TNF-{alpha}-induced cell death in ethanol-exposed cells depends on p38 MAPK signaling but is independent of Bid and caspase-8

John G. Pastorino, Nataly Shulga, and Jan B. Hoek

Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Submitted 15 October 2002 ; accepted in final form 14 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Alcoholic liver disease is associated with an increase in the number of necrotic and apoptotic liver parenchymal cells. Part of this injury is mediated by TNF-{alpha}. Ethanol exposure sensitizes cells to the cytotoxic effects of TNF-{alpha}. 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-{alpha} 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-{alpha} induction of the MPT and the resulting cytotoxicity. Specifically, ethanol-exposed cells display caspase-8- and Bid-independent cell killing during TNF-{alpha} 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-{alpha}-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


ALCOHOLIC LIVER DISEASE (ALD) in the United States is estimated to affect >2 million people (12). ALD consists of the three major pathological entities: alcoholic steatosis, alcoholic hepatitis, and cirrhosis. These lesions usually occur sequentially, although they may coexist in any combination and occur independently. Proinflammatory cytokines such as TNF-{alpha} 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-{alpha} are clinical manifestations of alcoholic hepatitis such as fever, neutrophilia, anorexia, and muscle wasting. TNF-{alpha} 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-{alpha} seems to play a critical role in the development of ALD. Depletion of TNF-{alpha}-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-{alpha} also prevented the onset of liver damage (23). There is an increase in spontaneous and LPS-mediated TNF-{alpha} bioactivity in the supernatants of monocytes from ALD patients (36). Patients with severe ALD and mortality had higher plasma TNF-{alpha} concentrations than those who survived, with a correlation between plasma TNF-{alpha} levels and serum bilirubin and creatinine values (26). Thus there is strong evidence that TNF-{alpha} contributes to liver injury in ALD.

TNF-{alpha} and its receptor (TNFR) belong to the large TNF-{alpha}-related ligand and TNF-{alpha}/nerve growth factor receptor families. The TNF-{alpha} 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-{alpha} 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-{alpha} also activates the stress kinase p38. TNF-{alpha} is known to activate p38 MAPK either through transforming growth factor-{beta}-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-{alpha} can cause mitochondrial damage. In particular, we demonstrate that TNF-{alpha} 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-{alpha}-induced mitochondrial dysfunction and the resulting cytotoxicity.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Cell culture and treatments. Hepatoma G2E47 (HepG2E47) cells that express cytochrome P-4502E1 (CYP2E1) (kindly provided by Dr. Arthur I. Cederbaum) were maintained in 25-cm2 polystyrene culture flasks with 5 ml MEM containing 100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.4 mg/ml G418, 1 mM pyruvate, and 10% heat-inactivated FBS (complete MEM) incubated under an atmosphere of 95% air-5% CO2 at 37°C. Cells were subcultured 1:5 once a week. The cells were treated with 25 mM ethanol for 48 h before treatment with TNF-{alpha}. 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-{alpha} addition. Thirty minutes after treatment with the above reagents, TNF-{alpha} was added. TNF-{alpha} 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-{alpha} 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.


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Ethanol exposure promotes caspase-8-independent cell killing by TNF-{alpha}. 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-{alpha} (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-{alpha} 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-{alpha} (Fig. 1, A and C). Inhibition of translation and transcription sensitizes many cells to TNF-{alpha} cytotoxicity. This is thought to be due to an inhibition of the protective response of the cell to TNF-{alpha} 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-{alpha} 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-{alpha} 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-{alpha}, the response of the cells to inhibition of caspase-8 differed markedly between the two treatment conditions. HepG2E47 cells treated with TNF-{alpha} and ethanol or TNF-{alpha} 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-{alpha} and CHX but was without effect on the loss of cell viability caused by TNF-{alpha} and ethanol. Similar results were seen in hepatocytes where the caspase-8 inhibitor prevented the cell killing induced by TNF-{alpha} and CHX (Fig. 1D) in control-fed animals but not that induced by TNF-{alpha} 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-{alpha} and CHX, with activity reaching a maximum of eightfold above control levels at 1.5 h. By contrast, HepG2E47 cells treated with TNF-{alpha} and ethanol displayed a lower level of caspase-8 activation compared with that of cells treated with TNF-{alpha} plus CHX, with the maximal level attained being only half of that seen in TNF-{alpha}- and CHX-treated cells.



