Increased cytochrome P-450 2E1 expression sensitizes hepatocytes to c-Jun-mediated cell death from TNF-alpha

Hailing Liu1, Brett E. Jones1, Cynthia Bradham2, and Mark J. Czaja1

1 Department of Medicine, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461; and 2 Department of Medicine and Department of Biochemistry and Biophysics, Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, North Carolina 27599


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The mechanisms underlying hepatocyte sensitization to tumor necrosis factor-alpha (TNF-alpha )-mediated cell death remain unclear. Increases in hepatocellular oxidant stress such as those that occur with hepatic overexpression of cytochrome P-450 2E1 (CYP2E1) may promote TNF-alpha death. TNF-alpha treatment of hepatocyte cell lines with differential CYP2E1 expression demonstrated that overexpression of CYP2E1 converted the hepatocyte TNF-alpha response from proliferation to apoptotic and necrotic cell death. Death occurred despite the presence of increased levels of nuclear factor-kappa B transcriptional activity and was associated with increased lipid peroxidation and GSH depletion. CYP2E1-overexpressing hepatocytes had increased basal and TNF-alpha -induced levels of c-Jun NH2-terminal kinase (JNK) activity, as well as prolonged JNK activation after TNF-alpha stimulation. Sensitization to TNF-alpha -induced cell death by CYP2E1 overexpression was inhibited by antioxidants or adenoviral expression of a dominant-negative c-Jun. Increased CYP2E1 expression sensitized hepatocytes to TNF-alpha toxicity mediated by c-Jun and overwhelming oxidative stress. The chronic increase in intracellular oxidant stress created by CYP2E1 overexpression may serve as a mechanism by which hepatocytes are sensitized to TNF-alpha toxicity in liver disease.

nuclear factor-kappa B; glutathione; apoptosis; necrosis; c-Jun NH2-terminal kinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ONE OF THE BIOLOGICAL EFFECTS of tumor necrosis factor-alpha (TNF-alpha ) is cytotoxicity, and TNF-alpha has been implicated as an in vivo mediator of hepatocyte death after injury from toxins, ischemia-reperfusion, and viruses (for review, see Ref. 4). Hepatocytes are normally resistant to TNF-alpha toxicity, and uninjured hepatocytes undergo proliferation rather than death in response to TNF-alpha stimulation (51). For hepatocytes to undergo TNF-alpha -induced cell death, they must somehow be sensitized to the TNF-alpha death signaling pathway. The fact that cells, including hepatocytes, are sensitized to TNF-alpha -induced death by RNA or protein synthesis arrest (28, 52) and that hepatotoxins inhibit cellular macromolecular synthesis has suggested that toxic liver injury in particular may sensitize hepatocytes to TNF-alpha -induced death by blocking the transcriptional upregulation of a protective proteins(s) (14). Supportive of this theory are recent studies (5, 24, 32, 51) implicating specific transcriptional activators as critical mediators of hepatocellular resistance to TNF-alpha cytotoxicity. Inhibition of the normal TNF-alpha induction of nuclear factor-kappa B (NF-kappa B) or c-Myc transcriptional activity in nontransformed hepatocytes sensitizes these cells to death from TNF-alpha (5, 24, 32, 51). One mechanism by which hepatocytes become susceptible to the toxic effects of TNF-alpha in vivo may therefore be through a failure of TNF-alpha -dependent transcriptional activation.

Alternatively, downstream factors may sensitize cells to TNF-alpha -induced death regardless of whether or not protective protein expression occurs. One such factor may be the redox state of the hepatocyte. TNF-alpha is known to induce cellular oxidative stress (20, 41), and the production of reactive oxygen species (ROS) has been implicated in many forms of cell death (8). A reduction in antioxidant defenses may sensitize cells to death from ROS, and depletion of the principal nonenzymatic cellular antioxidant GSH has been shown to increase hepatocyte TNF-alpha toxicity (13, 38, 52). However, it has been unclear whether GSH depletion merely worsens TNF-alpha -induced death in cells sensitized by other factors or if GSH depletion by itself sensitizes cells to TNF-alpha -induced death (13, 38, 52).

The ability of antioxidant depletion to increase TNF-alpha -induced death suggested that prooxidant factors may similarly promote TNF-alpha -induced death in hepatocytes. The cytochrome P-450 isoform 2E1 (CYP2E1) mediates the oxidative metabolism of numerous endogenous and foreign compounds (19), including hepatotoxins such as ethanol (25, 30). In addition, CYP2E1 has uncoupled NADPH oxidase activity that in the absence of substrate results in increased production of superoxide and H2O2 (23). CYP2E1 overexpression occurs in animals and humans with alcoholic liver disease (29, 43) and nonalcoholic steatohepatitis (47, 48). Oxidative stress mediated by overexpressed CYP2E1 has been shown to promote liver injury in both of these diseases (27, 35). Interestingly, TNF-alpha has also been implicated as a critical mediator of liver injury in alcoholic liver injury (22) and nonalcoholic steatohepatitis (31). The known role of oxidative stress in TNF-alpha cytotoxicity led to the hypothesis that CYP2E1-induced oxidant stress may act to sensitize hepatocytes to death from TNF-alpha . To test this hypothesis, the resistance of hepatocytes with differential levels of CYP2E1 expression to TNF-alpha cytotoxicity was examined. The present studies support the hypothesis by demonstrating that in vitro overexpression of CYP2E1 in rat hepatocytes is sufficient by itself to sensitize these cells to TNF-alpha -induced death.


