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J. Biol. Chem., Vol. 278, Issue 32, 29693-29700, August 8, 2003
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From the Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029
Received for publication, May 6, 2003 , and in revised form, May 23, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mitogen-activated protein kinases (MAPKs) are components of signaling cascades that respond to extracellular stimuli by targeting transcription factors, resulting in the modulation of gene expression. Three major MAP kinase subfamilies have been described; they are extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase, and p38. ERK was the first cascade to be elucidated; it is activated by mitogens and is primarily involved in the regulation of cell growth and proliferation. The remaining two cascades respond to cellular stress signals and are, hence, sometimes referred to as stress-activated protein kinases. There is a good deal of overlap among the cascades both in their targets and upstream activators. The signal transduction pathways that regulate HO-1 gene activation under the multiplicity of inducing conditions remain only partially understood. ERK and/or p38 MAPK have been reported to participate in the activation of the HO-1 gene by inducing xenobiotics (9–10).
Cytochrome P450 2E1 (CYP2E1), the ethanol-inducible form, metabolizes and activates many toxicologically important substrates including ethanol, carbon tetrachloride, acetaminophen, and N-nitrosodimethylamine to more toxic products (11–13). Induction of CYP2E1 by ethanol is one of the central pathways by which ethanol generates a state of oxidative stress in hepatocytes. To study the biochemical and toxicological effects of CYP2E1 induction, our laboratory established a HepG2 cell line that constitutively overexpresses CYP2E1 (E47 cells) or a control HepG2 cell line transfected with the empty vector (C34 cells) (14). CYP2E1-dependent toxicity in the presence of ethanol, arachidonic acid (AA), and AA plus iron has been characterized in these cell lines; these agents did not produce significant toxicity in C34 cells, whereas their addition to E47 cells decreased cell viability and caused either necrosis or apoptosis (14–17). Toxicity by these agents was enhanced when cellular glutathione (GSH) levels were lowered by treatment with L-buthionine-(S,R)-sulfoximine (BSO). Moreover, treatment of E47 cells with only BSO, an irreversible inhibitor of glutamate-cysteine ligase, resulted in apoptosis and necrosis (18), whereas no toxicity was found in C34 cells or HepG2 cells that expressed CYP3A4 instead of CYP2E1. Antioxidants such as GSH-ethyl ester and N-acetylcysteine partially prevented the apoptosis and necrosis after BSO treatment, whereas diallyl sulfide, a CYP2E1 inhibitor, was fully protective.
In response to the increased oxidative stress caused by induction of CYP2E1 activity in hepatocytes, antioxidant enzymes such as glutamate-cysteine ligase, catalase, and glutathione S-transferase were found to be up-regulated (19–20). In this study, we present data showing that HO-1 is transcriptionally activated in HepG2 cells overexpressing CYP2E1 and that the ERK MAPK pathway plays an important role in mediating this activation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40 g) and male
Sprague-Dawley rats (150 170 g) were housed in a facility approved by
The American Association for Accreditation of Laboratory Animal Care. Mice
were fed with commercially available ethanol (35%) and control diets
(Bio-Serv, Frenchtown, NJ), which were equicaloric and had the same
composition with respect to fat (42% of calories) and protein (16% of
calories) for 59 days. To induce liver CYP2E1, rats were injected
intraperitoneally with pyrazole, 200 mg/kg body weight, once per day for 2
days followed by overnight fast
(21). Cell Culture and Transfection Experiments—This study was performed using HepG2 cells, which constitutively express CYP2E1 (E47 cells) or control HepG2 cells (C34 cells), which have undetectable P450 activity (14). Cells were cultured in minimal essential medium containing 10% fetal bovine serum and 0.5 mg/ml of G418 supplemented with 100 units/ml of penicillin and 100 µg/ml of streptomycin and 2 mM L-glutamine in a humidified atmosphere in 5% CO2 at 37 °C. The expression of CYP2E1 was routinely monitored by assaying for the rate of oxidation of p-nitrophenol as described below. For each experiment, cells were plated and incubated in minimal essential medium overnight, the culture medium was replaced with fresh medium, and the different treatments were added. Plasmid pHHO-1 containing human HO-1 cDNA and pHHOSV15luc containing a 15-kilobase upstream promoter region of human HO-1 gene linked to a luciferase reporter gene were generous gifts from Dr. S. Shibahara (Tohoku University School of Medicine, Sendai, Japan). The CYP2E1 containing plasmid (pCI-2E1) and the empty vector pCI-neo have been previously described (22). The pRL-null plasmid was from Promega (Madison, WI). Transfections were carried out using FuGENE 6 (Roche Applied Science) as the DNA carrier.
