Delayed-onset caspase-dependent massive hepatocyte apoptosis upon fas activation in bak/bax-deficient mice

Authors


  • Potential conflict of interest: Nothing to report.

  • Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (to T. Takehara) and a Grant-in-Aid for Research on Hepatitis from the Ministry of Health, Labour, and Welfare of Japan.

Abstract

The proapoptotic Bcl-2 family proteins Bak and Bax serve as an essential gateway to the mitochondrial pathway of apoptosis. When activated by BH3-only proteins, Bak/Bax triggers mitochondrial outer membrane permeabilization leading to release of cytochrome c followed by activation of initiator and then effector caspases to dismantle the cells. Hepatocytes are generally considered to be type II cells because, upon Fas stimulation, they are reported to require the BH3-only protein Bid to undergo apoptosis. However, the significance of Bak and Bax in the liver is unclear. To address this issue, we generated hepatocyte-specific Bak/Bax double knockout mice and administered Jo2 agonistic anti-Fas antibody or recombinant Fas ligand to them. Fas-induced rapid fulminant hepatocyte apoptosis was partially ameliorated in Bak knockout mice but not in Bax knockout mice, and was completely abolished in double knockout mice 3 hours after Jo2 injection. Importantly, at 6 hours, double knockout mice displayed severe liver injury associated with repression of XIAP, activation of caspase-3/7 and oligonucleosomal DNA breaks in the liver, without evidence of mitochondrial disruption or cytochrome c–dependent caspase-9 activation. This liver injury was not ameliorated in a cyclophilin D knockout background nor by administration of necrostatin-1, but was completely inhibited by administration of a caspase inhibitor after Bid cleavage. Conclusion: Whereas either Bak or Bax is critically required for rapid execution of Fas-mediated massive apoptosis in the liver, delayed onset of mitochondria-independent, caspase-dependent apoptosis develops even in the absence of both. The present study unveils an extrinsic pathway of apoptosis, like that in type I cells, which serves as a backup system even in type II cells. (HEPATOLOGY 2011 )

Fas, also called APO-1 and CD95, is one of the death receptors that are potent inducers of apoptosis and constitutively expressed by every cell type in the liver.1 Dysregulation of Fas-mediated apoptosis is involved in several liver diseases.2 In the liver of patients with chronic hepatitis C, Fas is overexpressed in correlation with the degree of hepatitis, and Fas ligand can be detected in liver-infiltrating mononuclear cells.3, 4 Fas is also strongly expressed in the livers of patients with chronic hepatitis B, autoimmune hepatitis, and nonalcoholic steatohepatitis.4, 5 Moreover, in the liver of patients with fulminant hepatitis, Fas is up-regulated with strong detection of Fas ligand.6 In mice, injection of Jo2 agonistic anti-Fas antibody leads to massive hepatocyte apoptosis and lethality, suggesting that the hepatocyte is one of the most sensitive cell types to Fas stimulation.7 This model is considered to at least partly mimic human fulminant liver failure.

Fas, upon ligation by Fas ligand, activates caspase-8 through the recruitment of Fas-associated protein with a death domain and formation of the death-inducing signaling complex (DISC).1, 2 Whereas activated caspase-8 directly activates effector caspases such as caspase-3 and caspase-7 through the so-called extrinsic pathway, leading to apoptosis in type I cells, it activates caspase-3/7 through the mitochondrial pathway in type II cells. In type II cells, activated caspase-8 cleaves the BH3-only protein Bid into its truncated form, which in turn directly or indirectly activates and homo-oligomerizes Bak and/or Bax to form pores at the mitochondrial outer membrane, leading to the release of cytochrome c. After being released, cytochrome c assembles with Apaf-1 to form apoptosomes which promote self-cleavage of procaspase-9 followed by activation of caspase-3/7 to cleave a variety of cellular substrates such as poly(adenosine diphosphate ribose) polymerase (PARP) and finally to execute apoptosis.8, 9 Hepatocytes are considered to be typical type II cells, because Bid knockout (KO) mice were reported to be resistant to hepatocyte apoptosis upon Fas activation.10, 11 Although Bak and Bax are crucial gateways to apoptosis of the mitochondrial pathway, little information is available about their significance in hepatocyte apoptosis because most traditional Bak/Bax double knockout (DKO) mice (bak−/−bax−/−) die perinatally.12

In the present study, we tried to address this issue by generating hepatocyte-specific Bak/Bax DKO mice. We demonstrate that either Bak or Bax is required and sufficient to induce Fas-mediated early-onset hepatocyte apoptosis and lethal liver injury. Importantly, even if deficient in both Bak and Bax, Bak/Bax DKO mice still develop delayed-onset caspase-dependent massive hepatocyte apoptosis, suggesting that the mitochondria-independent pathway of apoptosis, as observed in type I cells, works as a backup system when the mitochondrial pathway of apoptosis in the liver is absent. This study is the first to demonstrate the significant but limited role of Bak and Bax in executing Fas-induced apoptosis in the liver.