    METHODS
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INTRODUCTION
METHODS
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Cells and culture conditions. The rat hepatocyte cell line RALA255-10G (11) was cultured in DMEM (GIBCO-BRL, Grand Island, NY) supplemented with 4% FCS (Gemini BioProducts, Calabasas, CA), 2 mM glutamine, and antibiotics (GIBCO-BRL), as previously described (51). These cells are conditionally transformed with a temperature-sensitive T antigen. At the permissive temperature of 33°C, the cells express T antigen, remain undifferentiated, and proliferate. Culture at the restrictive temperature of 37°C suppresses T antigen expression, slows growth, and allows differentiated hepatocyte gene expression (11, 12, 16). All experiments were performed with cells cultured at 37°C. Cells with differential CYP2E1 expression were established by transfecting RALA hepatocytes with pCI-neo expression vectors (Promega, Madison, WI) containing the human CYP2E1 cDNA in either a sense or antisense orientation (the kind gift of Dr. A. Cederbaum) (36). Stable transfectants were selected in G418. Experiments were conducted in sense-transfected S-CYP15 cells that have a 10 ± 0.4-fold increase in CYP2E1 protein by Western blotting compared with wild-type (WT) cells, and antisense-transfected AN-CYP10 cells in which CYP2E1 protein is undetectable (Czaja, unpublished data). One experiment was also conducted in additional RALA hepatocyte clones: 1) pCI-neo4, a polyclonal line stably transfected with vector alone; 2) S-CYP7, a sense-CYP2E1-transfected clone with a 5 ± 0.7-fold increase in CYP2E1 protein relative to WT cells; 3) S-CYP10, a sense-CYP2E1-transfected clone with no increase in CYP2E1 levels; 4) AN-CYP9, an antisense CYP2E1-transfected line with no detectable CYP2E1 protein; and 5) AN-CYP7, an antisense CYP2E1-transfected line with CYP2E1 expression equivalent to WT cells.

For all experiments, the cells were cultured at 33°C until confluent, trypsinized, and replated at 0.65 × 106 cells/dish on 35-mm plastic dishes (Falcon, Becton Dickinson, Lincoln Park, NJ). After 24 h, the medium was changed to DMEM supplemented with 2% FCS, glutamine, antibiotics, and 1 µM dexamethasone, and the cells were cultured at 37°C. After 3 days of culture at 37°C, the cells received fresh serum-free medium containing dexamethasone. The medium was supplemented with dexamethasone to optimize hepatocyte differentiation as previously described (11, 12). Twenty hours later, these cells received fresh serum-free medium, and some cells were treated with rat recombinant TNF-alpha (R & D Systems, Minneapolis, MN) at a concentration of 10 ng/ml. Every 24 h after TNF-alpha treatment, the cell medium was changed to fresh serum-free medium. Some cells were also treated at various times with polyethylene glycol-linked catalase (1,000 U/dish), 5 mM GSH ethyl ester, 50 ng/ml phorbol 12-myristate 13-acetate (PMA), H2O2 (1.25 µmol/106 cells; Sigma, St. Louis, MO), 100 µM Val-Ala-Asp-fluoromethylketone (ZVAD; Bachem, Torrance, CA), or 50 µM N-[(indole-2-carbonyl)-alaninyl]-3-amino-4-oxo-5-fluoropentanoic acid (IDN-1529; IDUN Pharmaceuticals, La Jolla, CA).

MTT assay. The amount of cell death was determined by examining cell number with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (15), as previously described (51). The relative cell number was calculated by taking the optical density of cells given a particular treatment, dividing that number by the optical density for the untreated, control cells, and multiplying that number by 100.

Microscopic determination of apoptosis. The numbers of apoptotic and necrotic cells were quantitated by fluorescence microscopy after costaining with acridine orange and ethidium bromide (17), as previously described (51). The percentage of cells with apoptotic morphology (nuclear and cytoplasmic condensation, nuclear fragmentation, membrane blebbing, and apoptotic body formation) was determined by examining >400 cells/dish. Cells were counted as necrotic if they stained positive with ethidium bromide.

Fluorescence-activated cell sorting analysis of DNA hypoploidy. The identification of hypoploid cells by fluorescence-activated cell sorting (FACS) detection of DNA loss after controlled extraction of low-molecular-weight DNA was performed as previously described (24). Cells were trypsinized and centrifuged, and the cell pellets were fixed in 70% ethanol at -20°C for a minimum of 17 h. The cells were washed and resuspended in Hanks' buffered saline solution and incubated in phosphate-citric acid buffer (0.2 M Na2HPO4 and 0.1 M citric acid, pH 7.8) for 5 min. The cells were then centrifuged, and the pellet was resuspended in Hanks' buffered saline solution containing propidium iodide (20 mg/ml) and RNase (100 µg/ml). After a 30-min incubation at room temperature, the cells were analyzed on a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA) at an excitation of 488 nm. DNA fluorescence pulse processing was used to discriminate between single cells and aggregates of cells (Doublet Discrimination) by evaluating the FL2-width vs. FL2-area scatterplot. Light scatter gating was used to eliminate smaller debris from analysis. An analysis gate was set to limit the measurement of hypoploidy to an area of 10-fold loss of DNA content.