General Methodology—CYP2E1 activity in microsomes (23) was measured by studying the oxidation of p-nitrophenol to p-nitrocatechol as described previously. HO activity was determined by detecting bilirubin production from heme oxidation by isolated microsomes (24). ROS was determined by flow cytometry with 2',7'-dichlorofluorescin-diacetate (25). Luciferase activity was determined using the dual luciferase reporter assay system (Promega).
Western Blotting—HO-1, HO-2, and CYP2E1 protein expression were detected by Western blotting as described previously (26). Each sample containing 30 µg of total protein was loaded on a 12% denaturing polyacrylamide gel and electroblotted onto 0.2-µm nitrocellulose membranes. Protein concentration was determined using the Protein DC-20 assay kit (Bio-Rad). Protein immunoblot analysis was carried out using the following: anti-human HO-1 (1:5000) and anti-rat HO-1 (1:5000) monoclonal antibody, anti-human HO-2 polyclonal antibody (1:2000) (StressGen Biotech, Victoria, BC, Canada); anti-human CYP2E1 polyclonal antibody (1:30,000) (kindly provided by Dr. J. M. Lasker, Hackensack Biomedical Research Institute, NJ); ERK, p38, c-Jun N-terminal kinase rat polyclonal antibody (1:1000) or phosphorylated ERK, p38, and c-Jun N-terminal kinase mouse monoclonal antibody (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA) as primary antibody; horseradish peroxidase-conjugated goat anti-mouse IgG (1:4000) or goat anti-rabbit IgG (1:10,000) (Sigma) as secondary antibody. Blots were developed using the enhanced chemiluminescence immunoblot-detecting reagent (Amersham Biosciences).
Northern Blotting—Total RNA was isolated using the TRIzol reagent (Invitrogen). 5 µg of RNA were electrophoresed under denaturing conditions in 0.9% agarose/formaldehyde gels, transferred onto nylon membranes, and hybridized to random-primed 32P-labeled HO-1 or ribosomal S3 cDNA probes as described previously (27). HO-1 cDNA was produced by digesting the plasmid pHHO1 with XhoI and XbaI.
Statistics—Data are presented as average ± S.E. (n = 3). Student's t test for unpaired data was used to evaluate the differences between the compared groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40 g) were fed
with a control diet or a diet containing 35% ethanol for 59 days. CYP2E1 and
HO-1 protein expression in liver was detected by Western blotting. CYP2E1
protein was significantly increased in mice fed ethanol compared with control
mice (4-fold, p < 0.001). Although HO-1 protein could not be
detected in the liver of control mice, it was detectable in mice fed ethanol
(Fig. 1A).
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Induction of CYP2E1 in Vivo by Pyrazole Increases HO-1 Protein Expression in Rat Liver—Pyrazole induces CYP2E1 by stabilizing the protein against degradation. Increased CYP2E1 activity results in increased oxidative stress (16, 28). To evaluate whether there were any changes in HO-1 levels by the increased CYP2E1 activity in hepatocytes, rats were treated with 0.9% NaCl (control) or pyrazole (200 mg/kg of body weight/day for 2 days followed by an overnight fast). After sacrificing the rats, the livers were removed, and CYP2E1 and HO-1 content were determined by Western blotting. CYP2E1 protein was increased about 5.6-fold, whereas HO-1 protein was induced about 3.2-fold in the liver of pyrazole-treated rats compared with the saline-treated rats (Fig. 1B).