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Fig. 1. Ethanol exposure promotes caspase-8-independent cell killing by TNF-{alpha}. Hepatoma G2E47 (HepG2E47) cells that express cytochrome P-4502E1 were exposed for 48 h to 25 mM ethanol in complete MEM. The medium was removed and the cells were washed once with PBS and placed in serum-free MEM. A: cells were then treated with 10 ng/ml (220 IU/ml) of TNF-{alpha}. Alternatively, the cells were not exposed to ethanol but pretreated for 30 min with cycloheximide (CHX) before treatment with TNF-{alpha}. Treatment of the cells with TNF-{alpha} alone resulted in little cell killing. B: cells were pretreated for 30 min with 1 µM of the caspase-8 inhibitor Ile-Glu(Ome)-Thr-Asp(Ome) fluoromethylketone (IETD-FMK) before beginning treatment with TNF-{alpha} in either ethanol-exposed cells or in the presence of CHX. C: hepatocytes isolated from control-fed or ethanol-fed animals were treated with TNF-{alpha} alone or treated with TNF-{alpha} in combination with CHX. D: hepatocytes isolated from ethanol-fed animals were pretreated with the caspase-8 inhibitor IETD-FMK and then exposed to TNF-{alpha} for the time indicated. Alternatively, control hepatocytes were pretreated with IETD-FMK and then exposed to TNF-{alpha} in combination with CHX for the time periods indicated. Data are means ± SD from 3 independent experiments.

 


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Fig. 2. TNF-{alpha}-induced caspase-3 activation is independent of caspase-8 in ethanol-exposed cells. HepG2E47 cells were either exposed for 48 h to 25 mM ethanol or left untreated. A: TNF-{alpha} was then added to the ethanol-exposed cells or in combination with CHX to naiïve cells. Cell lysates were prepared, and the levels of caspase-3 and-8 activities were determined as described in the EXPERIMENTAL PROCEDURES. B: cells were pretreated for 30 min with 1 µM of IETD-FMK before treatment with TNF-{alpha} in the ethanol-exposed cells or in combination with CHX in the naiïve cells. C and D: hepatocytes isolated from ethanol-fed animals were treated with TNF-{alpha}, and the levels of caspase-8 or caspase-3 were determined at the time points indicated. D: hepatocytes were pretreated with the caspase-8 inhibitor IETD-FMK 30 min before exposure to TNF-{alpha}. The data are the means ± SD from 3 independent experiments.

 

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-{alpha} and ethanol or TNF-{alpha} and CHX both lead to an increase in caspase-3 activity that was sixfold above control levels between 3 and 4 h after TNF-{alpha} treatment. However, as demonstrated in Fig. 2B, pretreatment of the cells with the caspase-8 inhibitor prevented caspase-3 activation in TNF-{alpha}- and CHX-treated cells but did not prevent the increase in caspase-3 activity brought about by TNF-{alpha} 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-{alpha} in CHX-treated cells is dependent on upstream caspase-8 activation but is independent of caspase-8 activity when brought about by TNF-{alpha} in ethanol-exposed cells. Importantly, similar results with regards to TNF-{alpha}-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-{alpha} 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-{alpha} 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-{alpha}- 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-{alpha} or of non-ethanol-exposed cells with TNF-{alpha} and CHX. As shown in Table 1, FLIP overexpression suppressed the cell killing brought about by TNF-{alpha} and CHX but not that caused by TNF-{alpha} 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-{alpha} and ethanol.


