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LIVER AND BILIARY TRACT
-induced cell death in ethanol-exposed cells depends on p38 MAPK signaling but is independent of Bid and caspase-8
Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Submitted 15 October 2002 ; accepted in final form 14 May 2003
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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. Ethanol exposure sensitizes cells to the cytotoxic
effects of TNF-. This may be due, in part, to the increased propensity
of the mitochondria in ethanol-exposed cells to induction of mitochondrial
permeability transition (MPT) by various agents, including the proapoptotic
protein Bax. This idea is supported by the observation that increased cell
death induced by TNF- in ethanol-exposed cells was dependent on
development of the MPT. In the present study, we elucidate the pathways
through which ethanol exposure enhances TNF- induction of the MPT and
the resulting cytotoxicity. Specifically, ethanol-exposed cells display
caspase-8- and Bid-independent cell killing during TNF- treatment.
Moreover, the ethanol-enhanced pathway is dependent on p38 MAPK signaling,
which brings about caspase-3 activation, mitochondrial depolarization,
accumulation of cytochrome c in the cytosol, and the translocation of
Bax to the mitochondria. Additionally, ethanol-exposed cells display a
blunting of TNF--induced Akt activation and Bcl-2 antagonist of cell
death phosphorylation that may account, in part, for the increased sensitivity
of the mitochondria to Bax-mediated damage.
alcoholic hepatitis; Bax; caspase-3; mitochondria; apoptosis
and IL-6 have been linked to many of the associated damage
and repair processes seen in ALD
(21,
35). Indeed many of the
effects exerted by TNF- are clinical manifestations of alcoholic
hepatitis such as fever, neutrophilia, anorexia, and muscle wasting.
TNF- is very pleiotropic in its effects on hepatocytes, causing mitosis
or cytotoxicity, depending on the metabolic state of the cell and the presence
of other cytokines and chemokines
(13). TNF- seems to
play a critical role in the development of ALD. Depletion of
TNF--producing Kupffer cells by gadolinium chloride prevented the
development of hepatocyte injury in a rat intragastric feeding model
(1). In addition, neutralizing
antibodies to TNF- also prevented the onset of liver damage
(23). There is an increase in
spontaneous and LPS-mediated TNF- bioactivity in the supernatants of
monocytes from ALD patients
(36). Patients with severe ALD
and mortality had higher plasma TNF- concentrations than those who
survived, with a correlation between plasma TNF- levels and serum
bilirubin and creatinine values
(26). Thus there is strong
evidence that TNF- contributes to liver injury in ALD.
TNF- and its receptor (TNFR) belong to the large TNF--related
ligand and TNF-/nerve growth factor receptor families. The TNF-
ligand binds to TNFR in a trimeric state, inducing receptor trimerization and
activation. There are 75- and 55-kDa TNFRs (TNFR75 and TNFR55, respectively).
Only TNFR55 possesses a cytoplasmic binding motif termed the death domain that
mediates caspase activation (5,
6). Caspases are capable of
activating each other and themselves, which has lent support to the existence
of a caspase cascade (48). The
pathway leading from the binding of TNF- to TNFR55 and caspase
activation involves the recruitment of cytosolic proteins that contain a death
domain. The TNFR55-associated death domain protein (TRADD) binds to TNFR55.
TRADD then induces the binding of Fas-associated protein with death domain
(FADD) that recruits caspase-8 (FLICE). The complex of proteins thus formed is
referred to as the death-inducing signaling complex (DISC)
(45,
53). Oligomerization of
caspase-8 in the DISC has been demonstrated to induce self processing and
activation of the enzyme, which is then capable of activating downstream group
II caspases, such as -3 and -7. The formation of the DISC and the activation
of caspase-8 is a critical decision point in the ultimate fate of the cell.
This is demonstrated by the existence of FLICE-inhibitory protein (FLIP)
(25,
55). FLIP acts as a decoy
protein of caspase-8, binding to FADD, thus becoming incorporated into the
DISC. However, FLIP is unable to become an active caspase and initiate the
caspase cascade because of a substitution of a tyrosine for an active site
cysteine.
The level of caspase-8 activation in the DISC has led to the categorization of cells into two general classes, types I and II (46, 47). In type I cells, caspase-8 is activated to a level ample to directly cleave and activate downstream caspases such as caspase-3. By contrast, in type II cells, the level of caspase-8 activation appears to be inadequate to set off the caspase cascade alone. However, the caspase-8 that is activated is sufficient enough to cleave Bid. Bid is a proapoptotic protein that belongs to the Bcl-2 family (28, 31). The cleavage product of Bid, truncated Bid (tBid), translocates to the mitochondria and induces the release of proapoptotic factors from the mitochondrial intermembrane space, such as apoptosis-inducing factor and cytochrome c. In turn, these agents can cause the activation of caspase-3, which can cause further mitochondrial damage, thus initiating a self-amplifying loop (49).
Besides caspase-8, TNF- also activates the stress kinase p38.
