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*
Amgen Institute and Ontario Cancer Institute and
Departments of Immunology and Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; and
Ontario Cancer Institute, Department of Oncologic Pathology, Toronto, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fas, a transmembrane receptor protein belonging to the TNF receptor family, contains a death domain mediating PCD. Fas is constitutively expressed on hepatocytes and has been implicated in many human liver diseases, including autoimmune hepatitis and hepatocellular carcinoma (6, 7). Fulminant hepatitis, an accelerated liver failure that is often fatal, is a complication of many human liver diseases. Injection of an animal with agonistic anti-Fas Ab in vivo causes an immediate and massive apoptotic death of hepatocytes, resulting in fulminant hepatitis and death within hours. The fact that this process is indeed Fas-mediated has been demonstrated by injecting anti-Fas Ab into mice with the lymphoproliferation mutation lpr. The lpr mice lack virtually all functional Fas and are completely resistant to liver failure induced by anti-Fas injection (8).
There is now much in vitro and some in vivo evidence that caspases are involved in the Fas-mediated cell death pathway (9, 10, 11). Pretreatment of cultured cells with the general caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z VAD FMK) has been shown to inhibit anti-Fas-mediated apoptosis (12). Similarly, treatment of hepatocytes with N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac DEVD CHO), an inhibitor that specifically blocks caspase-3-like caspases, also inhibits Fas-mediated apoptosis (13). When mice were injected systemically with caspase inhibitors before treatment with agonistic anti-Fas Ab, the mice failed to show significant apoptosis in the liver and were resistant to hepatic failure (14). In addition, we have previously shown that activated lymphocytes from caspase-3-deficient mice are resistant to apoptosis induced by in vitro treatment with anti-Fas Ab (10). Finally, a recent study of cultured hepatocytes showed that both caspase-3 and -7 are activated upon Fas ligation, implicating them as effector caspases for hepatocyte cell death (15).
During Fas activation, caspase-8 is recruited to the death-induced signaling complex (DISC) on the cell surface through interaction with Fas-associated death domain protein/mediator of receptor-induced toxicity-1 (FADD/MORT-1) (16, 17). Caspase-8 activates downstream caspases such as caspase-3, -6, and -7 by direct cleavage of their procaspase forms (18). In addition, caspase-8 can indirectly activate downstream caspases by inducing a release of cytochrome c from the mitochondria that triggers caspase activation through Apaf-1. Caspase-9 has been shown to form a complex with Apaf-1 and cytochrome c in the presence of dATP that triggers downstream caspase activation (19). The mechanism of cytochrome c release in response to Fas activation has been clarified with the recent discovery of Bid, a protein of undefined function, which, after cleavage and activation by caspase-8, facilitates the release of cytochrome c from mitochondria (20, 21). Furthermore, cytochrome c released in this way interacts with caspase-9 to activate additional caspase-8, thereby creating a positive amplification loop (22). Thus, the activation of caspase-8 initiates mitochondria-independent and -dependent pathways, both leading to the activation of downstream caspases (23). Which pathway is used to induce apoptosis in a given cell type under given circumstances has been shown to depend on the levels of active caspase-8 and its downstream substrates within the cell. Whether one or both of these pathways is required in the Fas-mediated cell death of the hepatocytes in vivo, and whether caspase-3 is required for either or both pathways, was the subject of this study.
Bcl-2 is an important antiapoptotic protein located in mitochondria. Mice expressing a liver-specific Bcl-2 transgene are protected from fulminant liver failure induced by injection of anti-Fas Ab, in vivo evidence that Bcl-2 plays a crucial role in protecting against apoptotic cell death (24). It has been demonstrated in vitro that Bcl-2 inhibits cell death by preventing the release of cytochrome c (25, 26). In addition, overexpression of Bcl-2 or the related antiapoptotic protein Bcl-xL in certain cell types can block the activity of caspase-8 and -3 (23). Interestingly, Bcl-2 and Bcl-xL are substrates for caspase-3, which cleaves the carboxyl-terminal portions of these proteins and abolishes their antiapoptotic activities (27, 28). Furthermore, the carboxyl-terminal Bcl-2 cleavage product has been reported to trigger cell death in a way similar to that of the Bax family of proteins (28). However, there is to date no evidence in vivo to show that caspase-3 is crucial for Bcl-2 or Bcl-xL processing.
