Toxicological Sciences 58, 109-117 (2000)
Copyright © 2000 by the Society of Toxicology
Molecular and Genetic Toxicology |
Protection against Fas Receptor–Mediated Apoptosis in Hepatocytes and Nonparenchymal Cells by a Caspase-8 Inhibitor in Vivo: Evidence for a Postmitochondrial Processing of Caspase-8
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* 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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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Lymphocytes can kill target cells including hepatocytes during various inflammatory diseases by Fas receptor–mediated apoptosis. Caspase-8 is activated at the receptor level, thereby initiating the processing of downstream effector caspases. The aim of this study was to investigate the time course of caspase-8 activation and to evaluate the efficacy of the caspase-8 inhibitor IETD-CHO in a model of Fas-induced apoptosis in vivo. C3Heb/FeJ mice were treated with the anti-Fas antibody Jo-2 (0.6 mg/kg). Western blot analysis demonstrated increased cytochrome c in the cytosol (20 min), which was followed by the progressive activation of caspase-3, -9 (40–120 min), and caspase-8 (120 min). At 90 and 120 min, extensive hemorrhage was observed, indicating damage to sinusoidal lining cells. In addition, high plasma ALT levels (997 ± 316 U/L) and histological evaluation indicated severe parenchymal cell injury. Parenchymal and nonparenchymal cells showed a similar increase in caspase-3 activity and DNA fragmentation. Treatment with IETD-CHO (10 mg/kg) attenuated the increase in caspase-3 activity and DNA fragmentation by 80–90% and completely prevented hemorrhage and parenchymal cell damage. IETD-CHO also prevented the early release of mitochondrial cytochrome c and the processing of caspase-3, -8, and -9. Thus, our data support the hypothesis that Fas-mediated apoptosis is dependent on caspase-8 activation in hepatocytes and nonparenchymal cells. However, the bulk of procaspase-8 is processed late, suggesting that only a small amount of procaspase-8 may actually be activated at the Fas receptor. This initial signal may be amplified by further activation of caspase-8 by effector caspases, i.e., after mitochondrial activation. Caspase-8 is a promising therapeutic target for inhibition of Fas-mediated apoptosis.
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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apoptosis mediated by the Fas receptor (CD95) has been implicated in a growing number of human liver diseases including viral hepatitis, Wilson's disease, alcoholic hepatitis, cholestatic liver disease, and autoimmune disease (Galle and Krammer, 1998
; Nanji, 1998; Patel and Gores, 1995; Strand et al., 1998). Mechanisms of activating the Fas receptors include ligation with Fas ligand expressed on cytotoxic T-lymphocytes (Ando et al., 1993) or on neighboring hepatocytes (Galle et al., 1995). Alternatively, a ligand-independent oligomerization of Fas has been described after exposure to toxic bile acids (Faubion et al., 1999).
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-2–inhibitable 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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Male C3Heb/FeJ mice (20–25 g body weight) were purchased from Jackson Laboratories (Bar Harbor, ME). The animals had free access to food (certified rodent diet #5002C; PMI Feeds, Inc., Richmond, IN) and water. The experimental protocols followed the criteria of Pharmacia & Upjohn, Inc., the University of Arkansas for Medical Sciences, and the National Research Council for the care and use of laboratory animals in research. Animals were treated intravenously with 600 µg/kg of the anti-mouse Fas antibody Jo-2 (PharMingen, San Diego, CA) (Ogasawara et al., 1993
). Some groups of animals were treated with 10 mg/kg of the caspase-8 inhibitor Ac-IETD-CHO (N-acetyl-Ile-Glu-Thr-Asp-aldehyde) (Biomol Research Laboratories, Inc., Plymouth Meeting, PA); the drugs were injected ip 30 min before Jo-2. Vehicle control animals received 2% DMSO in PBS (12 ml/kg) at the same time.