TNF- is known to activate p38 MAPK either through transforming growth
factor-
-activated kinase or apoptosis-stimulating kinase (ASK-1)
(22,
27,
44). In some instances the
activation of p38 MAPK may be important in mediating mitochondrial
dysfunction. Activation of p38 MAPK has been demonstrated to promote
cytochrome c release and Bax translocation to the mitochondria with
the subsequent release of deleterious intermembrane space proteins
(16). In the present study, we
demonstrate that ethanol exposure is capable of altering the pathway by which
TNF- can cause mitochondrial damage. In particular, we demonstrate that
TNF- induces an elevated and prolonged activation of p38 MAPK in
ethanol-exposed cells. Moreover, this p38 MAPK activity is responsible for
mediating a translocation of Bax to the mitochondria and subsequent induction
of mitochondrial dysfunction. Importantly, all of these effects are
independent and distinct from the pathway initiated by caspase-8 activation.
These findings suggest a mechanism through which ethanol exposure can
sensitize cells to TNF--induced mitochondrial dysfunction and the
resulting cytotoxicity.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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. The culture medium was replaced
every 24 h with fresh medium containing ethanol. To prevent evaporation of
ethanol, the flasks or plates were placed in a plastic dessicator in the
incubator containing a mixture of water and ethanol. After 24 h of ethanol
exposure, the cells (control or ethanol treated) were trypsinized, counted in
a hemocytometer, and plated at 1.0 x 105 cells into
1.88-cm2 wells of a 24-well plate for cell viability and membrane
potential measurements or into 6-well, 9.3-cm2 plates at 1.0
x 106 cells for determination of caspase-3 or -8 activity.
The cells were allowed to attach and spread for 24 h. When present, ethanol
was added back to the cells during this time (total time of ethanol exposure,
48 h). On the day of the experiment, the cells were washed and placed in MEM
without serum and in the absence of ethanol. Cells were pretreated for 30 min
with one of the following reagents. Ile-Glu(Ome)-Thr-Asp(Ome)
fluoromethylketone (IETD-FMK) and SB-203580 were dissolved in DMSO and added
to the wells in a 0.2% volume for a final concentration of 1 and 5 µM,
respectively. Control cultures received a comparable volume of DMSO.
Cycloheximide (CHX) was dissolved in water and added to the cell cultures at a
final concentration of 1 µM also 30 min before TNF- addition. Thirty
minutes after treatment with the above reagents, TNF- was added.
TNF- was dissolved in PBS and added to the wells in a 0.2% volume to
give a final concentration of 10 ng/ml (220 U/ml).
Preparation of hepatocytes. Male Sprague-Dawley rats (140-160 g)
were obtained from Charles River Laboratories (Raleigh, NC). Nutritionally
adequate liquid diets were formulated according to the method of Leiber and
DeCarli (30). The
ethanol-containing diet consisted of 18% total calories as protein, 35% as
fat, 11% as carbohydrate, and 36% as ethanol. In the control diet, ethanol was
replaced isocalorically with maltose-dextrin. Isolated hepatocytes were
prepared by collagenase perfusion
(20). Yields of 2-4 x
108 cells per liver with 90-95% viability as assessed by Trypan
blue exclusion were routinely obtained. Hepatocytes were either plated in 24-
or 6-well plates at 1.0 x 105 or 1.0 x 106
cells per well, respectively. The cells were treated with TNF- alone or
in combination with IETD-FMK or SB-203580 as indicated in Cell culture and
treatments for the HepG2E47 cells.
Measurements of cell viability. Cell viability was determined by Trypan blue exclusion and the ability of cells to reduce 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). For Trypan blue exclusion, 10 µl of a 0.5% solution of Trypan blue was added to the wells. Both viable and nonviable cells were counted for each data point in a total of eight microscopic fields. For the MTS assay, the reaction was started by the addition of MTS and phenazine methosulfate (PMS). The absorbance change obtained on reduction of MTS by viable cells was read 90 min later with a plate reader at 490 nm. One-hundred percent cell death was determined by the addition of Triton X-100 to a final concentration of 0.5%, 30 min before MTS and PMS addition. The MTS assay and Trypan blue exclusion gave identical results.
Measurement of mitochondrial energization. Mitochondrial energization was determined as the retention of the dye 3,3'-dihexyloxacabocyanine [DiOC6(3)] (Molecular Probes Eugene, OR). Cells were loaded with 40 nM of DiOC6(3) during the last 30 min of treatment. The cells were then washed twice in PBS. The level of retained DiOC6(3) was measured on a Cytofluor-2 fluorescence plate reader at 488-nm excitation and 500-nm emission.
Detection of caspase-3 and caspase-8 activity. The assay is based on the ability of the active enzymes to cleave the chromophore pNA from the enzyme substrates DEVD-pNA (caspase-3) or IETD-pNA (caspase-8). Cell extracts were prepared and diluted 1:1 with 2x reaction buffer (10 mM Tris, pH 7.4, 1 mM dithiothreitol, 2 mM EDTA, 0.1% CHAPS, 1 mM PMSF, 10 µg/ml pepstatin, 10 µg/ml leupeptin). DEVD-pNA or IETD-pNA were added to a final concentration of 50 µM, and the reaction incubated for1hat37°C. Samples were then transferred to a 96-well plate, and absorbance measurements were made in a 96-well plate reader at 405 nm.