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Table 1. FLIP fails to inhibit TNF-{alpha} cytotoxicity in ethanol-exposed cells

 

Activation of p38 MAPK is both necessary and sufficient for TNF-{alpha}- and ethanol-induced cytotoxicity. The p38 MAPK cascade is activated by a variety of cellular stresses including TNF-{alpha} (Fig. 3). Therefore, the effects of ethanol exposure on the time course of p38 MAPK activation induced by TNF-{alpha} 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-{alpha} alone or TNF-{alpha} 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-{alpha} 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-{alpha} 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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Fig. 3. TNF-{alpha} induced activation of p38 MAPK is potentiated by ethanol exposure in HepG2E47 cells (A) and primary hepatocytes (B). HepG2E47 cells were either exposed for 48 h to 25 mM ethanol or left untreated and then treated with either TNF-{alpha} alone or TNF-{alpha} and CHX (A). Alternatively, primary hepatocytes were isolated from ethanol-fed or control-fed rats and then treated with TNF-{alpha} either alone or in combination with CHX (B). At the indicated time points, cells were harvested. Samples of cell extracts were normalized for protein content, and ELISA was performed to measure phosphorylated p38 MAPK levels. Data are means from 3 independent experiments. OD, optical density.

 

We then wanted to determine whether p38 MAPK activation played a role in TNF-{alpha} and ethanol-induced cell killing. As before, HepG2E47 cells were exposed for 48 h to ethanol and then treated with TNF-{alpha}. However, in some cases the cells were pretreated for 30 min with the p38 MAPK inhibitor, SB-203580, before the addition of TNF-{alpha}. As demonstrated in Fig. 4A, TNF-{alpha} 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-{alpha} 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-{alpha}- 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-{alpha}- and ethanol-induced cell killing. Surprisingly, inhibition of p38 MAPK activity did not prevent the cell killing brought about by TNF-{alpha} 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-{alpha} and CHX alone. Similar results were found in hepatocytes isolated from control-fed vs. ethanol-fed animals, where TNF-{alpha}-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-{alpha} and CHX (Fig. 4C).



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Fig. 4. TNF-{alpha} induced cytotoxicity and caspase-3 activation are dependent on p38 MAPK signaling in ethanol-exposed cells. HepG2E47 cells were exposed to 25 mM ethanol for 48 h or left untreated. Cells were then washed and pretreated for 30 min with 5 µM of the p38 MAPK inhibitor SB-203580 or left untreated. The cells were then treated with 10 ng/ml of TNF-{alpha} alone in ethanol-exposed cells or in combination with CHX in nai

ïve cells. Cell viability (A) and caspases-3 and -8 activities (B) were determined as described in

EXPERIMENTAL PROCEDURES. Similarly, hepatocytes isolated from control-fed or ethanol-fed animals were pretreated for 30 min with 5 µM of the p38 MAPK inhibitor SB-203580 or left untreated. Cell viability (C) and caspases-3 and -8 activities (D) were determined as for HepG2E47 cells. Data are means ± SD from 3 independent experiments.

 

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Table 2. Effect of chemically distinct inhibitors of p38 MAPK on TNF-{alpha}-induced cell killing in HepG2E47 cells exposed to ethanol

 

