* Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205;
Department of Pharmacology and
Department of Preclinical Toxicology, Pharmacia & Upjohn, Inc., Kalamazoo, Michigan 49007
Received April 6, 2000; accepted July 14, 2000
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ABSTRACT |
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Key Words: liver cell apoptosis; Fas-induced liver failure; caspase cascade; mitochondria; cytochrome c; IETD-CHO; caspase-8 inhibitor.
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INTRODUCTION |
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The intracellular signaling events triggered by Fas-receptor ligation have been characterized in various lymphocyte cell lines (Peter and Krammer, 1998). Stimulation of Fas receptor results in the aggregation of its intracellular domains and the recruitment of FADD (Fas-associated death domain) and procaspase-8, which together with the receptor form the death-inducing signaling complex (DISC) (Peter and Krammer, 1998
). Procaspase-8 is proteolytically activated by association with the DISC (Peter and Krammer, 1998
). Caspase-8 can then directly activate downstream effector caspases such as caspase-3, -6 and -7 (Enari et al., 1996
; Fernandes-Alnemri et al., 1996
). In addition, caspase-8 can activate mitochondria, resulting in the release of cytochrome c (Liu et al., 1996
). Upon assembly of the apoptosome consisting of Apaf-1, cytochrome c, dATP, and procaspase-9, the active caspase-9 is formed, which processes procaspase-3 to the active enzyme (Li et al., 1997
). Caspase-3 cleaves a number of proteins, including an inhibitor protein of endonucleases (Sakahira et al., 1998
). This allows the active endonuclease to enter the nucleus and to initiate DNA degradation. Recently, the missing link between caspase-8 activation and mitochondrial cytochrome c release has been described (Bossy-Wetzel and Green, 1999
; Gross et al., 1999b
). BID, a member of the Bcl-2 family, is located in the cytosol as a 22-kD protein. Proteolytic removal of the N-terminal leaves a 15-kD protein, which inserts into the outer mitochondrial membrane and induces the release of cytochrome c from the mitochondria into the cytosol (Gross et al., 1999b
). The entire process of BID-induced cytochrome c release can be inhibited by Bcl-2 overexpression (Gross et al., 1999a
,b
). Based on the recent characterization of lymphocyte cell lines, the two pathways of caspase-3 activation may not be operating in the same cell simultaneously, but certain cell types may prefer one or the other pathway (Scaffidi et al., 1998
). In type I cells, large amounts of caspase-8 are generated at the DISC and are directly responsible for processing of procaspase-3. On the other hand, type II cells generate low amounts of caspase-8, which initiates the sequence of BID processing, mitochondrial cytochrome c release, caspase-9 activation, and subsequent procaspase-3 processing (Scaffidi et al., 1998
).
Administration of an anti-Fas antibody induces apoptosis in the liver in vivo (Ogasawara et al., 1993). This process involves activation of caspase-3 and caspase-7 (Hentze et al. 1999
; Inayat-Hussein et al. 1997; Jones et al., 1998
; Lawson et al., 1999
; Rodriguez et al., 1996a
). General inhibitors of caspases such as ZVAD-fmk inhibit apoptosis and prevent liver failure (Hentze et al. 1999
; Jones et al., 1998
; Lawson et al., 1999
; Rodriguez et al., 1996a
). The fact that overexpression of Bcl-2 protected against Fas-mediated apoptosis (Lacronique et al., 1996
; Rodriguez et al., 1996b
) supports the hypothesis that hepatocytes behave similar to type II lymphocyte cell lines (Scaffidi et al., 1998
). These cell lines are characterized by delayed caspase-8 activation and the Bcl-2inhibitable mitochondrial release of cytochrome c and caspase-3 activation (Scaffidi et al., 1998
). Despite the description of the protective effect of Bcl-2 overexpression, hepatic caspase-8 activation after Fas receptor stimulation has not been characterized in vivo. In addition, it is unclear how effective pharmacological inhibition of caspase-8 will protect against Fas-mediated hepatocellular apoptosis and fulminant liver failure in vivo. Therefore, the objectives of this investigation were to study the time course of caspase-8 activation in relationship to known postmitochondrial events, e.g., cytochrome c release, and caspase-9 and -3 processing. Furthermore, we tested the efficacy of the caspase-8 inhibitor IETD-CHO to prevent apoptosis and liver failure after Fas receptor activation in vivo.
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MATERIALS AND METHODS |
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Experimental protocol.
Groups of animals were killed by cervical dislocation under ketamine anesthesia (225 mg/kg ketamine; 11.4 mg/kg xylazine; 2.3 mg/kg acepromazine) at different times after injecting Jo-2 (t = 02 h). Blood was collected from the vena cava into a heparinized syringe. The blood was centrifuged and plasma was used for determination of alanine aminotransferase (ALT) activity with test kit DG 159-UV (Sigma Chemical Co., St. Louis, MO). Livers were sectioned transversely across the midportion of each lobe, and pieces of the liver were immediately homogenized for caspase activity measurements and Western blot analysis. Other parts of each liver were frozen in liquid nitrogen and stored at 80°C for analysis of DNA fragmentation, or fixed in phosphate buffered Formalin for histological analysis.