Isolation of cytosolic and mitochondrial fractions. Cells were plated in 25-cm2 flasks at 5.0 x 106 cells per flask. After treatments, the cells were harvested by trypsinization followed by centrifugation at 600 g for 10 min at 4°C. The cell pellets were washed once in PBS and then resuspended in 3 volumes of isolation buffer (in mM: 20 HEPES, pH 7.4, 10 KCl, 1.5 MgCl2, 1 sodium-EDTA, 1 dithiothreitol, 10 PMSF, and 1.0 µg/ml leupeptin, 1.0 µg/ml aprotinin in 250 mM sucrose). After being chilled on ice for 3 min, the cells were disrupted by 40 strokes of a glass homogenizer. The homogenate was centrifuged twice at 2,500 g at 4°C to remove unbroken cells and nuclei. The mitochondria were then pelleted by centifugation at 12,000 g at 4°C for 30 min. The supernatant was removed and filtered through 0.2 µm and then 0.1-µm Ultrafree MC filters (Millipore) followed by centrifugation at 100,000 g at 4°C to yield cytosolic protein. Cytosolic fractions (25 µg protein) were separated on 12% SDS-PAGE gels and electroblotted onto nitrocellulose membranes. Cytochrome c was detected by a monoclonal antibody (Pharmingen, San Diego, CA) at a dilution of 1:5,000. Secondary goat anti-mouse horseradish peroxidase-labeled antibody (Santa Cruz Biotechnology, Santa Cruz CA) diluted at 1:10,000 was detected by enhanced chemiluminescence.
Western blot analysis. After various treatments, equal numbers of cells were harvested by trypsinization and centrifugation at 500 g for 5 min and washed once with PBS. Cell pellets were then lysed in SDS sample buffer. Samples were separated on 12% SDS-PAGE and electroblotted onto nitro-cellulose membranes. Phospho-BAD-136 and BAD were detected by rabbit polyclonal antibody at a dilution of 1:500 (Cell Signaling Technology, Beverly, MA). Bax was detected with an antibody that binds to the NH2 terminus N-20 (Santa Cruz Biotechnology). Bid was detected with a rabbit polyclonal antibody capable of detecting tBid (Biosource, Camarillo, CA). In each case, the relevant protein was visualized by staining with the appropriate secondary horseradish peroxidase-labeled antibody (1:10,000) and was detected by enhanced chemiluminescence.
ELISA of Akt and p38 MAPK activation. Cells were collected in PBS by centrifugation. The cells were then lysed in cell extraction buffer (in mM: 10 Tris, pH 7.4, 100 NaCl, 1 EDTA, 1 EGTA, 1 NaF, 20 Na4P2O7,2Na3VO4, 1 PMSF, and protease inhibitor cocktail plus 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate). Standards, samples, or controls were added to microtiter wells coated with the appropriate antibody (anti-p38 MAPK or anti-Akt). The wells were covered and incubated for2hat room temperature. The wells were then decanted and thoroughly washed four times. One hundred microliters of an antibody, either anti-p38 MAPK (pTpy180/182), anti-p38 MAPK, anti-Akt (pS473), or anti-Akt was added to the wells and incubated for1hat room temperature. The solution was decanted, and the wells were washed four times. Afterward, 100 µl of horseradish peroxidase-conjugated anti-rabbit IgG was added to each well. The wells were incubated for an additional 30 min. The wells were decanted and washed four times. Then 100 µl stabilized chromogen was added to each well and incubated for 30 min in the dark; 100 µl stop solution was then added to each well. The absorbance of each well was then read at 450 nm on a Synergy HTS microplate reader.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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. HepG2E47, which express the ethanol-metabolizing enzyme
CYP2E1, were pretreated with 25 mM ethanol for 48 h. The cells were then
treated with 10 ng/ml TNF- (230 IU/ml)
(Fig. 1, A and
B). Alternatively, primary hepatocytes were isolated from
control or ethanol-fed animals. The cells were plated overnight and then
treated as indicated (Fig. 1, C
and D). As demonstrated in
Fig. 1, A and
C, TNF- treatment of ethanol-exposed HepG2E47
cells or primary hepatocytes obtained from ethanol-fed animals resulted in a
loss of cellular viability of >60 to 70% at 6 h. By contrast, hepatocytes
isolated from control-fed animals and HepG2E47 cells not exposed to ethanol
displayed only a 10% loss of cell viability after a 6-h exposure to
TNF- (Fig. 1, A and
C). Inhibition of translation and transcription
sensitizes many cells to TNF- cytotoxicity. This is thought to be due
to an inhibition of the protective response of the cell to TNF- and
depletion of proteins such as inhibitors of apoptosis and FLIP that bind to
and inhibit caspases. As demonstrated in
Fig. 1, A and
C, HepG2E47 cells or hepatocytes from control-fed animals
treated with TNF- in the presence of the translational inhibitor CHX
exhibited a loss of cell viability that was similar in time course and extent
to that of ethanol-exposed cells treated with TNF- or hepatocytes
isolated from ethanol-fed animals (Fig. 1,
A and C). However, despite their similarity in
the time course and extent of cell death induced by TNF-, the response
of the cells to inhibition of caspase-8 differed markedly between the two
treatment conditions. HepG2E47 cells treated with TNF- and ethanol or
TNF- and CHX were pretreated for 30 min with the caspase-8 inhibitor,
IETD-FMK. As shown in Fig.
1B, IETD-FMK prevented the cell killing brought about by
TNF- and CHX but was without effect on the loss of cell viability
caused by TNF- and ethanol. Similar results were seen in hepatocytes
where the caspase-8 inhibitor prevented the cell killing induced by
TNF- and CHX (Fig.