Protein isolation and Western blot analysis. For poly(ADP-ribose) polymerase (PARP) immunoblots, cells were washed in PBS, centrifuged, and resuspended in lysis buffer containing 20 mM Tris, pH 7.5, 1% SDS, 2 mM EDTA, 2 mM EGTA, 6 mM beta -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml of pepstatin A, leupeptin, and aprotinin. After a 10-min incubation on ice, the cell suspension was sonicated. Following centrifugation, the supernatant was collected and the protein concentration determined by the Bio-Rad protein assay (Hercules, CA). Fifty micrograms of protein were resolved on 10% SDS-PAGE as previously described (51). Membranes were stained with Ponceau red to ensure equivalent amounts of protein loading and electrophoretic transfer among samples. Then membranes were exposed to a rabbit anti-PARP polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1,000 dilution followed by a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (GIBCO-BRL) at a 1:20,000 dilution. Proteins were visualized by chemiluminescence (SuperSignal West Dura Extended, Pierce, Rockford, IL).

Electrophoretic mobility shift assays. Nuclear proteins were isolated by the method of Schreiber et al. (40), modified as previously described (50). Electrophoretic mobility shift assays (EMSA) were performed on 5 µg of protein with a 32P end-labeled oligonucleotide for the NF-kappa B consensus sequence (Santa Cruz Biotechnology). The DNA binding reaction was carried out as previously described (50), and the samples were resolved on a 4% polyacrylamide gel, dried, and subjected to autoradiography. Supershift assays with anti-p50 and anti-p65 NF-kappa B antibodies (Santa Cruz Biotechnology) were performed as previously described (51).

Transient transfections and reporter gene assays. RALA hepatocytes were transiently transfected using Lipofectamine Plus (GIBCO-BRL) with the NF-kappa B-driven luciferase reporter construct NF-kappa B-Luc (18) or the activator protein-1 (AP-1)-regulated reporter coll73-Luc (46). Cells were cotransfected with pRL-TK (Promega), a Renilla luciferase vector driven by a herpes simplex virus thymidine kinase promoter that served as a control for transfection efficiency. To assay luciferase activity, cells were washed in PBS and lysed in 1% Triton X-100, and the cell extract was assayed for firefly luciferase activity in a luminometer. Renilla luciferase was assayed in the same sample according to the manufacturer's instructions. Firefly luciferase activity was then normalized to Renilla luciferase activity.

Lipid peroxidation assay. Levels of lipid peroxidation were measured by the peroxidative degradation of cis-parinaric acid, as previously described (26). Cells were incubated in 5 µM cis-parinaric acid for 30 min in the dark. Cells were washed twice in PBS, trypsinized, and resuspended in PBS. The loss of fluorescence was read at an excitation of 325 nM and an emission of 413 nM.

GSH assay. Total cellular GSH was determined by the 5,5'-dithiobis(2-nitrobenzoic acid)-GSH disulfide recycling assay (1), as previously described (52). Protein concentrations were determined on the same lysates, and GSH levels were normalized to protein content.

c-Jun NH2-terminal kinase assays. c-Jun NH2-terminal kinase (JNK) activity was measured in cell lysates using a stress-activated protein kinase-JNK assay kit (Cell Signaling, Beverly, MA), according to the manufacturer's instructions. An NH2-terminal c-Jun (1-89) fusion protein bound to GSH sepharose beads was used to immobilize JNK from cell lystates containing 250 µg of total protein. After washing, the kinase reaction was performed in the presence of cold ATP using the c-Jun fusion protein as a substrate. Samples were resolved by 10% SDS-PAGE, and the amount of phosphorylated c-Jun was detected with an antibody specific for c-Jun phosphorylated at Ser63. As a control to ensure that equivalent amounts of protein were present in each sample, immunoblots for total c-Jun were also performed using an antibody that detects both phosphorylated and unphosphorylated c-Jun (Santa Cruz Biotechnology).

Adenovirus construction and infection. Ad5TAM was constructed from TAM-67, a truncation mutant of c-Jun missing amino acids 3-122 (6). TAM-67 dimerizes with other AP-1 subunits, forming inactive DNA binding dimers that function as a dominant-negative AP-1 (7). TAM-67 was NH2 terminally HA tagged using PCR amplification and then subcloned into pACCMV.PLPASR(+). Adenoviruses were generated by homologous recombination of the transfer vector with truncated adenovirus type 5 d1309 DNA in replication-permissive 293 cells. Plaques were screened by Western immunoblotting with an anti-HA antibody (Babco, Berkeley, CA). The selected Ad5TAM virus and a previously described beta -galactosidase-expressing control virus Ad5LacZ (51) were amplified in 293 cells, purified on CsCl gradients, and titered by plaque assay. RALA hepatocytes were infected at a multiplicity of infection of 20 as previously described (51).