Overexpression of CYP2E1 in HepG2 Cells Induces HO-1 Protein and HO Activity—To determine whether there is any link between the induction of CYP2E1 and HO-1 levels, HepG2 cells stably transfected with CYP2E1 (E47 cells) or with the empty vector (C34 cells) were used. HepG2 cells transfected with CYP3A4 (3A4 cells), another isoform of the cytochrome P450 family, were also studied as a parallel control. HO-2, the constitutive isoform of HO, was unchanged by expression of either CYP2E1 or CYP3A4 (Fig. 2A). HO-1 protein and HO activity were low in the control C34 and 3A4 cells (Fig. 2, A and B), which have undetectable CYP2E1 protein levels (Fig. 2A) and very low basal CYP2E1 activity (Fig. 2C). However, HO-1 protein and HO activity were increased (about 2.2- and 2.1-fold, respectively) in E47 cells (Fig. 2, A and B), which have high levels of CYP2E1 protein (Fig. 2A) and high CYP2E1 activity (Fig. 2C), suggesting a potential association between increased CYP2E1 expression and induction of HO-1 protein and activity in HepG2 cells.
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HO-1 Gene Is Transcriptionally Activated by CYP2E1-Overexpression—Because HO-1 protein was increased in HepG2 cells overexpressing CYP2E1, we examined whether such an increase was also present at the mRNA level. HO-1 mRNA was increased about 2-fold in E47 cells but not in 3A4 cells when compared with the control C34 cells (Fig. 3A), suggesting the possibility that CYP2E1 overexpression affects HO-1 gene expression at the transcription level. Increased mRNA levels may be because of an increase in transcription or to an increase in stability. To study whether this increase of HO-1 mRNA involves changes in mRNA stability, actinomycin D (10 µg/ml), a general inhibitor of mRNA transcription, was used to block HO-1 gene transcription, and the decline in the remaining HO-1 mRNA levels was followed as a function of time after the addition of actinomycin D. As shown in Fig. 3, B–C, the degradation rate of HO-1 mRNA in E47 cells was similar to that in C34 cells (p > 0.05 at all time points, half-life about 8 h), suggesting that increased HO-1 mRNA expression in E47 cells is not due to increased stability of the HO-1 mRNA. Transient transfection studies were carried out with a HO-1 promoter construct (pHHO15SVluc) that contained the 15-kilobase upstream region of the human HO-1 gene (29). Luciferase activity in E47 cells transfected with pHHO15SVluc was 1.8-fold higher (p < 0.05) than that in control C34 cells transfected with the same construct (Fig. 4A), an increase similar to the HO-1 mRNA levels (Fig. 3A). To rule out changes in clonal selection as an explanation for the increase in HO-1, the C34 cells were transfected with CYP2E1. Luciferase activity of C34 cells co-transfected with CYP2E1 (pCI-2E1) and pHHO15SVluc was 3.4-fold higher than that of C34 cells co-transfected with the empty vector pCI-neo and pHHO15SVluc (Fig. 4B), validating that CYP2E1 expression enhances HO-1 expression. Taken together, these data suggest that the HO-1 gene is transcriptionally activated by overexpression of CYP2E1 in HepG2 cells.
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Inhibitors of CYP2E1 Activity Prevent the Increase of ROS Production as Well as HO-1 Induction in E47 Cells—Increased CYP2E1 activity has been shown to increase ROS production (16, 28). To examine whether the induction of HO-1 in E47 cells was caused by an increase in ROS production, the effect of CYP2E1 inhibitors such as diallyl sulfide (DAS) (30), 4-methylpyrazole (4-MP) (31), and dimethyl sulfoxide (Me2SO) (32) on HO-1 induction was determined. ROS production was assayed by 2',7'-dichlorofluorescin fluorescence. E47 cells showed an elevated basal ROS production (1.8-fold, p < 0.05) compared with control C34 cells (Fig. 5A). Incubation with 1 mM DAS, 5 mM 4-MP, or 211 mM Me2SO for 3 days significantly inhibited the increase in ROS production in E47 cells (from 1.8-fold to 1.2-, 1.1-, and 0.7-fold, respectively), whereas these compounds did not significantly affect ROS production in C34 cells (p > 0.05) (Fig. 5A).