1D) in control-fed animals but not that induced by
TNF- alone in hepatocytes isolated from ethanol-fed animals
(Fig. 1D). Because of
these results, we examined the activity of caspase-8 in the two treatment
protocols. As shown in Fig.
2A, caspase-8 is quickly activated in HepG2E47 cells
treated with TNF- and CHX, with activity reaching a maximum of
eightfold above control levels at 1.5 h. By contrast, HepG2E47 cells treated
with TNF- and ethanol displayed a lower level of caspase-8 activation
compared with that of cells treated with TNF- plus CHX, with the
maximal level attained being only half of that seen in TNF-- and
CHX-treated cells.
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Caspase-8 can activate caspase-3 either directly as in type I cells or
through a mitochondrial pathway as in so-called type II cells. As shown in
Fig. 2A, treatment of
HepG2E47 cells with TNF- and ethanol or TNF- and CHX both lead
to an increase in caspase-3 activity that was sixfold above control levels
between 3 and 4 h after TNF- treatment. However, as demonstrated in
Fig. 2B, pretreatment
of the cells with the caspase-8 inhibitor prevented caspase-3 activation in
TNF-- and CHX-treated cells but did not prevent the increase in
caspase-3 activity brought about by TNF- and ethanol, despite the
ability of the inhibitor to eliminate caspase-8 activity under both treatment
conditions. These results clearly demonstrate that caspase-3 activation
triggered by TNF- in CHX-treated cells is dependent on upstream
caspase-8 activation but is independent of caspase-8 activity when brought
about by TNF- in ethanol-exposed cells. Importantly, similar results
with regards to TNF--induced caspase activation were obtained in
primary hepatocytes isolated from ethanol-fed rats. Caspase-8 and -3
activities reached four- and eightfold above control levels at 2 and 4 h,
respectively (Fig.
2C). TNF- did not bring about activation of
caspase-8 or -3 in hepatocytes isolated from control-fed animals, consistent
with the lack of cell killing under these conditions (results not shown).
Identical to HepG2E47 cells exposed to ethanol, the caspase-8 inhibitor
prevented the caspase-8 activation induced by TNF- in hepatocytes
isolated from ethanol-fed animals (Fig.
2D) but did not diminish the activity of caspase-3
(Fig. 2D).
The robust caspase-8 activity seen in TNF-- and CHX-treated cells is
probably due to a depletion of endogenous caspase inhibitors such as FLIP by
translational inhibition. As mentioned above, FLIP is an inactive decoy of
caspase-8 that has been shown to inhibit caspase-8 activation and prevent cell
killing. HepG2E47 cells were transfected with an expression construct for FLIP
24 h before treatment of ethanol-exposed cells with TNF- or of
non-ethanol-exposed cells with TNF- and CHX. As shown in
Table 1, FLIP overexpression
suppressed the cell killing brought about by TNF- and CHX but not that
caused by TNF- and ethanol. These results demonstrate that even an
endogenous inhibitor of caspase-8 such as FLIP does not inhibit the cell
killing induced by TNF- and ethanol.
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Activation of p38 MAPK is both necessary and sufficient for
TNF-- and ethanol-induced cytotoxicity. The p38 MAPK
cascade is activated by a variety of cellular stresses including TNF-
(Fig. 3). Therefore, the
effects of ethanol exposure on the time course of p38 MAPK activation induced
by TNF- were determined by ELISA. In non-ethanol-exposed HepG2E47 cells
or hepatocytes, by 15 min, p38 MAPK phosphorylation was twofold above basal
values (0 time point) in cells treated with TNF- alone or TNF-
and CHX and declined back down to basal levels by 45 min. HepG2E47 cells
exposed to ethanol and hepatocytes isolated from ethanol-fed animals exhibited
an increase of p38 MAPK activation that was similar initially to that of
non-ethanol-exposed cells. Treatment of both HepG2E47 cells exposed to ethanol
or hepatocytes isolated from ethanol-fed animals with TNF- produced an
increase of phosphorylated p38 MAPK of twofold above basal levels at 15 min
that then declined back down to basal levels by 45 min. However, in contrast
to non-ethanol-exposed cells, there was a robust elevation of p38 MAPK
activation starting at 60 min and reaching a maximum of three- to fourfold
above basal levels at 2 h and remaining elevated even after 4 h. Importantly,
it should be noted that treatment with TNF- under either condition did
not result in an increase in the absolute level of p38 MAPK protein as
determined by ELISA (results not shown).
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We then wanted to determine whether p38 MAPK activation played a role in
TNF- and ethanol-induced cell killing. As before, HepG2E47 cells were
exposed for 48 h to ethanol and then treated with TNF-. However, in
some cases the cells were pretreated for 30 min with the p38 MAPK inhibitor,
SB-203580, before the addition of TNF-. As demonstrated in
Fig. 4A, TNF-
and ethanol caused a 72% loss of cell viability over 6 h. By contrast, cells
pretreated with SB-203580 were still 95% viable after 6 h of TNF-
treatment of ethanol-exposed cells. Importantly, as shown in
Table 2, other inhibitors of
p38 MAPK that are chemically distinct from SB-203580 also protected against
TNF-- and ethanol-induced cell killing. In addition, SB-202474, a
compound with a similar chemical structure to SB-203580 but inactive against
p38 MAPK, did not inhibit TNF-- and ethanol-induced cell killing.
