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
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ABSTRACT |
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The mechanisms
underlying hepatocyte sensitization to tumor necrosis factor-
(TNF-
)-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-
death. TNF-
treatment of hepatocyte cell lines with
differential CYP2E1 expression demonstrated that overexpression of
CYP2E1 converted the hepatocyte TNF-
response from proliferation to
apoptotic and necrotic cell death. Death occurred despite the presence of increased levels of nuclear factor-
B transcriptional activity and was associated with increased lipid peroxidation and GSH
depletion. CYP2E1-overexpressing hepatocytes had increased basal and
TNF-
-induced levels of c-Jun NH2-terminal kinase (JNK) activity, as well as prolonged JNK activation after TNF-
stimulation. Sensitization to TNF-
-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-
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-
toxicity in liver disease.
nuclear factor-B; glutathione; apoptosis; necrosis; c-Jun NH2-terminal kinase
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INTRODUCTION |
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ONE OF THE BIOLOGICAL
EFFECTS of tumor necrosis factor- (TNF-
) is cytotoxicity,
and TNF-
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-
toxicity, and uninjured hepatocytes undergo proliferation rather than death in response to TNF-
stimulation (51). For hepatocytes to undergo TNF-
-induced cell
death, they must somehow be sensitized to the TNF-
death signaling
pathway. The fact that cells, including hepatocytes, are sensitized to TNF-
-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-
-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-
cytotoxicity. Inhibition of the normal TNF-
induction of
nuclear factor-
B (NF-
B) or c-Myc transcriptional activity in
nontransformed hepatocytes sensitizes these cells to death from TNF-
(5, 24, 32, 51). One mechanism by which hepatocytes become
susceptible to the toxic effects of TNF-
in vivo may therefore be
through a failure of TNF-
-dependent transcriptional activation.
Alternatively, downstream factors may sensitize cells to
TNF--induced death regardless of whether or not protective protein expression occurs. One such factor may be the redox state of the hepatocyte. TNF-
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-
toxicity (13, 38, 52). However, it has been unclear whether GSH depletion merely worsens TNF-
-induced death in cells sensitized by other factors or if GSH depletion by itself sensitizes cells to
TNF-
-induced death (13, 38, 52).
The ability of antioxidant depletion to increase TNF--induced death
suggested that prooxidant factors may similarly promote TNF-
-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-
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-
cytotoxicity led to the hypothesis that CYP2E1-induced oxidant stress
may act to sensitize hepatocytes to death from TNF-
. To test this
hypothesis, the resistance of hepatocytes with differential levels of
CYP2E1 expression to TNF-
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-
-induced death.
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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-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 -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-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-
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-B-driven luciferase reporter construct
NF-
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 -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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RESULTS |
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CYP2E1 overexpression sensitizes hepatocytes to TNF--induced
cell death.
To examine the effect of CYP2E1 overexpression on the hepatocellular
response to TNF-
, we determined the effects of TNF-
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-
cytotoxicity unless
sensitized by transcriptional arrest (24, 52). The WT cell
response to TNF-
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-
, 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-
treatment
despite the use of highly confluent cultures (Fig.
1A), consistent with TNF-
inducing a proliferative, nontoxic response in these cells. In
contrast, TNF-
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-
treatment, and this increase
remained stable at 72 h (Fig. 1A). Overexpression of
CYP2E1 therefore converts the hepatocyte phenotype from one of TNF-
resistance, to one of sensitivity to TNF-
-induced death.
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TNF--induced S-CYP15 cell death occurs by necrosis and
apoptosis.
TNF-
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-
treatment. Both
untreated WT and AN-CYP10 cells had low numbers of apoptotic and
necrotic cells that remained unchanged 48 h after TNF-
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-
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).
TNF--induced S-CYP15 cell death is secondary to increased
oxidative stress.
Both TNF-
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-
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-
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-
, S-CYP15 cells
therefore underwent an oxidative stress that resulted in significant
antioxidant depletion.
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S-CYP10 cells are sensitized to TNF--induced death despite
increased levels of NF-
B activation.
A critical determinant of hepatocyte resistance to TNF-
toxicity is
the ability of these cells to activate the transcription factor NF-
B
(5, 24, 51). NF-
B activation was examined in WT,
S-CYP15, and AN-CYP10 cells to determine whether S-CYP15 cell
sensitivity to TNF-
was a function of decreased NF-
B activity in
these cells. EMSA with an NF-
B consensus oligonucleotide
demonstrated that TNF-
induced equivalent levels of NF-
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-
B complex
was composed of p65/p50 heterodimers (data not shown). These results demonstrate that S-CYP15 cell sensitivity to TNF-
was not due to a
failure to activate NF-
B. They also indicate that AN-CYP10 cells
were responsive to TNF-
and therefore their TNF-
resistance was
not due to a loss of TNF receptor.
