1017–1032.[CrossRef][ISI][Medline]
  12. Yu,R., Lei,W., Mandlekar,S., Weber,M.J., Der,C.J., Wu,J. and Kong,A.N.T. (1999) Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals. J. Biol. Chem., 274, 27545–27552.[Abstract/Free Full Text]
  13. Kountouras,J., Boura,P. and Lygidakis,N.J. (2001) Liver regeneration after hepatectomy. Hepatogastroenterology, 48, 556–562.[ISI][Medline]
  14. Rastegar,M., Lemaigre,F.P. and Rousseau,G.G. (2000) Control of gene expression by growth hormone in liver: key role of a network of transcription factors. Mol. Cell. Endocrinol., 164, 1–4.[CrossRef][ISI][Medline]
  15. Liao,J., Piwien-Pilipuk,G., Ross,S.E., Hodge,C.L., Sealy,L., MacDougald,O.A. and Schwartz,J. (1999) CCAAT/enhancer-binding protein beta (C/EBPß) and C/EBP{delta} contribute to growth hormone-regulated transcription of c-fos. J. Biol. Chem., 274, 31597–31604.[Abstract/Free Full Text]
  16. Kang,K.W., Cho,I.J., Lee,C.H. and Kim,S.G. (2003) Essential role of phosphatidylinositol 3-kinase-dependent CCAAT/enhancer binding protein beta activation in the induction of glutathione-S-transferase by oltipraz. J. Nat. Cancer. Inst., 95, 53–66.[Abstract/Free Full Text]
  17. Hollman,P.C. and Katan,M.B. (1999) Health effects bioavailability of dietary flavonols. Free Radic. Res., 31, S75–S80.[ISI][Medline]
  18. Manthey,J.A. (2000) Biological properties of flavonoids pertaining to inflammation. Microcirculation, 7, S29–S34.[ISI][Medline]
  19. Kobayashi,S., Miyamoto,T., Kimura,I. and Kimura,M. (1995) Inhibitory effect of isoliquiritin, a compound in licorice root, on angiogenesis in vivo and tube formation in vitro. Biol. Pharm. Bull., 18, 1382–1386.[ISI][Medline]
  20. Wenzel,U., Kuntz,S., Brendel,M.D. and Daniel,H. (2000) Dietary flavone is a potent apoptosis inducer in human colon carcinoma cells. Cancer Res., 60, 3823–3831.[Abstract/Free Full Text]
  21. Reiners,J.J. Jr, Clift,R. and Mathieu,P. (1999) Suppression of cell cycle progression by flavonoids: dependence on the aryl hydrocarbon receptor. Carcinogenesis, 20, 1561–1566.[Abstract/Free Full Text]
  22. Ferry,D.R., Smith,A., Malkhandi,J., Fyfe,D.W., deTakats,P.G., Anderson,D., Baker,J. and Kerr,D.J. (1996) Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and evidence for in vivo tyrosine kinase inhibition. Clin. Cancer Res., 2, 659–668.[Abstract]
  23. Uda,Y., Price,K.R., Williamson,G. and Rhodes,M.J. (1997) Induction of the anticarcinogenic marker enzyme, quinone reductase, in murine hepatoma cells in vitro by flavonoids. Cancer Lett., 120, 213–216.[CrossRef][ISI][Medline]
  24. Sousa,R.L. and Marletta,M.A. (1985) Inhibition of cytochrome P-450 activity in rat liver microsomes by the naturally occurring flavonoid, quercetin. Arch. Biochem. Biophys., 240, 345–357.[ISI][Medline]
  25. Ahn,S., Olive,M., Aggarwal,S., Krylov,D., Ginty,D.D. and Vinson,C. (1998) A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus-dependent transcription of c-fos. Mol. Cell. Biol., 18, 967–977.[Abstract/Free Full Text]
  26. Schreiber,E., Harshman,K., Kemler,I., Malipiero,U., Schaffner,W. and Fontana,A. (1990) 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., 18, 5495–5503.[Abstract]
  27. Kim,S.G., Nam,S.Y., Kim,J.H., Cho,C.K. and Yoo,S.Y. (1997) Enhancement of radiation-inducible hepatic glutathione S-transferase Ya1, Yb1, Yb2, Yc1, and Yc2 expression by oltipraz: possible role in radioprotection. Mol. Pharmacol., 51, 225–233.[Abstract/Free Full Text]