Statistical analysis. All numerical results are reported as means ± SE and represent data from a minimum of three independent experiments with duplicate dishes in each experiment. Groups were compared by the Student's t-test. Statistical significance was defined as P < 0.05. Calculations were made with the Sigma Stat statistical package (Jandel Scientific, San Rafael, CA).


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ABSTRACT
INTRODUCTION
METHODS
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CYP2E1 overexpression sensitizes hepatocytes to TNF-alpha -induced cell death. To examine the effect of CYP2E1 overexpression on the hepatocellular response to TNF-alpha , we determined the effects of TNF-alpha on cell survival in RALA hepatocyte lines with differential levels of CYP2E1 expression. WT RALA hepatocytes express low levels of CYP2E1 protein by Western immunoblotting and are resistant to TNF-alpha cytotoxicity unless sensitized by transcriptional arrest (24, 52). The WT cell response to TNF-alpha was compared with the stably transfected S-CYP15 and AN-CYP10 lines, which relative to WT cells have increased and decreased CYP2E1 protein expression, respectively. Cells were treated with TNF-alpha , and cell number relative to untreated cells was determined by MTT assay over 72 h. WT hepatocytes had a slight increase in cell number over the 72 h following TNF-alpha treatment despite the use of highly confluent cultures (Fig. 1A), consistent with TNF-alpha inducing a proliferative, nontoxic response in these cells. In contrast, TNF-alpha treatment of S-CYP15 cells led to a slight decrease in cell number within 24 h and marked subsequent decreases in cell number at 48 (47% decrease) and 72 h (74% decrease) (Fig. 1A). AN-CYP10 cells had a significant 29% increase in cell number within 48 h after TNF-alpha treatment, and this increase remained stable at 72 h (Fig. 1A). Overexpression of CYP2E1 therefore converts the hepatocyte phenotype from one of TNF-alpha resistance, to one of sensitivity to TNF-alpha -induced death.


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Fig. 1.   Tumor necrosis factor-alpha (TNF-alpha ) induces apoptotic and necrotic cell death in S-CYP15 cells. A: wild-type (WT), S-CYP15 (S-CYP), and AN-CYP10 (AN-CYP) cells were cultured and treated with TNF-alpha as described in METHODS. The cell number as % of untreated cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 24, 48, and 72 h after TNF-alpha treatment as indicated. S-CYP15 cell number was significantly decreased relative to either WT or AN-CYP10 cells at all time points (all P < 0.00001). B: the % of apoptotic (Apo) and necrotic (Nec) cells was determined in untreated (Con) and 48-h TNF-alpha treated (TNF) cells by fluorescent costaining with acridine orange and ethidium bromide. The numbers of apoptotic and necrotic cells were significantly increased in both untreated and treated S-CYP15 cells, relative to either WT or AN-CYP10 cells (all P < 0.002). C: the % of hypoploid untreated and 48-h TNF-alpha -treated cells was determined by flow cytometric analysis of propidium iodide-stained cells. The % of hypoploid S-CYP15 cells was significantly increased after TNF-alpha treatment compared with WT or AN-CYP10 cells (both P < 0.001). D: aliquots of total cell lystates from untreated and 48-h TNF-alpha -treated cells were subjected to SDS-PAGE, and immunoblotting was performed with an anti-poly(ADP-ribose) polymerase (PARP) antibody. The intact 116-kDa PARP and its 85-kDa cleavage product are indicated.

To ensure that S-CYP15 cell sensitivity to TNF toxicity was related to increased CYP2E1 expression and not clonal differences, cell survival 48 h after TNF-alpha treatment was determined in additional cell lines. A second CYP2E1-overexpressing cell line, S-CYP7, was also sensitive to TNF-alpha toxicity and underwent 36% cell death at 48 h (Table 1). In contrast, four additional cell lines, with CYP2E1 expression equivalent to or less than that of WT cells, were resistant to TNF-alpha toxicity (Table 1). These included a line with vector alone (pCI-neo4 cells), a sense-CYP2E1-transfected line with no increase in CYP2E1 expression (S-CYP10 cells), and two antisense CYP2E1-transfected lines (ANCYP7 and ANCYP9 cells). Sensitization to TNF-alpha toxicity was therefore clearly a result of CYP2E1 overexpression.

                              
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Table 1.   Cell number 48 h after TNF-alpha treatment

TNF-alpha -induced S-CYP15 cell death occurs by necrosis and apoptosis. TNF-alpha can induce either apoptotic or necrotic hepatocyte death depending on the mechanism of sensitization. To assess the relative contributions of apoptosis and necrosis to S-CYP15 cell death, cells costained with acridine orange and ethidium bromide were examined under fluorescence microscopy 48 h after TNF-alpha treatment. Both untreated WT and AN-CYP10 cells had low numbers of apoptotic and necrotic cells that remained unchanged 48 h after TNF-alpha treatment (Fig. 1B). In contrast, untreated S-CYP15 cells had higher numbers of apoptotic and necrotic cells that then increased twofold and fourfold, respectively, after TNF-alpha treatment (Fig. 1B). The increased numbers of apoptotic and necrotic cells in untreated S-CYP15 cultures represented a higher basal rate of cell turnover, and not a dying cell population, because the numbers of trypan blue-excluding cells remained constant over the 72 h of culture in serum-free medium (data not shown).