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 = 0–2 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 5–10 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 4–20% 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 anti–caspase-3 polyclonal IgG, rabbit anti–caspase-8 polyclonal IgG, rabbit anti–caspase-9 polyclonal IgG, or rabbit anti–cytochrome 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 (400–700 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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fas Receptor Ligation Initiates Apoptosis in Both Hepatocytes and Sinusoidal Lining Cells
Intravenous administration of 600 µg/kg of the anti-murine Fas antibody Jo-2 caused severe liver injury in C3Heb/FeJ mice, as assessed by the release of ALT into the plasma and the development of severe hemorrhage, indicated by the elevated hemoglobin content in the liver, 90 min after treatment (Table 1
). However, extensive apoptosis preceded the release of ALT and hemorrhage, as determined by increased caspase-3 activity and DNA fragmentation, indicators of apoptotic cell death. These parameters increased significantly as early as 40 min after Jo-2 injection. In contrast, no increase in caspase-8 activity was observed at any time (data not shown). To evaluate whether apoptosis occurred selectively in parenchymal cells (PC) or in addition to affected nonparenchymal cells (NPC), cell fractions were isolated from controls and Jo-treated animals at 45 min. Both PC and NPC showed significantly elevated caspase-3 activity and DNA fragmentation (Fig. 1).
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Caspase-8 Activation Occurs Downstream of Mitochondria during Fas Receptor–Mediated Apoptosis in Vivo
To determine the sequence of apoptotic signaling in liver cells in vivo, Western blot analysis was performed on liver samples following Jo-2 antibody administration at 20–120 min. The earliest detectable change was a moderate increase in cytosolic cytochrome c at 20 min, suggesting early mitochondrial activation (Fig. 2
). Densitometric analysis showed levels of 170 ± 4% compared to baseline (100%). Cytosolic cytochrome c levels were increased to 259 ± 13% at 40 min and 350 ± 59% at 60 min, then started to decline (161 ± 55% at 90 min). At 120 min, little if any cytochrome c was detectable (not shown). In contrast, no processing of procaspase-8 (Fig. 3A) was observed during the first 90 minutes. However, by 120 min, there was clear activation of caspase-8, as indicated by the reduced amount of proenzyme and the appearance of the active fragments p18 and p11. The molecular weights of proenzyme and active fragments were obtained from Van de Craen et al. (1998). In addition, no processing of procaspase-3 was observed during the first 20 min (Fig. 3B). By 40 min, some processing of procaspase-3 was evident with the detection of the p10 active fragment. This observation was in agreement with the caspase-3 activity assay (Table 1
). Substantial processing of procaspase-3 was observed at 90 and 120 min. Similarly, at 90 and 120 min, activation of caspase-9 was observed as reflected by the reduction of the proenzyme (Fig. 3C). The molecular weight of the proenzyme was obtained from Fujita et al. (1999). We were unable to detect the active fragment of caspase-9 due to the inability of the anti–caspase-9 antibody to detect the processed subunit.
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Inhibition of the Caspase-8 Activity Protects against Fas Receptor–Mediated Apoptosis in Vivo
The role of caspase-8 was further evaluated using the caspase-8 inhibitor IETD-CHO. Animals were pretreated with IETD-CHO (10 mg/kg) 30 min prior to Jo-2 administration and liver injury was determined 120 min after Jo-2 treatment. IETD-CHO reduced Jo-2 mediated increase in caspase-3 activity by 90% to similar levels observed in control animals (Table 2
). In addition, Jo-2 mediated DNA fragmentation was attenuated by 80% following pretreatment with IETD-CHO. Moreover, further liver cell injury as indicated by increased plasma ALT activities and hemorrhage determined by tissue hemoglobin content were reduced to baseline values.