Surprisingly, inhibition of p38 MAPK activity did not prevent the cell killing
brought about by TNF- and CHX treatment, with a >70% loss of cell
viability seen after 6 h of treatment, in the presence of the p38 MAPK
inhibitor SB-203580 (Fig.
4A), levels of cell killing similar to that seen with
TNF- and CHX alone. Similar results were found in hepatocytes isolated
from control-fed vs. ethanol-fed animals, where TNF--induced cell
killing in hepatocytes isolated from ethanol-fed animals was prevented by the
inhibitor of p38 MAPK (Fig.
4C) but had no effect on the loss of cell viability in
hepatocytes isolated from control-fed animals caused by the combination of
TNF- and CHX (Fig.
4C).
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We then examined the effect of inhibiting p38 MAPK on caspase-8 and -3
activities. Pretreatment of the cells with SB-203580 had no effect on the time
of onset or level of activation of caspase-8 induced by TNF- in either
ethanol-exposed HepG2E47 cells or in hepatocytes isolated from ethanol-fed
animals (Fig. 4, B and
D). However, inhibition of p38 MAPK markedly prevented
caspase-3 activation in both ethanol-exposed HepG2E47 cells and hepatocytes
isolated from ethanol-fed animals (Fig. 4,
B and D). Importantly, the p38 MAPK inhibitor
did not prevent the caspase-3 activation induced by TNF- in combination
with CHX in either HepG2E47 cells or hepatocytes (results not shown),
consistent with the inability of p38 inhibition to prevent cell killing under
these conditions.
Inhibition of p38 MAPK prevents mitochondrial dysfunction in
TNF-- and ethanol-exposed cells. Mitochondrial injury has
been demonstrated to be a pivotal event in both apoptotic and necrotic cell
death. As shown in Fig. 5, A and
C, HepG2E47 cells exposed to ethanol or hepatocytes
obtained from ethanol-fed animals treated with TNF- displayed a loss of
mitochondrial membrane potential over 3 h. A similar loss of mitochondrial
energization was seen in HepG2E47 cells not exposed to ethanol or in
hepatocytes isolated from control-fed animals treated with TNF- and CHX
(Fig. 5, B and
D). Importantly, inhibition of p38 MAPK activity
prevented the onset of mitochondrial depolarization brought about by
TNF- in HepG2E47 cells exposed to ethanol and in hepatocytes isolated
from ethanol-fed animals (Fig. 5,
A and C) but not in those treated with
TNF- and CHX (Fig. 5, B and
C). Conversely, the caspase-8 inhibitor IETD-FMK
prevented mitochondrial denergization in TNF-- and CHX-treated cells
(Fig. 5, B and
D) but not in TNF-- and ethanol-exposed cells
(Fig. 5, A and
B). Mitochondrial dysfunction during cell injury is
associated with the release of injurious mitochondrial intermembrane space
proteins such as cytochrome c. As demonstrated in
Fig. 6, A and
C, lane 2, there was an accumulation of
cytochrome c in the cytosol of TNF-- and CHX-treated HepG2E47
cells (A) or hepatocytes obtained from control-fed animals
(C) that was maximal at 2 h, a time point coinciding with the onset
of caspase-3 activation. Accumulation of cytochrome c in the cytosol
of TNF--and CHX-treated cells was prevented by caspase-8 inhibition but
not by p38 MAPK inhibition (Fig. 6,
A and C, lanes 3 and 4,
respectively). Treatment with TNF- of ethanol-exposed HepG2E47 cells
(B) or hepatocytes isolated from ethanol-fed animals (D)
exhibited an accumulation of cytochrome c in the cytosol similar to
that of TNF-- and CHX-treated cells, with maximal accumulation seen at
2 h post-TNF- treatment (Fig. 6,
B and D, lane 2). However, the exact
opposite results were obtained in TNF-- and ethanol-exposed cells
compared with TNF-- and CHX-treated cells regarding the effects of
caspase-8 and p38 MAPK inhibition. Whereas caspase-8 inhibition prevented
cytochrome c release in TNF-- and CHX-treated cells, it had no
effect on the release of cytochrome c induced by TNF- in
ethanol-exposed HepG2E47 cells or in hepatocytes isolated from ethanol-fed
animals (Fig. 6, B and
D, lane 3). By contrast, p38 MAPK inhibition had
no effect on cytochrome c accumulation in the cytosol of
TNF--and CHX-treated cells (Fig. 6,
A and C, lane 4) but completely
prevented that induced by TNF- in ethanol-exposed cells
(Fig. 6, B and
D, lane 4).
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The role of reactive oxygen species in p38 MAPK activation.
HepG2E47 cells exposed to ethanol or hepatocytes isolated from ethanol-fed
animals were either treated with TNF- alone or pretreated for1hwiththe
antioxidant ebselen and then treated with TNF-. As demonstrated in
Fig. 7, pretreatment of either
ethanol-exposed HepG2E47 (A) or hepatocytes isolated from ethanol-fed
animals (B) with ebselen prevented the initial spike in p38 MAPK
activation induced by TNF-, suggesting that it may be mediated by the
generation of reactive oxygen species (ROS). Importantly, this initial
activation of p38 MAPK was also seen in non-ethanol-exposed cells treated with
TNF- (Fig. 3). However,
ebselen did not prevent the secondary and much greater and more sustained
increase of p38 MAPK activation characteristic of ethanol-exposed cells
treated with TNF- (Fig. 7,
A and B). Such results suggest that the
secondary increase of p38 MAPK induced by TNF- in ethanol-exposed cells
may be brought about by mechanisms other than ROS.