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-{alpha} 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-{alpha} 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-{alpha}- 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-{alpha} 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-{alpha} and CHX (Fig. 5, B and D). Importantly, inhibition of p38 MAPK activity prevented the onset of mitochondrial depolarization brought about by TNF-{alpha} 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-{alpha} and CHX (Fig. 5, B and C). Conversely, the caspase-8 inhibitor IETD-FMK prevented mitochondrial denergization in TNF-{alpha}- and CHX-treated cells (Fig. 5, B and D) but not in TNF-{alpha}- 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-{alpha}- 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-{alpha}-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-{alpha} 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-{alpha}- and CHX-treated cells, with maximal accumulation seen at 2 h post-TNF-{alpha} treatment (Fig. 6, B and D, lane 2). However, the exact opposite results were obtained in TNF-{alpha}- and ethanol-exposed cells compared with TNF-{alpha}- and CHX-treated cells regarding the effects of caspase-8 and p38 MAPK inhibition. Whereas caspase-8 inhibition prevented cytochrome c release in TNF-{alpha}- and CHX-treated cells, it had no effect on the release of cytochrome c induced by TNF-{alpha} 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-{alpha}-and CHX-treated cells (Fig. 6, A and C, lane 4) but completely prevented that induced by TNF-{alpha} in ethanol-exposed cells (Fig. 6, B and D, lane 4).



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Fig. 5. TNF-{alpha}-induced mitochondrial depolarization in ethanol-exposed cells is dependent on p38 MAPK signaling but is independent of caspase-8 activity. HepG2E47 were either exposed to 25 mM ethanol for 48 h or left untreated. Cells were then pretreated with 5 µM of SB-203580 or 1 µM of IETD-FMK. Cells were then treated with 10 ng/ml TNF-{alpha} either alone in the ethanol-exposed cells (A) or in combination with CHX in nai

ïve cells (B). Similarly, hepatocytes isolated from ethanol-fed (C) or control-fed (D) animals were pretreated with 5 µM of SB-203580 or 1 µM of IETD-FMK. Hepatocytes were then treated with 10 ng/ml TNF-{alpha} either alone in the hepatocytes from ethanol-fed animals (C) or in combination with CHX in control-fed hepatocytes (D). Mitochondrial energization was then estimated at the times indicated as the level of retention of the potential sensitive dye DiOC6(3), as described in EXPERIMENTAL PROCEDURES. Results are typical of 3 independent experiments. TMRM, tetramethylrhodamine methyl ester.

 


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Fig. 6. Differential effects of inhibiting p38 MAPK signaling vs. caspase-8 activity on TNF-{alpha} induced cytochrome c redistribution in ethanol- or CHX-treated cells. HepG2E47 cells were either exposed to 25 mM ethanol for 48 h or left untreated (A and B). Alternatively, hepatocytes were isolated from control or ethanol-fed animals (C and D). Cells were then pretreated with 5 µM of SB-203580 or 1 µM of IETD-FMK for 30 min. Cells were then treated with 10 ng/ml TNF-{alpha} either alone in the ethanol-exposed cells or in combination with CHX in nai

ïve cells. At 2 h posttreatment, cells were harvested and cytosolic fractions were then prepared as described in EXPERIMENTAL PROCEDURES. Samples were normalized for protein content and run out on 12% SDS-PAGE and then electroblotted onto nitrocellulose membranes. Cytochrome c was detected by using an anti-cytochrome c monoclonal antibody and a secondary horseradish peroxidase-labeled antibody. Blots were visualized by using enhanced chemiluminescence. Results are typical of 3 independent experiments.

 

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-{alpha} alone or pretreated for1hwiththe antioxidant ebselen and then treated with TNF-{alpha}. 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-{alpha}, 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-{alpha} (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-{alpha} (Fig. 7, A and B). Such results suggest that the secondary increase of p38 MAPK induced by TNF-{alpha} in ethanol-exposed cells may be brought about by mechanisms other than ROS.



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Fig. 7. The antioxidant ebselen inhibits an early increase of p38 MAPK activation induced by TNF-{alpha} in both control and ethanol-exposed cells but does not prevent a later and more sustained increase of p38 MAPK activation seen in ethanol-exposed cells only. HepG2E47 were exposed to 25 mM ethanol for 48 h (A). Where indicated, cells were pretreated for 30 min with 10 µM ebselen, followed by exposure to 10 ng/ml TNF-{alpha}. Similarly, hepatocytes isolated from ethanol-fed animals were either left untreated or pretreated with 10 µM ebselen for 30 min followed by treatment with 10 ng/ml TNF-{alpha} for the time periods indicated (B). The level of p38 activation was determined by ELISA as described in EXPERIMENTAL PROCEDURES.