To further characterize TNF-alpha -dependent cell death in S-CYP15 cells, we examined cells for DNA fragmentation by FACS analysis for the presence of DNA hypoploidy. All three cell types had low percentages of hypoploid untreated cells (Fig. 1C). The numbers of hypoploid cells were unchanged by TNF-alpha treatment in WT and AN-CYP10 cells (Fig. 1C). However, S-CYP15 cells had a more than fourfold increase in the percentage of hypoploid cells after 48 h of TNF-alpha treatment (Fig. 1C).

The induction of DNA fragmentation in TNF-alpha -treated S-CYP15 cells implies the presence of caspase activation, which is necessary for DNase activation. As an additional measure of whether caspase activation occurred in CYP2E1-dependent TNF-alpha toxicity, cells were examined for PARP cleavage. PARP cleavage was not observed in WT or AN-CYP10 cells untreated or 48 h after TNF-alpha treatment (Fig. 1D). Low levels of PARP cleavage were present in untreated S-CYP15 cells (Fig. 1D), consistent with the higher level of constitutive cell death in these cells (Fig. 1B). After 48 h of TNF-alpha treatment, PARP cleavage further increased in S-CYP15 cells (Fig. 1D).

TNF-alpha -induced S-CYP15 cell death is secondary to increased oxidative stress. Both TNF-alpha stimulation (20, 41) and CYP2E1 overexpression (30) induce the cellular generation of ROS, suggesting that these two factors combined may kill cells through a synergistic increase in oxidative stress. To examine this possibility, levels of lipid peroxidation were measured in the cell lines by the fluorescent peroxidative degradation of cis-parinaric acid. In this assay, decreased levels of fluorescence reflect increased amounts of lipid peroxidation. Levels of lipid peroxidation in untreated S-CYP15 cells were only slightly increased compared with WT and AN-CYP10 cells (Fig. 2A). After 48 h of TNF-alpha treatment, levels of lipid peroxidation decreased significantly in both WT and AN-CYP10 cells but increased in S-CYP15 cells, resulting in a 40% increase in lipid peroxidation relative to WT cells (Fig. 2A). Increased lipid peroxidation was accompanied by antioxidant depletion in S-CYP15 cells. After 48 h of TNF-alpha treatment, GSH content decreased 36% in S-CYP15 cells (from 24.3 to 15.6 nmol/mg protein), while no decrease in GSH levels occurred in WT or AN-CYP10 cells. In response to TNF-alpha , S-CYP15 cells therefore underwent an oxidative stress that resulted in significant antioxidant depletion.


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Fig. 2.   TNF-alpha -induced, S-CYP15 cell death results from oxidative stress and is blocked by antioxidants. A: relative fluorescence intensity of WT, S-CYP15, and AN-CYP10 untreated cells (control) or cells treated for 48 h with TNF-alpha (TNF) and incubated with cis-parinaric acid for 30 min. The decrease in S-CYP15 cell fluorescence intensity after TNF-alpha treatment was significant relative to the other 2 cell lines (both P < 0.002). B: relative cell number as determined by MTT assay in S-CYP15 untreated cells (control) and 48-h TNF-alpha -treated cells (TNF). The cells received no additional treatment (Ø) or 2 doses (given at the time of TNF-alpha treatment and 24 h later) of polyethylene glycol-linked catalase (Cat), glutathione ethyl ester (GSH), Val-Ala-Asp-fluoromethylketone (ZVAD), or N-[(indole-2-carbonyl)-alaninyl]-3-amino-4-oxo-5-fluoropentanoic acid (IDN-1529; 1529). Cat, GSH, and IDN-1529 significantly increased cell survival after TNF-alpha treatment (P < 0.001). C: relative cell number in S-CYP15 cells 48 h after TNF-alpha administration and no Cat treatment (No Cat Tx), a single Cat treatment (single Tx), or double Cat treatment (double Tx). The time of the initial Cat treatment is indicated as the number of hours after TNF-alpha administration. Cells treated with 2 doses of Cat received the same initial treatment and a second one 24 h after TNF-alpha administration. All of the Cat treatments resulted in a significant increase in cell number (all P < 0.004).

To determine whether oxidative stress played a causal role in S-CYP15 cell sensitivity to TNF-alpha toxicity, the effects of antioxidants on cell death were investigated. Treatment with polyethylene glycol-linked catalase to neutralize H2O2 completely blocked cell death and led to an increase in cell number in untreated and 48-h TNF-alpha -treated S-CYP15 cells (Fig. 2B). GSH ethyl ester also inhibited 70% of TNF-alpha -induced cell death (Fig. 2B). In contrast, the pancaspase inhibitor ZVAD was toxic to the cells and failed to inhibit cell death from TNF-alpha (Fig. 2B). Lower concentrations and more frequent administration of ZVAD also failed to block cell death (data not shown). However, the nontoxic pancaspase inhibitor IDN-1529 decreased cell death by 62% (Fig. 2B).