  28. Kang,K.W., Ryu,J.H. and Kim,S.G. (2000) The essential role of phosphatidylinositol 3-kinase and of p38 mitogen-activated protein kinase activation in the antioxidant response element-mediated rGSTA2 induction by decreased glutathione in H4IIE hepatoma cells.Mol. Pharmacol., 58, 1017–1025.[Abstract/Free Full Text]
  29. Cho,M.K., Suh,S.H. and Kim,S.G. (2002) JunB/AP-1 and NF-{kappa}B-mediated induction of nitric oxide synthase by bovine type I collagen in serum-stimulated murine macrophages. Nitric Oxide, 6, 319–332.[CrossRef][ISI][Medline]
  30. Pickett,C.B., Telakowski-Hopkins,C.A., Argenbright,L. and Lu,A.Y. (1984) Regulation of glutathione S-transferase mRNAs by phenobarbital and 3-methylcholanthrene: analysis using cDNA probes. Biochem. Soc. Trans., 12, 71–74.[ISI][Medline]
  31. Rushmore,T.H., King,R.G., Paulson,K.E. and Pickett,C.B. (1990) Regulation of glutathione S-transferase Ya subunit gene expression: identification of a unique xenobiotic-responsive element controlling inducible expression by planar aromatic compounds. Proc. Natl Acad. Sci. USA, 87, 3826–3830.[Abstract]
  32. Servant,M.J., Giasson,E. and Meloche,S. (1996) Inhibition of growth factor-induced protein synthesis by a selective MEK inhibitor in aortic smooth muscle cells. J. Biol. Chem., 271, 16047–16052.[Abstract/Free Full Text]
  33. Davies,S.P., Reddy,H., Caivano,M. and Cohen,P. (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J., 351, 95–105.[CrossRef][ISI][Medline]
  34. Reiners,J.J. Jr, Lee,J.Y., Clift,R.E., Dudley,D.T. and Myrand,S.P. (1998) PD98059 is an equipotent antagonist of the aryl hydrocarbon receptor and inhibitor of mitogen-activated protein kinase kinase. Mol. Pharmacol., 53, 438–445.[Abstract/Free Full Text]
  35. Reiners,J.J. Jr, Clift,R. and Mathieu,P. (1999) Suppression of cell cycle progression by flavonoids: dependence on the aryl hydrocarbon receptor. Carcinogenesis, 20, 1561–1566.[Abstract/Free Full Text]
  36. McMahon,M., Itoh,K., Yamamoto,M., Chanas,S.A., Henderson,C.J., McLellan,L.I., Wolf, C.R., Cavin,C. and Hayes,J.D. (2001) 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., 61, 3299–3307.[Abstract/Free Full Text]
  37. Enomoto,A., Itoh,K., Nagayoshi,E., Haruta,J., Kimura,T., O’Connor,T., Harada,T. and Yamamoto,M. (2001) High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes. Toxicol. Sci., 59, 169–177.[Abstract/Free Full Text]
  38. Kang,K.W., Lee,S.J., Park,J.W. and Kim,S.G. (2002) Phosphatidylinositol 3-kinase regulates nuclear translocation of NF-E2-related factor 2 through actin rearrangement in response to oxidative stress. Mol. Pharmacol., 62, 1001–1010.[Abstract/Free Full Text]
  39. Mink,S., Haenig,B. and Klempnauer,K.H. (1997) Interaction and functional collaboration of p300 and C/EBPß. Mol. Cell. Biol., 17, 6609–6617.[Abstract]
  40. Lee,J.M., Hanson,J.M., Chu,W.A. and Johnson,J.A. (2001) Phosphatidylinositol 3-kinase, not extracellular signal-regulated kinase, regulates activation of the antioxidant-responsive element in IMR-32 human neuroblastoma cells. J. Biol. Chem., 276, 20011–20016.[Abstract/Free Full Text]
Received November 19, 2002; revised November 26, 2002; accepted December 6, 2002.