 

Ethanol exposure promotes a p38-mediated translocation of Bax to the mitochondria during TNF-{alpha} 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-{alpha} 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-{alpha}- and CHX-treated cells (Fig. 8A, lane 2). By contrast, cells exposed to ethanol and subsequently treated with TNF-{alpha} 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-{alpha}- 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-{alpha} 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-{alpha}- and ethanol-exposed cells, reaching a maximum at 2 h post-TNF-{alpha} 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-{alpha} (Fig. 8B, lane 6). The cell killing brought about by TNF-{alpha} 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-{alpha} in ethanol-exposed cells. Importantly, treatment with the sense oligonucleotide had no effect on Bax expression or TNF-{alpha}- and ethanol-induced cytotoxicity.



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Fig. 8. TNF-{alpha} induces a p38 MAPK-dependent translocation of Bax to the mitochondria in ethanol-exposed cells. HepG2E47 cells were either exposed to 25 mM ethanol for 48 h or left untreated. Cells were then pretreated with 5 µM of SB-203580 or 1 µM of IETD-FMK. Cells were subsequently treated with 10 ng/ml TNF-{alpha} either alone in the ethanol-exposed cells or in combination with CHX in nai

ïve cells. At the indicated posttreatment times, the cells were harvested and mitochondrial fractions were then prepared as described in EXPERIMENTAL PROCEDURES. Samples were normalized for protein content and run out on 15% SDS-PAGE and electroblotted onto nitrocellulose membranes. Bax was detected by using an anti-Bax monoclonal antibody and an anti-mouse horseradish peroxidase-labeled secondary antibody. Bid was detected with a rabbit polyclonal antibody capable of detecting the truncated form. Blots were visualized by using enhanced chemiluminescence. The results are typical of 3 independent experiments. tBid, truncated Bid.

 


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Fig. 9. Bax expression is necessary for TNF-{alpha}-induced cell killing in ethanol-exposed cells HepG2E47 cells were transfected with a 1 µM concentration of an antisense oligonucleotide directed against the first 20 nucleotides of the mRNA for Bax (BaxAS). The sense strand of Bax (BaxS) was used as a control. HepG2E47 cells were incubated for 5 days. On day 3 a subset of the cells was concomitantly exposed to 25 mM ethanol. On day 5, the medium was changed, and the cells were treated with 10 ng/ml TNF-{alpha} for 6 h with cell viability at 6 h determined as described in EXPERIMENTAL PROCEDURES. Data are the means ± SD from 3 independent experiments. Bax was detected by using an anti-Bax monoclonal antibody and an anti-mouse horseradish peroxidase-labeled secondary antibody. Blots were visualized by using enhanced chemiluminescence. Results are typical of 3 independent experiments.

 

Inhibition of TNF-{alpha}-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-{alpha} brings about the phosphorylation of BAD and its resultant sequestration in the cytosol. Therefore, we determined whether the effects of ethanol on TNF-{alpha}-induced cell killing could be explained, at least in part, by ethanol having an effect on TNF-{alpha}-induced BAD phosphorylation. As demonstrated in Fig. 10A, treatment of HepG2E47 cells with TNF-{alpha} 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-{alpha}-induced BAD phosphorylation. Notably, the total level of BAD was unchanged by TNF-{alpha} or ethanol treatment in either condition. A similar inhibition of TNF-{alpha}-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-{alpha} or TNF-{alpha} and CHX induced a marked increase of Akt activation that reached a maximum at 60 min. By contrast, TNF-{alpha}-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-{alpha}-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-{alpha} and ethanol exposure.