The timing of oxidative stress in TNF-alpha -induced S-CYP15 cell death was investigated by varying the initial time of catalase administration. Delay of catalase treatment up to 6 h after TNF-alpha administration did not alter the effectiveness of this antioxidant in blocking cell death (Fig. 2C). The initiation of catalase treatment even as late as 12 and 24 h after TNF-alpha administration was partially effective in blocking TNF-alpha -induced cell death (Fig. 2C). TNF-alpha treatment of S-CYP15 cells resulted in a sustained oxidative stress in keeping with the prolonged period of cell death.

S-CYP10 cells are sensitized to TNF-alpha -induced death despite increased levels of NF-kappa B activation. A critical determinant of hepatocyte resistance to TNF-alpha toxicity is the ability of these cells to activate the transcription factor NF-kappa B (5, 24, 51). NF-kappa B activation was examined in WT, S-CYP15, and AN-CYP10 cells to determine whether S-CYP15 cell sensitivity to TNF-alpha was a function of decreased NF-kappa B activity in these cells. EMSA with an NF-kappa B consensus oligonucleotide demonstrated that TNF-alpha induced equivalent levels of NF-kappa B nuclear translocation in WT, S-CYP15, and AN-CYP10 cells (Fig. 3A). Identical to prior reports in WT cells (51), the S-CYP15 cell NF-kappa B complex was composed of p65/p50 heterodimers (data not shown). These results demonstrate that S-CYP15 cell sensitivity to TNF-alpha was not due to a failure to activate NF-kappa B. They also indicate that AN-CYP10 cells were responsive to TNF-alpha and therefore their TNF-alpha resistance was not due to a loss of TNF receptor.


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Fig. 3.   TNF-alpha induces nuclear factor-kappa B (NF-kappa B) activation in S-CYP15 cells. A: nuclear extracts were isolated from untreated or 2-h TNF-alpha -treated WT, S-CYP15, and AN-CYP10 cells and used for electrophoretic mobility shift assays with an NF-kappa B consensus oligonucleotide. Solid arrow, NF-kappa B binding complex. B: all 3 cell lines were transiently cotransfected with the NF-kappa B-regulated reporter construct NF-kappa B-Luc and the constitutive Renilla luciferase vector pRL-TK. Untreated (control) and 48-h TNF-alpha -treated (TNF) cells were assayed for firefly and Renilla luciferase activity. Firefly luciferase activity was then normalized to Renilla luciferase activity. Luciferase activity was significantly increased in untreated and TNF-alpha -treated S-CYP15 cells relative to either WT or AN-CYP10 cells (all P < 0.01).

DNA binding does not always reflect actual changes in NF-kappa B transcriptional activity, which depends on additional variables such as phosphorylation state and the presence of coactivating factors (37). Levels of NF-kappa B-dependent transcription were measured in the three cell lines by means of transient transfections with an NF-kappa B-driven luciferase reporter gene. Levels of NF-kappa B-dependent transcription normalized to a cotransfected, constitutive reporter were increased twofold in S-CYP15 cells compared with WT cells and markedly increased over the levels in AN-CYP10 cells (Fig. 3B). At 6 h after TNF-alpha treatment, all three cell lines had increased levels of NF-kappa B activation, but levels in S-CYP15 cells were still increased 10-fold over those in AN-CYP10 cells (Fig. 3B). S-CYP15 cells therefore undergo TNF-alpha -induced cell death despite the presence of elevated NF-kappa B activation.

S-CYP15 cell death from TNF-alpha is mediated by c-Jun. Another signaling cascade activated by TNF-alpha in hepatocytes is the mitogen-activated protein kinase JNK (49). Although the function of JNK in hepatocyte death is unknown, JNK activation has been reported in a variety of forms of death in nonhepatic cells (for review, see Ref. 9). Because oxidants as well as TNF-alpha induce JNK (10, 34, 49), the combination of CYP2E1 overexpression and TNF-alpha stimulation may induce a sustained and therefore death-promoting activation of JNK. To address this possibility, JNK activity was assayed in S-CYP15 and AN-CYP10 cells. JNK activity was markedly increased in S-CYP15 cells untreated and 30 min after TNF-alpha treatment compared with AN-CYP10 cells (Fig. 4A). Overinduction of JNK was not specific to TNF-alpha stimulation since S-CYP15 cells also had increased JNK activity relative to AN-CYP10 cells from other stimuli such as PMA and H2O2 (Fig. 4A). In addition to its increased magnitude, JNK activation was also more prolonged in S-CYP15 cells. Although JNK activity returned to baseline 1 h after TNF-alpha stimulation in AN-CYP10 cells, activity was still increased at 4 h in S-CYP15 cells (Fig. 4B).


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Fig. 4.   S-CYP15 cell death is associated with sustained c-Jun NH2-terminal kinase (JNK) activation and inhibited by expression of a c-Jun dominant negative. A: JNK activity was determined by an in vitro kinase assay in cell lysates from S-CYP15 and AN-CYP10 cells either untreated or treated for 30 min with TNF-alpha , phorbol 12-myristate 13-acetate (PMA), or H2O2. Extracts were immunoblotted with an antibody specific for the phosphorylated c-Jun substrate (P-c-Jun) and an antibody that detects total (phosphorylated and unphosphorylated) c-Jun (T-c-Jun). B: immunoblot of phosphorylated and total c-Jun from cells untreated or treated with TNF-alpha for the indicated number of hours. C: S-CYP15 cells were infected with Ad5LacZ or Ad5TAM and left untreated (Con) or treated with TNF-alpha for 48 h (TNF). The % of apoptotic (Apo) and necrotic (Nec) cells was then determined by fluorescent costaining with acridine orange and ethidium bromide. The numbers of apoptotic and necrotic cells were significantly decreased in TNF-alpha -treated Ad5TAM-infected cells compared with cells infected with Ad5LacZ (all P < 0.0001).