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These biochemical findings were confirmed histologically. Liver sections from control mice were morphologically unremarkable (Fig. 4A
). In contrast, livers from Jo-2 antibody treated mice showed extensive apoptotic necrosis of hepatocytes in midzonal locations. The nuclear chromatin of affected hepatocytes was generally condensed, clumped, and frequently fragmented, morphologically consistent with apoptosis (Fig. 4B). The extent of necrosis was characterized as marked to severe in all six treated mice, resulting in a mean severity score of 4.67 ± 0.21 (scale 0–5). A thin rim of viable hepatocytes, sometimes only a single layer thick, persisted around central veins, whereas a thicker zone was present around portal triads. Sinusoidal spaces in the necrotic regions were dilated and filled with red blood cells. Apoptotic necrosis did not occur in livers from two of the five IETD-CHO/Jo-2–treated mice. In the remaining animals, liver necrosis was characterized as minimal in one and mild in the two other mice in this group. The mean severity score for hepatocellular necrosis was 1.0 ± 0.45 (p < 0.05 compared to Jo-2 antibody–treated animals). Necrotic hepatocytes tended to be isolated or in small clusters, but almost always had midzonal location (Fig. 4C). Dilated sinusoids and pooling of red blood cells did not occur in the affected areas.
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Effect of IETD-CHO on Caspase Processing and Mitochondrial Cytochrome C Release
To further substantiate the protective effect of IETD-CHO in Fas–mediated apoptosis, Western blot analysis was performed on liver samples 120 min following Jo-2 treatment (Fig. 5
). Treatment with Jo-2 induced activation of caspase-8 and caspase-3, as indicated by the reduced amount of proenzyme and the appearance of the active fragments (Figs. 5A and 5B). Similarly, activation of caspase-9 was observed as reflected by the reduction of the proenzyme (Fig. 5C). In contrast, IETD-CHO pretreatment reduced Fas-mediated processing of caspase-8, caspase-3, and caspase-9 to baseline levels. To investigate the effect of IETD-CHO treatment on mitochondrial cytochrome c release, cytosolic cytochrome c was determined by Western blotting at 60 min after Jo-2 injection, i.e., at the time of the highest levels in the cytosol. Jo-2 treatment substantially increased cytosolic cytochrome c levels (Fig. 6). Densitometric analysis showed values of 235 ± 22% compared to controls (100 ± 22%). IETD-CHO treatment reduced the Jo-induced increase in cytochrome c levels to baseline values (87 ± 3%). In this experiment, Jo-2 treatment did not enhance ALT values (19 ± 3 U/L). However, caspase-3 activities were significantly increased (320 ± 30 pmol/min/mg protein) compared to controls (23 ± 3). IETD-CHO treatment completely prevented this increase (14 ± 2 pmol/min/mg protein). A similar effect of IETD-CHO on cytosolic cytochrome c was observed 30 min after Jo-2 treatment. Densitometric analysis of cytochrome c Western blots showed that the Jo-induced increase in cytosolic cytochrome c levels was reduced by 75 ± 5% (n = three animals per group). At this time point caspase-3 activity was not significantly increased (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The main objective of this investigation was to evaluate caspase-8 activation and the efficacy of a caspase-8 inhibitor in an experimental model of Fas-induced apoptosis and liver failure in vivo. Our results demonstrated that IETD-CHO effectively prevented hepatocellular apoptosis, hemorrhage, and liver failure after Fas receptor activation in vivo. It is generally accepted that Fas-mediated apoptosis depends on the activation of the caspase cascade (Enari et al., 1996