Abbreviations

ALT, alanine aminotransferase; CypD, cyclophilin D; DISC, death-inducing signaling complex; DKO, double knockout; DMSO, dimethylsulfoxide; IAP, inhibition of apoptosis protein; KO, knockout; PARP, poly(adenosine diphosphate ribose) polymerase; RIP, receptor-interacting protein; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; WT, wild-type.

Materials and Methods

Mice.

Heterozygous Alb-Cre transgenic mice expressing Cre recombinase gene under the promoter of the albumin gene were described.13 We purchased Bak KO mice (bak−/−), Bax KO mice (bax−/−), and Bak KO mice carrying the bax gene flanked by 2 loxP sites (bak−/−baxflox/flox) from the Jackson Laboratory (Bar Harbor, ME). Traditional cyclophilin D (CypD) KO mice have been described.14 All mice strains that we used were created from a mixed background (C57BL/6 and 129). We generated hepatocyte-specific Bak/Bax DKO mice (bak−/−baxflox/floxAlb-Cre) or hepatocyte-specific CypD/Bak/Bax triple KO mice (cypd−/−bak−/−baxflox/floxAlb-Cre) by mating the strains. Mice were injected intraperitoneally with 1.5 or 0.5 mg/kg Jo2 anti-Fas antibody (BD Bioscience, Franklin Lakes, NJ) or intravenously with 0.25 mg/kg recombinant Fas ligand (Alexis Biochemicals, San Diego, CA) cross-linked with 0.5 mg/kg anti-Flag M2 antibody (Sigma-Aldrich, St. Louis, MO) to induce apoptosis. In some experiments, mice were intraperitoneally injected with 2 mg/kg necrostatin-1 (Sigma-Aldrich) or 40 mg/kg Q-VD-Oph (R&D Systems, Minneapolis, MN). They were maintained in a specific pathogen-free facility and treated with humane care with approval from the Animal Care and Use Committee of Osaka University Medical School.

Apoptosis Assay.

Measurement of serum alanine aminotransferase (ALT) levels, hematoxylin and eosin staining, and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) of liver sections have been described.15 Analysis of cytochrome c release from isolated mitochondria have also been described.16 To detect DNA fragmentation, 1.5 μg DNA extracted from 30 mg liver tissue by Maxwell16 (Promega, Madison, WI) was incubated with 0.5 μg RNase A (Qiagen, Tokyo, Japan) and separated by way of electrophoresis on a 1.5% agarose gel.

Western Blot Analysis.

For western immunoblotting, the following antibodies were used: anti–full-length Bid, anti–Cox IV, anti–cleaved caspase-3, anti–caspase-7, anti–caspase-8, anti–caspase-9, anti-PARP, anti-Bax, anti-cIAP1, and anti-XIAP antibodies were obtained from Cell Signaling Technology (Beverly, MA); anti-Bax and anti-cIAP2 antibodies were obtained from Millipore (Billerica, MA); anti-Bid antibody, which detects truncated Bid, was generously provided by Xiao-Ming Yin (Indiana University School of Medicine, Indianapolis, IN)17; and anti–β-actin antibody was obtained from Sigma-Aldrich. For isolation of the mitochondria-rich fraction, a Mitochondrial Isolation Kit (Thermo Scientific, Rockford, IL) was used. The isolation of hepatocytes from whole liver has been described.13

Detection of Bax Oligomerization.

Liver tissue was lysed with HCN buffer (25 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 300 mM NaCl, 2% CHAPS, protease inhibitor cocktail, phosphatase inhibitor cocktail, 100 μM BOC-Asp(OMe)CH2F [MP Biomedicals, Solon, OH]; pH 7.5). After the liver lysate was sonicated and centrifuged, the supernatant was collected and the concentration was adjusted. For cross-linking, 100 μL of the lysate was incubated with 5 μL 100 mM bis(maleimido)hexane (Thermo Scientific) and 5 μL 100 mM BS3 (Thermo Scientific) for 30 minutes at room temperature as described.18 After quenching the cross-linkers by way of incubation with 12 μL 1 M Tris-HCl (pH 7.5) for 15 minutes at room temperature, the lysate was boiled with sample buffer followed by western blot analysis for Bax.

Electron Microscopy.

Livers were fixed by perfusion of phosphate-buffered saline with 2.5% glutaraldehyde solution buffered at pH 7.4 with 0.1 M Millonig's phosphate, postfixed in 1% osmium tetroxide solution at 4°C for 1 hour, dehydrated in graded concentrations of ethanol, and embedded in Quetol 812 epoxy resin (Nisshin EM, Tokyo, Japan). Ultrathin sections (80 nm) cut on ultramicrotome were stained with uranyl acetate and lead citrate and examined with an H-7650 electron microscope (Hitachi Ltd., Tokyo, Japan) at 80 kV.

Statistical Analysis.

Data are presented as the mean ± SE. Differences between two groups were determined using the Mann-Whitney U test for unpaired observations. The survival curves were estimated using the Kaplan-Meier method and were tested by way of log-rank test. P < 0.05 was considered statistically significant.

Results

Bak Deficiency Partially Ameliorates Fas-Induced Hepatocellular Apoptosis but Fails to Prevent Animal Death.