To assess the specific role of caspase-3 in Fas-mediated hepatocyte cell death, we examined hepatocyte apoptosis in response to agonistic anti-Fas mAb treatment of caspase-3-deficient mice. We show in vivo that, in the absence of caspase-3, the death of animals due to fulminant hepatitis induced by anti-Fas treatment is delayed. Analysis of histological sections revealed that caspase-3 deficiency allowed hepatocytes to remain viable following Fas ligation. Intracellular levels of Bcl-2 did not decrease as in the wild-type hepatocytes and cytochrome c release was impaired in caspase-3-/- hepatocytes. Bcl-2, Bcl-xL, caspase-9, caspase-8, and Bid were all proteolytically processed in wild-type hepatocytes upon Fas ligation but not in caspase-3-/- hepatocytes. These results suggest the presence of a positive feedback loop required for the apoptosis of hepatocytes, which is initiated by caspase-3-mediated cleavage of Bcl-2 and Bcl-xL. In the absence of caspase-3, Bcl-2 and Bcl-xL remain intact and are able to prevent the apoptotic machinery from proceeding. This work provides the first in vivo evidence that caspase-3 plays a critical role in the initial upstream steps of Fas-mediated hepatocyte PCD that lead to fulminant hepatic failure causing death.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of caspase-3 knockout mice has been described previously (10). 129J/C57BL/6 chimeric caspase-3-/- mice were backcrossed to C57BL/6 for at least four generations. Although the knockout mice were not born at the expected Mendelian ratio, at least half of those surviving the postnatal stage lived to be healthy adults of more than 6 wk of age. Mice used in this study were adults of between 6 and 12 wk of age.
Anti-Fas mAb injection and histologic examinations
Mice were injected i.p. with 10 or 100 µg of a purified hamster monoclonal agonistic anti-Fas Ab (Jo2 mAb, PharMingen, San Diego, Ca). Mice were examined closely for signs of clinical compromise, including decreased mobility, tachypnea, and hypothermia. At specified time points, mice were sacrificed and organs were fixed in freshly prepared 4% paraformaldehyde in PBS lacking Ca2+ and Mg2+. Paraffin sections (5 µm) were stained with hematoxylin and eosin (H&E). For the survival time course, mice were sacrificed when they appeared moribund. Because 100 µg anti-Fas mAb is an extremely high dose causing rapid death, 10 µg of the Ab were used in most experiments.
In situ analysis of liver apoptosis using TUNEL
Paraffin-embedded sections were dewaxed in xylene and rehydrated by passage through a graded series of ethanol solutions, ending with PBS. Sections were permeabilized with proteinase K (20 µg/ml in 10 mM Tris-HCl, pH 7.4–8.0) at 37°C for 15 min. After washing, sections were stained with fluorescent anti-TdT using the In Situ Cell Death Detection kit from Boehringer Mannheim (Mannheim, Germany). Sections were viewed and photographed using standard fluorescent microscopic techniques.