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S-CYP15 cell death from TNF- is mediated by c-Jun.
Another signaling cascade activated by TNF-
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-
induce JNK (10, 34, 49), the combination of CYP2E1 overexpression and TNF-
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-
treatment compared with AN-CYP10 cells (Fig.
4A). Overinduction of JNK was
not specific to TNF-
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-
stimulation in AN-CYP10 cells, activity was
still increased at 4 h in S-CYP15 cells (Fig. 4B).
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DISCUSSION |
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The cytotoxic effects of TNF- 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-
-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-
B (5, 24, 51) sensitize hepatocytes to TNF-
toxicity in vitro, the in vivo mechanism(s) of hepatocellular TNF-
sensitization remains unclear. The present studies examined the effect
of overexpression of the prooxidant enzyme CYP2E1 on the hepatocyte
response to TNF-
. These investigations were motivated by the fact
that the antioxidant status of the hepatocyte has been shown to affect
TNF-
-induced cell death (13, 38, 52), and CYP2E1
overexpression occurs in association with in vivo TNF-
-dependent
liver injury (29, 43, 47, 48). CYP2E1 overexpression in a
nontransformed hepatocyte cell line sensitized these cells to death
from TNF-
. WT and antisense-CYP2E1-transfected cells had a
proliferative response to TNF-
, whereas CYP2E1-overexpressing cells
underwent cell death. This sensitization to cell death was specific for
the TNF-
death pathway, because in contrast to our findings for
TNF-
, 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-
-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-
treatment. Thus despite the known rapidity of the
TNF-
death signaling cascade, TNF-
had long-term effects on cell
homeostasis, leading to cell death days after ligand-receptor interaction.
According to morphological criteria, TNF- 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- occurred by oxidant stress. This
conclusion is supported by the findings that 1) S-CYP15
cells had higher levels of lipid peroxidation after TNF-
treatment, 2) S-CYP15 cells underwent GSH depletion with TNF-
treatment, and 3) antioxidants blocked TNF-
-induced cell
death. Previous studies (20, 41) have demonstrated that
TNF-
causes a rapid generation of ROS in cells, but the long-term
effects of TNF-
on the cellular redox state have not been examined.
In WT and AN-CYP10 cells 48 h after TNF-
treatment, levels of
lipid peroxidation decreased, suggesting that the initial oxidant
stress caused by TNF-
resulted in a lasting upregulation of cellular
antioxidant defenses in these cells. TNF-
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-
resulted in
a level of oxidative stress sufficient to initiate cell death pathway signaling.
NF-B activation is a critical signal that blocks the TNF-
death
pathway in both nonhepatic cells and hepatocytes (5, 24, 33, 44,
45, 51). The mechanism of S-CYP15 cell sensitivity to
TNF-
-induced death was not decreased NF-
B activity because S-CYP15 cells had higher levels of NF-
B transcriptional activity than either WT or AN-CYP10 cells. Acute oxidant stress created by ROS
administration is well known to increase NF-
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-
B activation. Interestingly, increased NF-
B activation occurred in the absence of any significant change in DNA binding. This finding suggests that the mechanism of
NF-
B activation is related to factors known to regulate NF-
B activity but not binding, such as p65 phosphorylation or the levels of
coactivators (37). The ability of S-CYP15 cells to
undergo TNF-
-induced cell death implies that the effects of CYP2E1
overexpression overrode or acted downstream of the NF-
B-dependent
protective response. Alternatively, S-CYP15 cell death from TNF-
was
predominantly necrotic, and NF-
B activation may only protect against
TNF-
-induced apoptosis. Ethanol-induced hepatocyte
sensitization to TNF-
necrosis has also been described
(13) in the presence of NF-
B activation.
S-CYP15 cell death from TNF- 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-
stimulation. Although JNK may be essential for cell proliferation, abnormally sustained JNK may promote cell death. Sensitization of rat
mesangial cells to TNF-
toxicity by transcriptional arrest has been
reported (21) to be associated with prolonged JNK
activation. The present studies demonstrate that the mechanism of
TNF-
-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-
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- in
the pathogenesis of both alcoholic and nonalcoholic steatohepatitis
(42). The in vivo mechanism of TNF-
hepatotoxicity has
remained unclear. The ability of CYP2E1 overexpression to sensitize
hepatocytes to TNF-
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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