; Peter and Krammer, 1998). Consistent with this hypothesis, pan-caspase inhibitors, e.g., Z-VAD-fmk, and inhibitors of caspase-3, e.g., DEVD-CHO, effectively prevented Fas-induced apoptosis in the liver (Hentze et al., 1999; Jones et al., 1998; Lawson et al., 1999; Rodriguez et al., 1996b). Caspase-8 is considered the most upstream caspase of the cascade. Caspase-8 activation, i.e., processing of the proenzyme, is initiated at the death-inducing signaling complex (DISC) on the cytosolic side of the Fas receptor (Peter and Krammer, 1998). The active enzyme can then either directly process effector caspases such as procaspase-3 (Fernandes-Alnemri et al., 1996), or indirectly cause activation of caspase-3 through mitochondrial cytochrome c release and caspase-9 processing (Li et al., 1997; Liu et al., 1996). The pathway involving the mitochondria is considered an amplification loop, enabling limited amounts of caspase-8 to rapidly process large amounts of procaspase-3 (Bossy-Wetzel and Green, 1999; Gross et al., 1999b; Kuwana et al., 1998). Cells that have limited processing of caspase-8 at the DISC and require mitochondria for the activation of the caspase cascade are called type II cells (Scaffidi et al., 1998). Our results in the liver indicate that mitochondrial cytochrome c release is a very early event, followed by the activation of caspase-3 and -9. The apparent earlier processing of caspase-3 than caspase-9 (Fig. 3) may due to the fact that reduction of proenzyme levels are difficult to detect. Unfortunately, the available anti-mouse caspase-9 antibody detects only the proenzyme. In contrast, processing of the bulk of caspase-8 appeared to be a later event. One complication with the detection of changes in caspase-8 versus caspase-3 might be that the cells contain much more procaspase-3 than the proenzyme of the regulatory caspase-8. However, even if we can not detect the initial minor activation of caspase-8, the clear effect of the caspase-8 inhibitor IETD-CHO on mitochondrial cytochrome c release at 30 and 60 min after Jo-2 treatment provides indirect evidence for early caspase-8 activation. Together with the demonstrated processing of procaspase-8 at later time points, these results suggest a progressive activation of this regulatory caspase over the 2-h period of the experiment. In addition, the caspase-8 inhibitor IETD-CHO prevented not only the release of mitochondrial cytochrome c and the processing of procaspase-3 and -9, but also the processing of procaspase-8 at 2 h. As a suicide substrate, IETD-CHO can inhibit the active caspase-8. Furthermore, processing of procaspase-8 at the DISC is an autocatalytic process (Peter and Krammer, 1998). Consequently, IETD-CHO may also inhibit part of the processing of caspase-8 at the DISC. Based on the time course and the results of the inhibitor experiments, our data suggest that in liver cells most of the procaspase-8 processing does not occur at the DISC but that postmitochondrial caspases are involved in the activation process. Recent experiments using cell-free extracts or recombinant proteins demonstrated that caspase-9 and caspase-3 can process procaspase-8 (Slee et al., 1999; Van de Craen et al., 1999). Based on these data, our results suggest that during Fas-mediated apoptosis in liver cells in vivo some processing of procaspase-8 may occur at the DISC, but the majority of the proenzyme appears to be processed by postmitochondrial activated caspases, e.g., caspase-3 and -9. The postmitochondrial activation of caspase-8 may function as part of a feedback amplifying loop, which further activates mitochondria, stimulates more cytochrome c release and activates even more effector caspases. The high efficacy of a caspase-8 inhibitor in preventing Fas-mediated apoptosis may be mainly due to these amplification loops. There is a close correlation between Fas receptor–mediated caspase-8 activation in liver cells in vivo compared to type II lymphocyte cell lines (Scaffidi et al., 1998). Our data complement previous reports showing that Fas-induced hepatocellular apoptosis is critically dependent on mitochondrial activation (Lacronique et al., 1996; Rodriguez et al., 1996a; Yin et al., 1999). Together, these data demonstrate that liver cells in vivo are type II cells.
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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This work was supported in part by National Institutes of Health Grant ES-06091.
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NOTES |
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1 To whom correspondence should be addressed at Department of Pharmacology & Toxicology, University of Arkansas for Medical Sciences, 4301 W. Markham St., Mailslot 638, Little Rock, AR 72205–7199. Fax: (501) 686-8970. E-mail: JaeschkeHartmutW{at}exchange.uams.edu.
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