Immunohistochemistry
Paraffin-embedded sections were dewaxed in toluene for 10 min and rehydrated through a graded series of ethanol solutions. Peroxidase blocking was performed in 3% H2O2/H2O for 10 min followed by rinses in dH2O and PBS. Sections were incubated with anti-Bcl-2 (clone 124; Dako, Glostrup, Denmark) or anti-p53 (clone D07; Novo Castra, Newcastle-upon-Tyne, U.K.) in a moist chamber for 1 h at room temperature (RT). The treated sections were then incubated with biotin-conjugated secondary Ab for 20 min at RT, then streptavidin-HRP for 20 min at RT, followed by development in aminoethylcarbazole (AEC) for 20 min. For cytochrome c staining, sections were dewaxed and rehydrated as described above. Sections were preblocked (3% BSA, 5% goat serum, and 0.3% Tween 20) for 30 min before incubation with cytochrome c Ab (diluted 1:50 in PBS with 3% BSA and 5% goat serum; PharMingen) at RT for 1.5 h followed by washes in PBS. The secondary Ab, an FITC-labeled goat anti-mouse IgG, was diluted in PBS (1:10, Southern Biotechnology Associates, Birmingham, AL) and sections incubated for 45 min at RT. Sections were then washed in PBS and mounted using Vectashield antifade mounting medium (Vector Labs, Burlingame, CA).
Western blotting
Cells were lysed in 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), and 1 mM sodium vanadate) supplemented with protease inhibitor mixture (0.1 mM PMSF and 2 µg/ml of leupeptin and apoprotein) for 15 min on ice. Lysates were centrifuged at 10,000 rpm for 5 min at 4°C, and protein concentration was estimated by the Pierce Protein Assay (Pierce, Rockford, IL) using BSA as the standard. Forty micrograms of total protein was loaded onto 14% SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with the appropriate Abs. Abs reactive to Bcl-2 (gift of D. Andrews, Hamilton, Ontario, Canada), Bcl-xL (Calbiochem, La Jolla, CA), Bid (gift of X. Wang, Dallas, TX), caspase-6 and -7 (Santa Cruz Biotechnology, Santa Cruz, CA), caspase-8 and -9 (gift of R. Hakem, Toronto, Canada), or ß-actin (Sigma, St. Louis, MO) were used in this study. Western blot analysis was conducted according to standard procedures using enhanced chemiluminescence detection (Amersham, Arlington Heights, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Within 2 h of i.p. injection with 10 µg anti-Fas mAb,
wild-type mice displayed signs of clinical compromise, including
tachypnea, shallow breathing, prostration, and progressive deep
hypothermia, consistent with rapid liver failure. Death occurred within
3–6 h. In contrast, the time of death of
caspase-3-/- mice was delayed to between 15 and
24 h (Fig. 1
). Wild-type mice
injected with 100 µg anti-Fas mAb became moribund within 2–4 h,
whereas caspase-3-/- mice became ill only much
later, at 6–9 h (Fig. 1).
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To examine the induction of hepatocyte apoptosis in vivo,
wild-type and caspase-3-/- mice were sacrificed
3 h after administration of 10 µg anti-Fas mAb and the
livers were examined by H&E staining. The livers of additional
caspase-3-/- mice were examined 6 h after
anti-Fas mAb injection. At the 3 h time point, the livers of
wild-type mice appeared grossly hemorrhagic. The normal lobular
microarchitecture of the liver was maintained but hepatocytes showed
characteristic signs of apoptosis, including condensation of chromatin
at the nuclear membrane and fragmentation of the cell into subcellular
bodies, which accumulated in the sinusoidal lumens. Injury of
sinusoidal endothelial cells was also observed, leading to peilosis and
panlobular sinusoidal congestion (Fig. 2,
B and C). The absence of inflammatory cells was
consistent with the noninflammatory nature of apoptotic cell death.
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, E and
F). A few mutant livers did show some degree of sinusoidal
congestion but with far less apoptosis than was observed in
anti-Fas injected wild-type livers. At 6 h post-anti-Fas
injection, the livers of caspase-3-/- mice
remained intact and most hepatocytes were viable. Minimal apoptosis was
observed, although sinusoidal congestion was more prominent (Fig. 2, G and H). No comparison to wild-type littermates
was made because very few wild-type mice survived until the 6-h time
point. Increased TUNEL staining in wild-type hepatocytes compared with caspase-3-/- hepatocytes following anti-Fas mAb injection of mice
The presence or absence of apoptosis in mouse livers following
injection of 10 µg anti-Fas mAb was confirmed by TUNEL staining.