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In a similar manner to the inhibition of CYP2E1 activity and decrease in ROS production, HO-1 induction in E47 cells was also prevented by these inhibitors. Incubation of E47 cells with 1 mM DAS, 5 mM 4-MP, or 211 mM Me2SO for 3 days significantly inhibited the HO-1 mRNA induction in E47 cells (from 2.2-fold to 1.2-, 0.8-, and 0.7-fold, respectively) when compared with control C34 cells (Fig. 5, B–C). E47 cells also had an elevated HO-1 protein level (about 3-fold) compared with control C34 cells in the absence of any addition (Fig. 6, A–B). This induction of HO-1 protein was also prevented by DAS, 4-MP, or Me2SO (from 3.2-fold to 1.58-, 0.65-, and 0.70-fold, respectively) compared with control C34 cells. These three inhibitors did not show significant effects on HO-1 protein expression in C34 cells (Fig. 6, A–B), validating their specificity for effects on CYP2E1.
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Catalase Inhibits the Induction of HO-1 by CYP2E1
Overexpression—Increased CYP2E1 activity by overexpressing CYP2E1
increases production of ROS such as H2O2,
,
and lipid peroxidation end products
(14,
32). Catalase effectively
decomposes hydrogen peroxide, whereas vitamin E, the principal lipid-soluble
antioxidant in biological tissues, is widely used to prevent the onset of cell
damage due to the induction of lipid peroxidation
(33). To further characterize
the role of ROS in the mechanism of HO-1 induction by CYP2E1, 5000
units/ml catalase or 100 µM vitamin E were added to the culture
medium for 48 h. Both the increases in HO-1 mRNA and protein in E47 cells were
almost completely inhibited by catalase (93 and 89%, respectively), whereas
vitamin E had no effect (Fig. 7,
A–B), suggesting a potential role for hydrogen
peroxide in the induction of the HO-1 gene by CYP2E1
overexpression.
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ERK MAPK Is Activated by Overexpression of CYP2E1—To determine the role, if any, of MAPKs in HO-1 gene activation by overexpression of CYP2E1, we first examined the effect of overexpression of CYP2E1 on MAPK activities. MAPKs are activated by dual phosphorylation of threonine and tyrosine residues located in the "activation lip" of the conserved core kinase sequence (34), and the activated species can be detected by antibodies directed against phosphorylated peptides encompassing these residues. Untreated C34 or E47 cells were incubated overnight, and cell extracts were analyzed for phosphorylated and total MAPKs by Western blotting. Although the phosphorylated form of all three MAPKs (ERK, p38, c-Jun N-terminal kinase) can be detected in both C34 and E47 cells, only the phosphorylated ERK was increased in E47 cells compared with C34 cells (Fig. 8, A–B), suggesting that ERK MAPK is activated by overexpression of CYP2E1. The increase in the levels of phosphorylated ERK was not due to a concomitant elevation in the amount of ERK since the level of ERK (ERK1 or ERK2) in E47 cells was unchanged compared with C34 cells (Fig. 8, A–B), suggesting that ERK MAPK is activated by overexpression of CYP2E1.