To determine whether this sustained JNK activation promoted S-CYP15 cell death from TNF-alpha , the effects of blocking the activity of c-Jun, a downstream effector of JNK, on cell death were examined. S-CYP15 cells were infected with the adenovirus Ad5LacZ as a control for nonspecific viral effects or Ad5TAM, which expresses TAM-67, a dominant-negative c-Jun (6, 7). The effectiveness of Ad5TAM in blocking AP-1 transcriptional activity was determined by measuring AP-1-dependent gene expression using the transiently transfected coll73-Luc luciferase reporter. After 6 h of TNF-alpha treatment, AP-1-driven luciferase activity was decreased 48% in Ad5TAM-infected cells compared with Ad5LacZ-infected cells. The effect of Ad5TAM-mediated inhibition of AP-1 activity on cell survival was determined by MTT assay. Similar to uninfected cells, Ad5LacZ-infected S-CYP15 cells underwent a 31.8 ± 3% decrease in cell number after 48 h of TNF-alpha treatment. In contrast, TNF-alpha -treated, Ad5TAM-infected cells had a 13.8 ± 3.1% increase in cell number. To further demonstrate that inhibition of c-Jun function affected cell death, the numbers of apoptotic and necrotic cells were quantitated by fluorescence microscopy. Again similar to uninfected cells, Ad5LacZ-infected cells had a modest increase in the number of apoptotic cells, and a marked increase in necrotic cells after 48 h of TNF-alpha (Fig. 4C). Ad5TAM-infected cells had significantly decreased numbers of apoptotic and necrotic untreated and TNF-alpha -treated cells (Fig. 4C). The numbers of apoptotic and necrotic cells 48 h after TNF-alpha treatment were decreased 65% and 75%, respectively, compared with Ad5LacZ-infected cells. TNF-alpha -induced death in CYP2E1-overexpressing cells therefore occurred by a c-Jun-dependent mechanism.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The cytotoxic effects of TNF-alpha mediate a number of forms of liver injury (4), and an understanding of the mechanism by which hepatocytes convert from resistance to sensitivity to TNF-alpha -induced death may be critical to the prevention of liver disease. Although global arrest of RNA/protein synthesis (28, 52) or the inactivation of specific transcription factors such as NF-kappa B (5, 24, 51) sensitize hepatocytes to TNF-alpha toxicity in vitro, the in vivo mechanism(s) of hepatocellular TNF-alpha sensitization remains unclear. The present studies examined the effect of overexpression of the prooxidant enzyme CYP2E1 on the hepatocyte response to TNF-alpha . These investigations were motivated by the fact that the antioxidant status of the hepatocyte has been shown to affect TNF-alpha -induced cell death (13, 38, 52), and CYP2E1 overexpression occurs in association with in vivo TNF-alpha -dependent liver injury (29, 43, 47, 48). CYP2E1 overexpression in a nontransformed hepatocyte cell line sensitized these cells to death from TNF-alpha . WT and antisense-CYP2E1-transfected cells had a proliferative response to TNF-alpha , whereas CYP2E1-overexpressing cells underwent cell death. This sensitization to cell death was specific for the TNF-alpha death pathway, because in contrast to our findings for TNF-alpha , CYP2E1 overexpression led to cellular resistance to menadione-induced apoptosis (Jones BE, unpublished data). In contrast to the relatively rapid 6- to 24-h time course of TNF-alpha -induced death after transcriptional inactivation (5, 24, 28, 32, 51, 52), CYP2E1 overexpression sensitized cells to a prolonged cell death. S-CYP15 cells had low levels of cell death at 24 h, but cell death increased markedly at 48-72 h after a single TNF-alpha treatment. Thus despite the known rapidity of the TNF-alpha death signaling cascade, TNF-alpha had long-term effects on cell homeostasis, leading to cell death days after ligand-receptor interaction.

According to morphological criteria, TNF-alpha induced primarily necrotic cell death in S-CYP15 cells, although apoptosis was also apparent. The findings of PARP cleavage and high levels of DNA hypoploidy are also compatible with apoptotic cell death, suggesting that the visualized necrosis may have been partly secondary to apoptosis and not primary necrosis. Cell death was partially caspase dependent, providing further evidence of apoptotic death. S-CYP15 cells also had higher basal levels of necrotic and apoptotic cells and detectable PARP cleavage, demonstrating that CYP2E1 overexpression by itself caused some toxicity.