Apoptosis assays.
For DNA fragmentation analysis, the Cell Death Detection ELISA (Boehringer Mannheim, Indianapolis, IN) was used. A 20% liver homogenate in 50 mM Na-phosphate buffer (120 mM NaCl, 10 mM EDTA; pH 7.0) was prepared and centrifuged at 14,000 x g. Diluted supernatant was used for the ELISA. In this test, the kinetics of product generation (vmax) is a measure of DNA fragmentation. The vmax values obtained for untreated controls (100%) are compared to those in treated groups. The assay allows the specific quantitation of histone-associated DNA fragments (mono- and oligonucleosomes) in the cytoplasmic fraction of cell lysates. Although not specific for apoptosis, the DNA fragmentation assay can be used to quantitate apoptosis if the mechanism of cell death has been verified by morphology and other parameters (Hentze et al., 1999; Lawson et al., 1998
, 1999
; Leist et al., 1994
; Jaeschke et al., 1998
). Results with the ELISA were shown to correlate with those of the TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) in the Fas antibody (Lawson et al., 1998
; 1999) and TNF-induced apoptosis model (Jaeschke et al., 1998
; Lawson et al., 1998
) in the liver. For determination of caspase activities, freshly excised liver was homogenized in 25 mM HEPES buffer (pH 7.5) containing 5 mM EDTA, 2 mM DTT and 0.1 % CHAPS. After centrifugation at 14,000 x g, the diluted supernatant was assayed for caspase activity using synthetic fluorogenic substrates: Ac-DEVD-MCA (Ac-Asp-Glu-Val-Asp-MCA) (Peptide Institute, Inc., Osaka, Japan) for caspase 3 (CPP32)/caspase 7 (Mch3), and Ac-IETD-MCA (N-acetyl-Ile-Glu-Thr-Asp-MCA) (California Peptide Research Institute, Inc., Napa, CA) for caspase-8 at concentrations of 50 µM. The kinetics of the proteolytic cleavage of the substrates was monitored in a fluorescence microplate reader (Fmax; Molecular Devices, Corp., Sunnyvale, CA) using an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The fluorescence intensity was calibrated with standard concentrations of MCA and the caspase activity was calculated from the slope of the recorder trace and expressed in pmol/min/mg protein. Protein concentrations in the supernatant were assayed using the bicinchoninic acid kit (Sigma).
Isolation of mouse liver cells.
Parenchymal and nonparenchymal cells were isolated as described previously (Jaeschke et al., 1998). Briefly, the liver was perfused free of blood in an open system for 510 min using an oxygenated Ca2+-free Hanks buffer. A collagenase supplemented (25 mg/100 ml buffer) Hanks buffer was used to digest the liver. When good digestion was obtained (approximately 10 min), the liver was removed, minced, and strained through a tissue sieve. Cells were then centrifuged at 50 x g for 3 min. The supernatant (nonparenchymal cells) was removed and saved. The pellet (parenchymal cells) was resuspended in Hanks buffer and spun at 50 x g for 3 min. The supernatant was combined with the supernatant from the first spin and the pellet resuspended. Cell fractions were then spun at 600 x g for 10 min. The supernatants were discarded and the nonparenchymal pellet was resuspended in pronase buffer (200 mg/50 ml buffer) and stirred for 10 min to remove any hepatocytes in the suspension. This solution was then spun at 600 x g for 10 min and the pellet washed once. Both cell fractions were exposed to an ammonium chloride lysing solution for 10 min to lyse contaminating red blood cells. Cells were washed again, resuspended, and counted. Cell fractions were > 98% pure as assessed microscopically (cell size) and > 95% viable as judged by Trypan blue exclusion. Cell concentrations were adjusted to 4 x 106 cells/ml with either caspase buffer or 50 mM phosphate buffer (DNA fragmentation ELISA).
Western blot analysis.
Liver tissue was homogenized in 25 mM HEPES (pH 7.5) containing 5 mM EDTA, 2 mM DTT, 0.1% CHAPS, 1 µg/ml pepstatin, leupeptin, and aprotinin. Homogenates were centrifuged at 14,000 x g at 4°C for 20 min. Protein concentrations on the cytosolic extracts were determined using the bicinchoninic acid kit (Sigma). Cytosolic extracts (50 µg per lane) were resolved by 420% SDS-polyacrylamide gel electrophoresis under reducing conditions. Separated proteins were transferred to polyvinylidine difluoride membranes (PVDF, Immobilin-P, Millipore, Bedford, MA). The membranes were first blocked with 5% milk in TBS (20 mM Tris, 0.15 M NaCl, 0.1% Tween 20, and 0.1% bovine serum albumin) overnight at 4 °C followed by incubation with primary antibody for 2 h at room temperature. A goat anticaspase-3 polyclonal IgG, rabbit anticaspase-8 polyclonal IgG, rabbit anticaspase-9 polyclonal IgG, or rabbit anticytochrome c polyclonal IgG (Santa Cruz Biotechnology) was used as a primary antibody. The membranes were washed and then incubated with the secondary antibody anti-rabbit IgG-HRP or anti-goat IgG-HRP (Santa Cruz Biotechnology). Proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech. Inc., Piscataway, NJ) according to the manufacturer's instructions. Densitometric analysis of some gels was performed with a GS170 Calibrated Imaging Densitometer (Biorad, Hercules, CA) using Quantity One 4.0.3 software (Biorad).