No TUNEL staining was observed in either wild-type or
caspase-3-/- livers at 90 min after treatment
(data not shown). However, at 3 h postinjection, intense
fluorescence was observed in livers of anti-Fas mAb-injected
wild-type mice, indicating the occurrence of massive apoptosis (Fig. 3, A and B). In
contrast, livers from caspase-3-/- mice showed
minimal TUNEL staining at 3 h postinjection (Fig. 3, C
and D). Even at 6 h postinjection, the livers of
caspase-3-/- mice showed less TUNEL staining
than livers of wild-type mice at 3 h postinjection (Fig. 3, E and F).
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The cleavage of Bcl-2 by caspase-3 has been shown to abolish the
antiapoptotic function of the Bcl-2 molecule and to convert it to a
Bax-like proapoptotic molecule (28). Livers of wild-type
and caspase-3-/- mice that had been treated
with 10 µg anti-Fas mAb were therefore examined for Bcl-2
expression using immunohistochemistry. At 90 min post-anti-Fas mAb
injection, no differences in staining pattern were observed between
wild-type and caspase-3-/- hepatocytes (data
not shown). However, 3 h after anti-Fas mAb treatment, Bcl-2
levels were dramatically decreased compared with the baseline in
wild-type liver (Fig. 4, B and
C), presumably due to Bcl-2 processing during apoptosis. In
contrast, the intensity of Bcl-2 expression did not decrease in livers
of injected caspase-3-/- littermates. Instead,
Bcl-2 was maintained in mutant cells in a punctate pattern consistent
with localization in the mitochondria (Fig. 4, F and
G). To ensure that the Bcl-2 expression pattern was not
merely a characteristic inherent to livers of
caspase-3-/- mice, levels of p53 expression
were measured as a control in wild-type (Fig. 4D) and
caspase-3-/- (Fig. 5H) livers 3 h after
anti-Fas mAb injection. No differences in the p53 expression
pattern were noted. These results suggest that Bcl-2 is processed in
vivo in response to Fas ligation and that this processing requires
caspase-3.
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To determine whether caspase-3 is required for cytochrome
c release during Fas-mediated apoptosis in vivo, the
localization of cytochrome c in hepatocytes of wild-type and
caspase-3-/- mice treated with anti-Fas mAb
was determined in liver sections using immunofluorescence. In untreated
livers of both wild-type and caspase-3-/- mice,
cytochrome c appeared in the cytoplasm in a punctate manner
indicative of mitochondrial localization (Fig. 5A). When
livers were examined 3 h after injection of 10 µg anti-Fas
mAb, hepatocytes from wild-type mice showed dramatic pan-fluorescence,
consistent with a massive release of cytochrome c into the
cytoplasm (Fig. 5B). However, most hepatocytes from livers
of injected caspase-3-/- littermates did not
show this pattern of staining but rather retained the punctate pattern
of fluorescence, suggesting that cytochrome c release to the
cytoplasm was blocked (Fig. 5C). The punctate pattern of
immunofluorescence was still prominent in hepatocytes of
caspase-3-/- livers even at 6 h
post-anti-Fas mAb injection (Fig. 5D).
Requirement for caspase-3 in processing of regulatory anti- and proapoptotic proteins
Studies in vitro have suggested that caspase-3 is a downstream
member of the apoptotic protease cascade triggered by either activated
caspase-9 or -8 (16, 21). However, in hepatocytes from
caspase-3-/- mice injected with anti-Fas
mAb, we observed decreased processing of both Bcl-2 and
Bcl-xL, considered to be upstream regulatory
proteins of apoptosis initiation (Fig. 6A). The processing of
caspase-9, caspase-8, and Bid was also decreased in
anti-Fas-treated caspase-3-/- mice compared
with the wild type (Fig. 6B). Interestingly, cleavage of the
downstream effector enzyme caspase-7 was also impaired in the absence
of caspase-3. Caspase-6, another downstream effector, was not cleaved
in either anti-Fas-treated wild-type or
caspase-3-/- hepatocytes (Fig. 6C).