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ERK MAPK Pathway Is Important for the Induction of HO-1 by Overexpression of CYP2E1—To address the role of individual MAPK pathways in HO-1 gene activation by overexpression of CYP2E1, we examined the effects of PD98059, an ERK pathway inhibitor, and SB203580, an inhibitor of p38, on pHHOSV15luc expression. The effect of wortmannin, an inhibitor of the phosphatidylinositol 3-kinase pathway, on pHHOSV15luc expression was also studied. C34 and E47 cells were transfected with the HO-1 reporter gene construct (pHHO15SVluc) and then treated with 10 µM SB203580, 10 µM PD98059 (Calbiochem), or 10 µM Wortmannin (Sigma) for 6 h. Luciferase activity was determined using the dual luciferase reporter assay system. Consistent with higher expression of HO-1 mRNA, E47 cells have about a 1.8-fold higher pHHO15SVluc activity than C34 cells (Fig. 8C). PD98059 blocked the increased HO-1 reporter activity in E47 cells (from 178 to 101%), whereas it did not have any effect on the reporter activity of C34 cells (from 100 to 101%) (Fig. 8C), suggesting that ERK MAPK plays an important role for the activation of HO-1 by overexpression of CYP2E1. SB203580 significantly and comparably attenuated HO-1 reporter activity in both C34 and E47 cells (from 100 and 178% to 41 and 45%, respectively) (Fig. 8C), suggesting that the p38 MAPK pathway may be important for the basal HO-1 expression in HepG2 cells. In contrast, the phosphatidylinositol 3-kinase inhibitor wortmannin did not have any effects on HO-1 reporter activity either in C34 or E47 cells (Fig. 8C), suggesting that this pathway is not important for HO-1 gene expression in HepG2 cells.
HO-1 mRNA and Protein Are further Induced after Treatment with AA and BSO in the E47 Cells—AA and BSO have been shown to increase oxidative stress and cause toxicity in E47 cells but not in C34 cells (14, 16, 18, 22). To evaluate the response of HO-1 to these CYP2E1-dependent pro-oxidants, E47 and C34 cells were incubated with or without 50 µM AA or 100 µM BSO for 48 h. Both HO-1 mRNA and protein were further elevated about 3-fold in the E47 cells after treatment with AA or BSO (Figs. 9A and 10A). HO-1 mRNA and protein were only slightly increased by AA or BSO in the C34 cells (1.6- and 1.2-fold, respectively) (Figs. 9B and 10B). HO-2 protein expression was not affected by AA or BSO treatment (Fig. 10, A–B). Because HO-1 induction is a good indicator of a response to oxidative stress, the further increases of HO-1 expression in E47 cells most likely reflect the increased oxidative stress generated by AA and BSO.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study we have used a HepG2 cell line that overexpresses CYP2E1 to determine the mechanism of induction of HO-1. CYP2E1, when reduced by NADPH-cytochrome P450 reductase, is a loosely coupled enzyme that displays high NADPH oxidase activity and is very reactive in catalysis of lipid peroxidation and production of ROS relative to other P450 isoforms (38–39). Increased CYP2E1 activity is usually accompanied by increased ROS production and lipid peroxidation (14, 30). E47 cells were found to have increased HO-1 protein and mRNA expression as well as elevated HO activity. Up-regulation of HO-1 by CYP2E1 may be an adaptive response of cells to the increased oxidative stress caused by the increased CYP2E1 activity. Effective inhibitors of CYP2E1 activity, such as DAS, 4-MP, and Me2SO, which inhibited the increased ROS production in E47 cells, also inhibited HO-1 induction in these cells. Other antioxidant genes such as glutamate-cysteine ligase, catalase, and glutathione S-transferase are also up-regulated by overexpression of CYP2E1 (19–20) and, like HO-1, may reflect adaptive responses to the overexpression of CYP2E1.
HO-1 is transcriptionally activated in HepG2 cells by overexpression of CYP2E1. E47 cells have increased HO-1 mRNA expression, whereas HO-1 mRNA degradation rates are unchanged compared with control HepG2 cells (C34 cells). Transfection of a human HO-1 promoter-reporter gene construct (pHHO15SVluc) into E47 and C34 cells further confirmed that the HO-1 promoter is activated by expression of CYP2E1. Hydrogen peroxide may possibly play a role in mediating this transactivation of the HO-1 gene by CYP2E1, since the addition of catalase, which decomposes hydrogen peroxide, blocked the induction of HO-1 mRNA and protein in E47 cells. It is not clear whether the added catalase is operating intracellularly or extracellularly; uptake of catalase by hepatocytes by an endocytosis-dependent mechanism has been reported (40). Alternatively, because hydrogen peroxide is diffusible, extracellular catalase may function as an extracellular sink, removing intracellular hydrogen peroxide down its concentration gradient (17, 25). Hydrogen peroxide has been reported to increase the DNA binding activity of the cadmium-response element on the promoter region of the human HO-1 gene (41) and activate HO-1 gene transcription in HepG2 cells (42). Increased CYP2E1 activity also increases lipid peroxidation in E47 cells (14). However, lipid peroxidation reactions do not appear to play an important role in explaining the up-regulation of the HO-1 gene in the untreated E47 cells, since the addition of the classical inhibitor of lipid peroxidation, vitamin E, does not prevent the induction of HO-1 protein and mRNA.