Histology.
Formalin-fixed portions of the liver were paraffin embedded and 5-µm thick sections were cut. Liver damage was evaluated in H&E stained sections and assigned a score based on the extent of apoptotic necrosis: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe. We use the term necrosis at the later time points to indicate "dead cells" irrespective of how they died. Because in this study most of these cells have morphological features consistent with apoptosis, it is called apoptotic necrosis. The pathologist (SLV) performing the histological evaluation was blinded as to the treatment of animals.
Tissue hemoglobin as indicator for hemorrhage was determined with the Total Hemoglobin Kit (Sigma Diagnostics, St. Louis, MO) as described (Lawson et al., 2000). Briefly, a 20% liver homogenate was prepared in 50 mM Na-phosphate buffer (120 mM NaCl, 10 mM EDTA). After centrifugation at 16,000 x g for 10 min at 4°C, the supernatant was diluted in Drabkin's reagent and the absorbance measured at 550 nm. To account for different background absorbance, the absorbance at 550 nm was obtained from a spectrum (400700 nm). The hemoglobin concentration was determined with a calibration curve and calculated as micrograms hemoglobin/mg liver protein.
Statistics.
Data are given as mean ± SE. Differences between two groups were evaluated with Student's t-test. Comparisons between multiple groups were performed with one-way ANOVA followed by Bonferroni t test. p < 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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A general problem with using peptide inhibitors is the limited specificity (Talanian et al., 1997). Because the concentrations of IETD-CHO achieved in liver cells in vivo are not known, we can not rule out that IETD-CHO may also be able to directly inhibit caspase-3 and other caspases under these in vivo conditions. However, IETD-CHO treatment prevented processing of caspase-3, -8 and -9. Procaspase-8 is the only procaspase that can be activated in vivo without other caspases. This would suggest that inhibition of the active caspase-8 generated at the DISC prevented activation of effector caspases. Thus, potential effects of the inhibitor on other active caspases was not a relevant factor in these experiments. Consequently, one would conclude that even with limited specificity, the hepatoprotective effect of IETD-CHO was due mainly to the inhibition of active caspase-8 generated initially at the DISC.
An interesting aspect of our investigation is the fact that both hepatocytes and nonparenchymal cells show equal susceptibility for Fas-mediated apoptosis, as indicated by similar activation of caspase-3 and DNA fragmentation. Hepatic parenchymal and nonparenchymal cells express Fas receptors (Muschen et al., 1998). Apoptosis in nonparenchymal cells preceded hemorrhage, i.e., the accumulation of red blood cells in the space of Disse. In addition, IETD-CHO inhibited not only hepatocellular apoptosis, but also effectively prevented hemorrhage. This would suggest that apoptotic cell death of sinusoidal lining cells could be the major reason for hemorrhage and the recently described extensive microcirculatory disturbances (Wanner et al., 1999
). In addition to the direct initiation of apoptotic cell death in hepatocytes by the anti-Fas antibody, the secondary microcirculatory problems with potential lack of oxygen, may be a contributing factor for the ultimate severe cell injury and total liver failure in this model.
In summary, our data showed that inhibition of caspase-8 with IETD-CHO effectively prevented Fas-mediated apoptotic cell death, hemorrhage, and liver failure. Interestingly, IETD-CHO did not only prevent activation of downstream effector caspases, but also prevented the processing of the bulk of caspase-8 itself. Because IETD-CHO is a suicide substrate of the active caspase-8, these results suggest that only a small fraction of caspase-8 may have been actually activated at the Fas receptor. Thus, our results together with data in the literature support the hypothesis that the bulk of caspase-8 was activated by effector caspases. These findings suggest that the amplification of the Fas receptor signal in liver cells may not merely involve one passage through mitochondria. In contrast, the initial signal (caspase-8 processing at the receptor) may lead to mitochondrial activation and processing of downstream caspases, which in turn may process more procaspase-8. By going through multiple amplification loops, the activation of the caspase cascade and apoptotic cell death can be maximally accelerated. As shown by our in vivo data, this system makes caspase-8 a highly effective target for therapeutic interventions.
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ACKNOWLEDGMENTS |
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NOTES |
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