These results show that caspase-3 does not function only as apoptotic
enzyme downstream of caspase-8 and -9, but rather that caspase-3 also
plays an important role at the earliest steps of the initiation of
apoptosis, serving to modify crucial upstream caspases and pro- and
antiapoptotic regulatory proteins.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Caspase-3 is usually thought of as the downstream effector protease most important for the classic nuclear changes associated with apoptosis (10, 32). However, data derived from in vitro studies have indicated that caspase-3 can cleave a number of other substrates. It is now known that cleavage of the anti-apoptotic protein Bcl-2 into a proapoptotic form is a necessary step in Fas-mediated PCD, and that caspase-3 mediates this processing (28). In this report, we provide direct in vivo evidence that caspase-3 is essential for the initial steps of the massive hepatocyte apoptosis that occurs in response to Fas ligation and leads to the rapid death of wild-type animals. Caspase-3-/- mice were resistant to death induced by injection of agonistic anti-Fas mAb, and caspase-3-/- hepatocytes showed evidence of a block in apoptosis. Bcl-2 expression was maintained at control levels in anti-Fas-treated mutant hepatocytes, and cytochrome c release from mitochondria was limited. Proteolytic processing of the upstream proteins Bcl-2, Bcl-xL, caspase-8, caspase-9, and Bid could not proceed in the absence of caspase-3. The precise mechanism of delayed hepatocyte apoptosis remains unclear. However, it is clear that caspase-3 is essential for the initiation of apoptosis in response to Fas ligation. A delay in the initiation of apoptosis of hepatocytes would result in slower deterioration of the liver and prolonged survival of the mutant mice.
From our data, we speculate that the primary role of caspase-3 during Fas-mediated apoptosis in vivo is to cleave Bcl-2 and Bcl-xL, abrogating their ability to block the apoptotic cascade. Although Bcl-2 and Bcl-xL are CED-9 homologues and well established as antiapoptotic proteins, their precise functions during the cell death process are unclear. The structure of the Bcl-xL protein is reminiscent of the pore-forming proteins of bacterial toxins such as diphtheria toxin and the colicins, and it has been hypothesized that Bcl-xL may function as an ion channel regulating the permeability of the mitochondria (33). Such an ion channel could minimize osmotic stress and in so doing prevent the mitochondrial matrix swelling and outer membrane disruption that lead to cytochrome c release. A concise mechanism for Bcl-2 function has yet to be reported. Nevertheless, many laboratories have shown that Bcl-2 is capable of inhibiting cytochrome c release from mitochondria (34). In addition, both Bcl-2 and Bcl-xL have been shown to directly block caspase-8 activity (35), which may also contribute to the prevention of cytochrome c release.
The roles of Bcl-2 and Bcl-xL in Fas signaling have yet to be clarified. Several groups have investigated whether mammalian CED-9 homologues can inhibit Fas-mediated apoptosis, but conflicting data have been obtained. The results vary from no inhibition (36, 37), to partial inhibition (38, 39), to substantial inhibition (33). It has been postulated that the level of caspase-8 activity generated by the Fas DISC complex and the levels of downstream substrates may determine whether Bcl-2 or Bcl-xL can inhibit Fas-mediated death (23). In cell types in which abundant caspase-8 activity is present in the Fas-DISC complex, Bcl-2 and Bcl-xL are capable of inhibiting apoptosis following Fas ligation. However, in cell types that have little caspase-8 activity in the DISC complex, Bcl-2 and Bcl-xL are not able to inhibit apoptosis (23).