MAPK pathways, including ERK and/or p38 MAPK, have been reported to participate in the activation of the HO-1 gene by inducing xenobiotics (9–10). In this study we found that the ERK MAPK pathway possibly mediates the HO-1 gene activation by overexpression of CYP2E1 in HepG2 cells, whereas p38 MAPK is important for the basal HO-1 expression in HepG2 cells. ERK is activated by overexpression of CYP2E1 in HepG2 cells. Compared with the control C34 cells, E47 cells have higher levels of phosphorylated ERK, whereas the levels of phosphorylated p38 and c-Jun N-terminal kinase are not changed. Inhibition of the ERK MAPK pathway by PD98059 blocked the increase of a HO-1 reporter gene (pHHOSV15luc) activity in E47 cells but had no effect in the control C34 cells, providing a correlation between the HO-1 promoter activity and ERK MAPK pathway. Inhibition of the p38 MAPK pathway by the specific inhibitor SB203580 significantly attenuated the HO-1 reporter basal activity in the control HepG2 cells (C34 cells) as well as HO-1 reporter activity in E47 cells, suggesting an essential role of the p38 MAPK pathway in maintaining the basal activity of the HO-1 promoter and supporting subsequent response to HO-1 inducers in HepG2 cells. We hypothesize that CYP2E1-derived oxidative stress results in activation of the ERK MAPK pathway, which is then followed by increased transcription of the HO-1 gene by a signaling mechanism also dependent on p38 MAPK activity. Increased production of ROS by overexpression of CYP2E1 is likely the link between activation of ERK and the up-regulation of HO-1 expression.
As shown in previous studies (18, 22, 21), AA and BSO caused significant increases in ROS production and lipid peroxidation and toxicity in the E47 cells but had little effect in C34 cells. AA and BSO also significantly increased HO-1 mRNA and protein expression in the E47 cells in contrast to a slight increase in C34 cells. HO-1 induction is a good indicator of oxidative stress. Induction of HO-1 in E47 cells by AA and BSO may reflect the further increase in oxidative stress caused by AA and BSO over the basal stress generated by CYP2E1 expression, since AA and BSO significantly increase ROS production and lipid peroxidation in E47 cells. The functional significance of this induction of HO-1 is currently under investigation; preliminary studies show that inhibitors of HO-1 potentiate the CYP2E1 plus AA- or CYP2E1 plus BSO-dependent toxicities in E47 cells, in association with an increase in oxidative stress.2
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Pharmacology and
Biological Chemistry, Mount Sinai School of Medicine, One Gustave L. Levy
Place, New York, NY 10029. Tel.: 212-241-7285; Fax: 212-996-7214; E-mail:
arthur.cederbaum{at}mssm.edu.
1 The abbreviations used are: HO, heme oxygenase; AA, arachidonic acid; BSO,
L-buthionine-(S,R)-sulfoximine; CYP2E1, cytochrome P450
2E1; C34 cells, HepG2 cell line established after transfection with pCI-neo;
E47 cells, HepG2 cell line established after transfection with pCI-neo-CYP2E1;
DAS, diallyl sulfide; ERK, extracellular signal-regulated kinase; MAPK,
mitogen-activated protein kinase; ROS, reactive oxygen species; 4-MP,
4-methylpyrazole.
2 P. Gong, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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