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Fig. 10. Ethanol inhibits TNF-{alpha}-induced Bcl-2 antagonist of cell death (BAD) phosphorylation. HepG2E47 cells were exposed to 25 mM ethanol for 48 h or left alone. The cells were then treated with 10 ng/ml TNF-{alpha} (A). Alternatively, primary hepatocytes isolated from chronically ethanol-fed rats or their pair-fed littermates were treated with 10 ng/ml TNF-{alpha} (B). At the times indicated, the cells were harvested, and extracts were prepared and run out on 15% SDS-PAGE. BAD phosphorylated on serine 136 and total BAD were detected by using rabbit polyclonal antibodies at a dilution of 1:500 and anti-mouse horseradish peroxidase-labeled secondary antibody diluted to 1:10,000. Blots were visualized by using enhanced chemiluminescence. Results are typical of 3 independent experiments.

 


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Fig. 11. Ethanol exposure blunts the TNF-induced activation of Akt/PKB HepG2E47 cells either exposed to 25 mM ethanol for 48 h or left untreated (A). Alternatively, primary hepatocytes were isolated from control or ethanol-fed animals (B). The ethanol-naiïve HepG2E47 or hepatocytes were treated with 10 ng/ml TNF-{alpha} alone or in combination with CHX. Similarly, hepatocytes isolated from ethanol-fed animals or HepG2E47 cells exposed to ethanol were treated with TNF-{alpha} alone. At the time points indicated, the level of activated Akt/PKB was measured by ELISA as described in EXPERIMENTAL PROCEDURES.

 

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Table 3. Expression of constitutively active Akt protects against TNF-{alpha}-induced cytotoxicity in ethanol-exposed cells

 


    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
In the present studies, we show that ethanol exposure promotes a TNF-{alpha}-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-{alpha}- 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-{alpha}-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-{alpha} 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-{alpha} 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-{alpha} (11).

However, despite the finding that ethanol exposure sensitized cells to TNF-{alpha}-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-{alpha}- 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-{alpha} family member TNF-{alpha}-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-{alpha}-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-{alpha}-induced cell killing (19). Alternatively, ethanol may enhance TNF-{alpha}-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-{alpha} 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-{alpha}-induced activation of ASK-1 and the downstream p38 MAPK. However, it must be pointed out that ethanol exposure also potentiates TNF-{alpha}-induced cytotoxicity in HepG2 cells not expressing CYP2E1, albeit over a longer time course of TNF-{alpha} treatment (39). In addition, the antioxidant ebselen, although preventing an initial TNF-{alpha}-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-{alpha} 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-{alpha}-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-{alpha}-treated cells are most likely compounded by its ability to inhibit BAD phosphorylation induced by TNF-{alpha}. BAD is phosphorylated by multiple kinases including PKA, Raf-1, and Akt. We have demonstrated that TNF-{alpha} 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-{alpha}-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-{alpha}-induced Akt activation and BAD phosphorylation on serine 136. Moreover, the toxicity of TNF-{alpha} 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-{alpha}. In turn, the increased p38 MAPK activity seen in an ethanol-exposed cell treated with TNF-{alpha} 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-{alpha}-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-{alpha}-induced cytotoxicity and perhaps provide a better understanding of the pathogenesis of hepatocyte injury in ALD.



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Fig. 12. Ethanol promotes Bax-induced mitochondrial dysfunction by enhancing p38 activation and inhibiting the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. TNFR, TNF receptor; ASK, apoptosis-activated kinase; ROS, reactive oxygen species.

 


    DISCLOSURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This study was supported by National Institute on Alcohol Abuse and Alcoholism Grants KO1-AA-00330-1 and 1-R01-AA-12897-01A2.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. G. Pastorino, Dept. of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Rm. 269, Jefferson Alumni Hall, Philadelphia, PA 19107 (E-mail: John.Pastorino{at}jefferson.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.


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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
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