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Ethanol exposure promotes a p38-mediated translocation of Bax to the
mitochondria during TNF- treatment. A number of
proapoptotic proteins of the Bcl-2 family have been demonstrated to mediate
mitochondrial injury. Bid is a BH3-domain protein that is cleaved and
truncated by caspase-8 to a more active form, tBid. As demonstrated in
Fig. 8A, lane
1, mitochondria isolated from HepG2E47 cells treated with TNF- and
CHX show an accumulation of tBid that reached a maximum at 1 h. Importantly,
the accumulation of mitochondrial tBid was prevented by caspase-8 inhibition,
thus suggesting that caspase-8 activation is a prerequisite for Bid
translocation to the mitochondria in TNF-- and CHX-treated cells
(Fig. 8A, lane
2). By contrast, cells exposed to ethanol and subsequently treated with
TNF- displayed no mitochondrial accumulation of tBid
(Fig. 8A, lane
4). This observation may be due to the low level of caspase-8 activity
detected under these treatment conditions. Interestingly, p38 MAPK inhibition
had no effect on tBid accumulation in TNF-- and CHX-treated cells, in
agreement with the inability of the p38 MAPK inhibitor to prevent caspase-8
activation under these treatment conditions
(Fig. 8A, lane
3). Bax is another member of the Bcl-2 family that is proapoptotic and
induces mitochondrial injury. Moreover, the translocation of Bax to the
mitochondria has been shown to be induced by p38 MAPK under some
circumstances. Because TNF- treatment of ethanol-exposed cells
generated no accumulation of tBid in the mitochondria, we wanted to determine
whether Bax could account for the mitochondrial dysfunction observed. As shown
in Fig. 8B, Bax
accumulates markedly in the mitochondria of TNF-- and ethanol-exposed
cells, reaching a maximum at 2 h post-TNF- treatment. This accumulation
of Bax at the mitochondria was dependent on p38 MAPK activation as
demonstrated by the ability of SB-203580 to inhibit it at 2 h
(Fig. 8B, lane
5). By contrast, inhibition of caspase-8 had no effect on mitochondrial
Bax accumulation in ethanol-exposed cells treated with TNF-
(Fig. 8B, lane
6). The cell killing brought about by TNF- and ethanol was
dependent on Bax. We suppressed the expression of Bax in the HepG2E47 cells by
using an antisense construct. As demonstrated in
Fig. 9A, a 5-day
treatment with the Bax antisense eliminated Bax expression. As shown in
Fig. 9B, the cells
treated with the Bax antisense oligonucleotide exhibited resistance to the
cell killing brought about by TNF- in ethanol-exposed cells.
Importantly, treatment with the sense oligonucleotide had no effect on Bax
expression or TNF-- and ethanol-induced cytotoxicity.
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Inhibition of TNF--induced Akt activation and BAD
phosphorylation in ethanol-exposed cells. BAD is a proapoptotic member of
the Bcl-2 family that does not induce mitochondrial dysfunction in and of
itself. However, BAD does increase the susceptibility of the mitochondria to
proteins like Bax, by binding to and inhibiting the ability of antiapoptotic
proteins such as Bcl-2 and Bcl-XL to prevent Bax-induced damage. We
have previously demonstrated
(41) that TNF- brings
about the phosphorylation of BAD and its resultant sequestration in the
cytosol. Therefore, we determined whether the effects of ethanol on
TNF--induced cell killing could be explained, at least in part, by
ethanol having an effect on TNF--induced BAD phosphorylation. As
demonstrated in Fig.
10A, treatment of HepG2E47 cells with TNF- alone
resulted in an increase in the levels of phosphorylated BAD over a 120-min
time course. In contrast, cells exposed to ethanol for 48 h exhibited a
significant blunting of TNF--induced BAD phosphorylation. Notably, the
total level of BAD was unchanged by TNF- or ethanol treatment in either
condition. A similar inhibition of TNF--induced BAD phosphorylation was
seen in hepatocytes isolated from ethanol-fed rats
(Fig. 10B). BAD is
phosphorylated on serines 112 and 136 by AKT/PKB. It is possible that ethanol
treatment may inhibit BAD phosphorylation and thereby potentiate cell killing
by affecting the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway.
Similar to p38 MAPK, we measured the activation of Akt by ELISA. As shown in
Fig. 11, both HepG2E47 cells
exposed to ethanol (A) and hepatocytes isolated from ethanol-fed
animals (B) exhibited an increase of basal Akt activation over that
of non-ethanol-exposed cells (0 time point). Treatment of ethanol-naiïve
HepG2E47 cells or hepatocytes isolated from control-fed animals with
TNF- or TNF- and CHX induced a marked increase of Akt activation
that reached a maximum at 60 min. By contrast, TNF--induced Akt
activation was markedly blunted in HepG2E47 cells exposed to ethanol and in
hepatocytes isolated from ethanol-fed animals. To further demonstrate the
importance of ethanol exposure in hindering TNF--induced Akt
activation, HepG2E47 cells were transiently transfected with a cDNA encoding a
constitutively active form of Akt (myr-Akt). As demonstrated in
Table 3, HepG2E47 cells
expressing constitutively active myr-Akt were rescued from the cell killing
induced by TNF- and ethanol exposure.