S-CYP15 cell death from TNF-alpha occurred by oxidant stress. This conclusion is supported by the findings that 1) S-CYP15 cells had higher levels of lipid peroxidation after TNF-alpha treatment, 2) S-CYP15 cells underwent GSH depletion with TNF-alpha treatment, and 3) antioxidants blocked TNF-alpha -induced cell death. Previous studies (20, 41) have demonstrated that TNF-alpha causes a rapid generation of ROS in cells, but the long-term effects of TNF-alpha on the cellular redox state have not been examined. In WT and AN-CYP10 cells 48 h after TNF-alpha treatment, levels of lipid peroxidation decreased, suggesting that the initial oxidant stress caused by TNF-alpha resulted in a lasting upregulation of cellular antioxidant defenses in these cells. TNF-alpha treatment of CYP2E1-overexpressing cells led to a slight increase in the levels of cellular lipid peroxidation in contrast to the decreases that occurred in WT and AN-CYP10 cells. S-CYP15 cells were unable to sufficiently upregulate or maintain their antioxidant levels and underwent GSH depletion and oxidant-mediated cell death. Presumably, the combined oxidant stress created by CYP2E1 overexpression and TNF-alpha resulted in a level of oxidative stress sufficient to initiate cell death pathway signaling.

NF-kappa B activation is a critical signal that blocks the TNF-alpha death pathway in both nonhepatic cells and hepatocytes (5, 24, 33, 44, 45, 51). The mechanism of S-CYP15 cell sensitivity to TNF-alpha -induced death was not decreased NF-kappa B activity because S-CYP15 cells had higher levels of NF-kappa B transcriptional activity than either WT or AN-CYP10 cells. Acute oxidant stress created by ROS administration is well known to increase NF-kappa B DNA binding and transcriptional activity (39), but these data demonstrate for the first time that the chronic, prooxidant state created by CYP2E1 overexpression led to stable NF-kappa B activation. Interestingly, increased NF-kappa B activation occurred in the absence of any significant change in DNA binding. This finding suggests that the mechanism of NF-kappa B activation is related to factors known to regulate NF-kappa B activity but not binding, such as p65 phosphorylation or the levels of coactivators (37). The ability of S-CYP15 cells to undergo TNF-alpha -induced cell death implies that the effects of CYP2E1 overexpression overrode or acted downstream of the NF-kappa B-dependent protective response. Alternatively, S-CYP15 cell death from TNF-alpha was predominantly necrotic, and NF-kappa B activation may only protect against TNF-alpha -induced apoptosis. Ethanol-induced hepatocyte sensitization to TNF-alpha necrosis has also been described (13) in the presence of NF-kappa B activation.

S-CYP15 cell death from TNF-alpha was dependent on c-Jun. JNK is an upstream activator of c-Jun, and JNK activation occurs in a number of forms of cell death where it may function as a pro- or antiapoptotic factor (9). CYP2E1 overexpression led to both constitutive and stimulus-induced increases in JNK activity. In addition, the time period of JNK activation was prolonged after TNF-alpha stimulation. Although JNK may be essential for cell proliferation, abnormally sustained JNK may promote cell death. Sensitization of rat mesangial cells to TNF-alpha toxicity by transcriptional arrest has been reported (21) to be associated with prolonged JNK activation. The present studies demonstrate that the mechanism of TNF-alpha -induced death in S-CYP15 cells was c-Jun activation that resulted from prolonged JNK activity. The only previous identification to our knowledge of a downstream effector of mitogen-activated protein kinase signaling is that of c-Jun mediating JNK3-dependent excitotoxic neuronal apoptosis (3, 53). The present study demonstrates that c-Jun mediates a proapoptotic function of JNK in TNF-alpha death signaling in hepatocytes, a cell type with JNK1/2 but lacking JNK3. Because c-Jun is a transcriptional activator, its effect on cell death is likely mediated through the upregulation of a proapoptotic gene(s). Although the identity of this gene(s) remains to be determined, its ultimate effect was the induction of a lethal oxidative stress.

The genetic and environmental factors leading to the development of steatohepatitis are undoubtedly multifactorial. However, considerable experimental evidence points to a critical involvement for TNF-alpha in the pathogenesis of both alcoholic and nonalcoholic steatohepatitis (42). The in vivo mechanism of TNF-alpha hepatotoxicity has remained unclear. The ability of CYP2E1 overexpression to sensitize hepatocytes to TNF-alpha death receptor-mediated cytotoxicity may be a mechanism of liver cell injury and death in liver diseases such as alcoholic and nonalcoholic steatohepatitis in which CYP2E1 overexpression occurs.


    ACKNOWLEDGEMENTS

We thank A. Bobe for secretarial assistance, J. Chou for providing RALA255-10G cells, A. Cederbaum for CYP2E1 expression vectors, P. Webb for the coll73-Luc construct, P. Aisen for help with the lipid peroxidation assay, D. Gebhard for assistance with FACS analysis, and IDUN Pharmaceuticals for the generous gift of IDN-1529.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-44234 (M. J. Czaja), a grant from the Alcoholic Beverage Medical Research Foundation (M. J. Czaja), an Australian National Health and Medical Council Research Scholarship (B. E. Jones), and an American Digestive Health Foundation Astra-Merck Fellowship-Faculty Transition Award (B. E. Jones).

Address for reprint requests and other correspondence: M. J. Czaja, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: czaja{at}aecom.yu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpgi.00304.2001

Received 9 July 2001; accepted in final form 22 October 2001.


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