We propose a model in which a positive amplification loop is required for Fas-mediated cell death, and that this loop depends on caspase-3 activity. In the presence of caspase-3, Bcl-2 and Bcl-xL are converted to their proapoptotic forms. Caspase-8, caspase-9, and Bid are also processed and activated. Cytochrome c is released from the mitochondria, either because Bcl-2 and Bcl-xL are no longer intact and able to protect the mitochondria from osmotic stress, and/or because caspase-8 and Bid, which promote cytochrome c release, are freed from inhibition by unprocessed Bcl-2 and/or Bcl-xL. In the presence of dATP, Apaf-1 and caspase-9 complex with the released cytochrome c and caspase-9 is activated, triggering the mitochondrial pathway of PCD. The cascade of enzymatic activation that follows includes the cleavage of additional procaspase-3 by activated caspase-9. Thus, a positive feedback loop is created in which activated caspase-3 acts upstream to irreversibly commit the cell to PCD and downstream to trigger its execution.
In the absence of caspase-3, Bcl-2 and Bcl-xL are not processed and remain in the intermembrane space of the mitochondria. Similarly, caspase-8 and Bid are not activated. Both events may effectively block the release of cytochrome c. Without abundant cytochrome c in the cytoplasm, the positive feedback loop is broken, the activation of caspase-9 is impaired, downstream caspases such as caspase-7 are not cleaved, the initial amplification of caspase cascade is aborted, and the cells remain viable. Although our results in caspase-3-/- mice are consistent with published reports of the effects of caspase inhibitors on hepatocyte PCD in vivo (40), they stand in contradiction to a recently published report (41) in which the viability of hepatocytes of wild-type and caspase-3-/- mice did not differ in response to Fas ligation. However, Fas ligation in this case was induced in vitro rather than in vivo in an experimental setting that has been documented to affect the kinetics of apoptosis and the sensitivity of cells to Fas-mediated PCD (15).
Caspase-3 deficiency did not completely rescue the mutant mice from death due to liver failure. There was no evidence of inflammation in mutant livers, a finding that would have suggested cell death by necrosis rather than apoptosis. It is possible that the death of the animals could be due to a slower PCD pathway that depends on other known caspases (12), as yet unknown caspases, or perhaps no caspases at all (29). For example, PCD mediated by the apoptosis-inducing factor (AIF) may operate independently of caspase-mediated pathways (16). The existence of such pathways and their physiological relevance in vivo remain to be determined.
In conclusion, this study provides in vivo evidence that caspase-3 is required for the initial events that occur in hepatocyte cell death following Fas ligation. The data are consistent with a scenario in which caspase-3 modifies Bcl-2 and/or Bcl-xL expression such that their antiapoptotic functions are abrogated, the integrity of the mitochondrial membrane is compromised, and cytochrome c crucial for an irreversible commitment to PCD is released. The caspase-3-/- mice provide a unique animal model for dissecting Fas-mediated PCD in vivo, thereby providing insight into the pathophysiology of fulminant hepatitis. The delayed death of caspase-3-/- mice has important clinical implications because fulminant liver failure is a very accelerated process that rapidly leads to death. A means of inhibiting caspase-3 so that liver failure is slowed may gain the physician a critical window of time for interventions promoting hepatocyte regeneration. Thus, the elucidation of the precise roles of caspases in mouse liver failure may lead to the development of therapeutic drugs designed to alleviate liver damage in humans.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tak W. Mak, Amgen Institute, 620 University Avenue, Suite 706, Toronto, Ontario, Canada M5G 2C1. E-mail address:
3 Abbreviations used in this paper: PCD, programmed cell death; Apaf-1, apoptotic protease activating factor-1; DISC, death-induced signaling complex; H&E, hematoxylin and eosin.
Received for publication December 3, 1998. Accepted for publication August 24, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) died 2–4 h after
injection of 100 µg anti-Fas Jo2 Ab, whereas
caspase-3-/- mice (•) survived until 6–9 h after
injection. Similarly, all wild-type mice died 3–6 h after injection of
10 µg Jo2 Ab, whereas the caspase-3-/- mice died 15–24
h after injection. Seven mice per group were used for each dose of the
experiment.