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-induced cytotoxicity that is mediated by sustained p38 MAPK
activation and is independent of caspase-8 and Bid activation. Inhibition of
p38 MAPK signaling prevented the translocation of Bax to the mitochondria in
TNF-- and ethanol-treated cells, with a resultant prevention of cell
death. Moreover, the effects of ethanol are further compounded by the ability
of ethanol exposure to blunt TNF--induced Akt activation and
phosphorylation of BAD. Expression of a constitutively active form of Akt/PKB
ameliorated the loss of cell viability induced by TNF- and ethanol.
The effects of ethanol on mitochondrial function are mutifactorial.
Mitochondria isolated from ethanol-fed rats exhibit an increase in
acetaldehyde protein adducts and ROS production and a decrease in GSH levels,
mitochondrial DNA, and complex I and III activities, among other alterations
(3,
7,
14). HepG2E47 cells, which
express the ethanol-inducible and -metabolizing enzyme CYP2E1 display similar
alterations in mitochondrial function and a propensity for induction of the
mitochondrial permeability transition (MPT)
(39,
56,
57). Such alterations leave
the mitochondria vulnerable to secondary insults. Indeed, we have demonstrated
that mitochondria isolated from ethanol-fed rats are more susceptible to
induction of the MPT induced by a wide variety of agents, including Bax
(40). This increased
sensitivity of the mitochondria is reflected in the ability of TNF- to
induce cytotoxicity in HepGE47 cells exposed to ethanol and in primary
hepatocytes isolated from ethanol-fed rats in the absence of transcriptional
or translational inhibitors
(39). Similar results have
been reported in cultured astrocytes, in which exposure of the cells to 5 mM
ethanol increased their sensitivity to TNF-
(11).
However, despite the finding that ethanol exposure sensitized cells to
TNF--induced cell death, the signaling pathways through which this was
mediated remained unclear. As mentioned, the assembly of the DISC at the TNFR
results in the activation of caspase-8. In type I cells, caspase-8 activation
is sufficient to activate downstream effector caspases such as caspase-3.
However, in type II cells such as HepG2E47, the amount of caspase-8 activated
is inadequate to cleave and initiate caspase-3 activity to a level sufficient
to induce cell death. In type II cells it has been shown that a mitochondrial
pathway augments the ability of caspase-8 to bring about cell killing.
Caspase-8 cleaves the BH3-protein Bid at an internal aspartate site to yield a
truncated 15-kDa protein (tBid) that translocates to the mitochondria
(29). tBid has been shown to
release cytochrome c in isolated mitochondria and intact cells
(31,
34). However, tBid is also
capable of interacting with Bax. In nonapoptotic cells, Bax is located in the
cytosol and in loose association with mitochondria
(17). On an apoptotic
stimulus, the COOH-terminal domain of Bax is unmasked, which promotes
translocation and the insertion of Bax into the mitochondrial membrane
(17,
37,
38,
43,
51). tBid is capable of
interacting with Bax to induce this conformational transformation and
subsequent induction of mitochondrial injury
(10). Indeed, it has been
demonstrated that a tBid-dependent conformational change of Bax is suppressed
by E1B-19K, an adenovirus gene product capable of binding Bax and inhibiting
apoptosis in TNF-- and CHX-treated HeLa cells
(42). However, E1B-19K did not
prevent caspase-8 activation or cleavage of Bid. Such results suggest that,
although caspase-8 and tBid are necessary for Bax to assume an active
conformation, caspase-8 and tBid are not sufficient under all circumstances by
themselves to induce cell death. Indeed, the TNF- family member
TNF--related apoptosis-inducing ligand induced robust caspase-8
activation and formation of tBid in Bax-negative cells, despite its inability
to induce cell death in these same cells
(9).
Such results point to the fact that Bax in and of itself can bring about mitochondrial damage and cell death. However, to accomplish this, Bax must assume an active conformation. As mentioned, the binding of tBid can bring this about. However, Bax can also undergo translocation to the mitochondria by a p38 MAPK-dependent mechanism. Indeed, it has been demonstrated that p38 MAPK kinase activity plays a critical role in nitric oxide-mediated cell death in neurons by stimulating Bax translocation to the mitochondria (16). Similarly, T-cell receptor stimulation of thymocytes resulted in a p38 MAPK-mediated translocation of Bax to the mitochondria with a resultant disruption in mitochondrial membrane potential, release of cytochrome c, and caspase-3 activation (58). All of these effects were independent of caspase-8 activity and tBid. Inhibition of p38 MAPK activity also protects against the cell killing induced by singlet oxygen, ultraviolet B radiation, cadmium, doxorubicin, and ischemia (2, 15, 24, 32, 33, 60) However, at present, the mechanism by which p38 MAPK brings about Bax translocation is unknown. Direct phosphorylation of Bax by p38 MAPK has not been demonstrated. Active p38 MAPK may phosphorylate an inhibitory binding partner of Bax such as Bcl-2. Indeed, nerve growth factor withdrawal in B-lymphocytes induces a p38 MAPK-dependent phosphorylation of Bcl-2 that was required for apoptosis (50).
The mode by which ethanol exposure increases the duration and intensity of
TNF--induced p38 MAPK activation is a matter under current
investigation. Ethanol is known to have an inhibitory effect on the activity
of some phosphatases. Ethanol may inhibit the MAPK phosphatase activity (MKP-1
and -2) that dephosphorylates and inactivates p38 MAPK. Overexpression of
MKP-1 protected rat mesangial cells from TNF--induced cell killing
(19). Alternatively, ethanol
may enhance TNF--induced apoptosis-activated kinase-1 (ASK-1)
activation, a MAPKKK. ASK-1 binds to thioredoxin
(44). Thioredoxin is a direct
inhibitor of ASK-1 signaling. Thioredoxin binding and inhibition of ASK-1 is
redox sensitive. TNF- or hydrogen peroxide treatment of cells resulted
in a dissociation of thioredoxin from ASK-1, with the resultant activation of
ASK-1. As mentioned, ethanol exposure promotes an oxidative stress in cells;
such a state of oxidative stress may potentiate TNF--induced activation
of ASK-1 and the downstream p38 MAPK. However, it must be pointed out that
ethanol exposure also potentiates TNF--induced cytotoxicity in HepG2
cells not expressing CYP2E1, albeit over a longer time course of TNF-
treatment (39). In addition,
the antioxidant ebselen, although preventing an initial TNF--induced
spike of p38 MAPK stimulation, did not prevent the more sustained and greater
increase of p38 MAPK activation seen in ethanol-exposed cells. These results
suggest that ethanol may have effects on the ability of TNF- to elicit
the p38 MAPK pathway independent of those mediated by oxidative stress.
It has been demonstrated that Akt downregulates p38 MAPK activity
(4,
18). Therefore, the
possibility exists that ethanol exposure may potentiate p38 MAPK signaling by
inhibiting the PI3-kinase/Akt pathway. Indeed, in the present study, we have
demonstrated that ethanol exposure inhibits TNF--induced Akt activation
and BAD phosphorylation, a phosphorylation that most likely is mediated by
Akt.
By whatever mechanism that ethanol exposure potentiates p38 MAPK
activation, ethanol's deleterious effects on mitochondria in
TNF--treated cells are most likely compounded by its ability to inhibit
BAD phosphorylation induced by TNF-. BAD is phosphorylated by multiple
kinases including PKA, Raf-1, and Akt. We have demonstrated that TNF-
induces phosphorylation of BAD on serine 136 and that this is mediated by Akt
(41). Indeed, inhibition of
the PI3-kinase/Akt pathway potentiates TNF--induced cytotoxicity.
Others (59) have also
demonstrated that ethanol exposure inhibits the PI3-kinase signaling pathway
induced by receptor activation. As demonstrated in Figs.
10 and
11, both HepG2E47 cells
exposed to ethanol and hepatocytes isolated from ethanol-fed animals displayed
an inhibition of TNF--induced Akt activation and BAD phosphorylation on
serine 136. Moreover, the toxicity of TNF- in ethanol-exposed cells was
ameliorated by expression of constitutively active Akt. Phosphorylation of BAD
promotes its binding to the cytosolic protein, 14-3-3
(54). When unphosphorylated,
BAD is localized to the mitochondria. However, unlike Bax, BAD is not directly
damaging to the mitochondria
(8,
28). Rather, its association
with Bcl-2 and/or Bcl-XL is thought to increase the susceptibility
of the mitochondria to damage by proteins such as Bax. By binding to
Bcl-2/Bcl-XL, BAD neutralizes their ability to bind and inhibit
Bax. Additionally, it has recently been demonstrated that the PI3-kinase/Akt
pathway can also suppress the translocation of Bax to the mitochondria
(52). This result suggests
that ethanol exposure may have a three-tier effect on the ability of Bax to
promote mitochondrial dysfunction, by increasing p38 MAPK's and decreasing
PI3-kinase/Akt's ability to promote and inhibit, respectively, Bax
translocation to the mitochondria and by enhancing Bax's ability to cause
mitochondrial injury by inhibiting BAD phosphorylation.
In summary, ethanol exposure potentiates p38 MAPK activation induced by
TNF-. In turn, the increased p38 MAPK activity seen in an
ethanol-exposed cell treated with TNF- is necessary and sufficient to
mediate the translocation of Bax to the mitochondria in which it induces
mitochondrial damage resulting in the release of deleterious intermembrane
space proteins such as cytochrome c. Moreover, the translocation of
Bax occurs through a pathway that is independent and distinct from that
mediated by caspase-8 activation and Bid cleavage
(Fig. 12). Because p38 MAPK
activity has been demonstrated to be necessary and sufficient for cell death
in other contexts, it will be important to determine the mechanism by which
ethanol potentiates p38 MAPK activity and, in turn, how this brings about Bax
activation. Additionally, the mechanism by which ethanol exposure prevents
TNF--stimulated Akt activation and phosphorylation of BAD and the
effects that this has on Bax's ability to induce mitochondrial dysfunction
will need to be clarified. The elucidation of ethanol's effects on these
pathways will help decipher how it potentiates TNF--induced
cytotoxicity and perhaps provide a better understanding of the pathogenesis of
hepatocyte injury in ALD.
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DISCLOSURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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in rat mesangial cells. J Biol Chem
273: 10362-10366,
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Hepatology 31:
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Cell 6: 53-63,
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