First, to examine the significance of Bak in hepatocellular apoptosis induced by Fas stimulation, Bak KO mice (bak−/−) and wild-type (WT) littermates (bak+/+) were intraperitoneally injected with 1.5 mg/kg Jo2 anti-Fas antibody and analyzed 3 hours later. Consistent with previous reports,10, 19 WT mice showed severe elevation of serum ALT levels with massive hepatocellular apoptosis (Fig. 1A,B). Bak KO mice also developed liver injury, but the levels of serum ALT and the number of TUNEL-positive hepatocytes were significantly lower in Bak KO mice than in WT mice (Fig. 1A-C). Western blotting for cleaved caspase-3, caspase-7, and PARP revealed that activation of effector caspases were partially inhibited in KO livers compared with WT livers (Fig. 1D). Cleavage of procaspase-9, which is initiated by mitochondrial release of cytochrome c, was also suppressed in Bak KO livers compared with WT liver (Fig. 1D). The cleaved form of caspase-8, a direct downstream target of Fas activation, was detected in both mice, but its levels were reduced in Bak KO mice compared with WT mice (Fig. 1D). This reduction may be explained by the lesser activation of caspase-3/7, because it has been reported that caspase-3/7 could activate caspase-8 through an amplification loop during apoptosis.20 Collectively, these findings demonstrated that Bak deficiency partially ameliorated Fas-induced hepatocellular apoptosis associated with reduced cleavage of caspase-9, caspase-3/7, and PARP. We then compared survival of mice after Jo2 injection but found that Bak KO mice also rapidly died with kinetics similar to those of WT mice, suggesting that partial amelioration of hepatocellular apoptosis induced by Bak deficiency did not lead to survival benefit under our experimental conditions (Fig. 1E). Because Bax residing in the cytosol moves to the mitochondria upon activation, where it undergoes oligomerization,21 we analyzed its translocation and oligomerization in the liver at 3 hours after Jo2 injection. Western blot analysis revealed that the levels of Bax expression clearly increased in the mitochondrial fraction in both WT livers and Bak KO livers (Fig. 1F). Signals for the Bax dimer were also detected in both livers (Fig. 1F). These findings indicate that Bax is also activated after Fas stimulation, raising the possibility of its involvement in hepatocellular apoptosis.

Figure 1.

Bak KO mice are partially resistant to Fas-induced hepatocellular apoptosis. Bak KO mice (Bak−/−) or control WT littermates (Bak+/+) were analyzed at 3 hours after intraperitoneal injection of 1.5 mg/kg Jo2 anti-Fas antibody. (A) Serum ALT levels (n = 10 or 11, respectively). (B) Hematoxylin and eosin (HE) and TUNEL staining of the liver sections. (C) Number of TUNEL-positive cells (n = 8 or 9, respectively). (D) Western blot analysis for the expressions of cleaved caspase-8, 9, -3, -7 and PARP. (E) Bak KO mice or control WT littermates were intraperitoneally injected with 1.5 mg/kg Jo2 anti-Fas antibody (n = 8 or 11, respectively). Survival rates after Jo2 injection are shown. (F) Bak KO mice or control WT littermates were analyzed 3 hours after intraperitoneal injection of Jo2 anti-Fas antibody (1.5 mg/kg) or vehicle. Left: Western blot analysis of the mitochondrial fraction of the liver for the expression of Bax. Right: Western blot analysis for the expression of Bax monomer and dimer in the liver.

Bax Deficiency Fails to Ameliorate Fas-Induced Hepatocellular Apoptosis.

Next, to examine the significance of Bax in hepatocellular apoptosis induced by Fas stimulation, Bax KO mice (bax−/−) and WT littermates (bax+/+) were injected with Jo2 and examined 3 hours later. There was no significant difference in the levels of serum ALT or the number of TUNEL-positive hepatocytes between the two groups (Fig. 2A-C), which is consistent with a previous report.22 The levels of the cleaved forms of caspase-8, -9, -3, -7, and PARP in Bax KO livers did not differ from those of WT livers (Fig. 2D). These findings demonstrate that, in contrast to Bak deficiency, Bax deficiency was not able to inhibit Fas-induced hepatocellular apoptosis.

Figure 2.

Bax KO mice are not resistant to Fas-induced hepatocellular apoptosis. Bax KO mice (Bax−/−) or control WT littermates (Bax+/+) were analyzed 3 hours after intraperitoneal injection of Jo2 anti-Fas antibody (1.5 mg/kg). (A) Serum ALT levels (n = 11 per group). (B) Hematoxylin and eosin (HE) and TUNEL staining of the liver sections. (C) Number of TUNEL-positive cells (n = 8 per group). (D) Western blot analysis for the expressions of cleaved caspase-8, -9, -3, -7, and PARP.

Bax Deficiency Completely Blocks Fas-Induced Early-Onset Hepatocellular Apoptosis in a Bak-Deficient Background.

To examine the impact of Bax in a Bak-deficient background, hepatocyte-specific Bak/Bax DKO mice (bak−/−baxflox/floxAlb-Cre) and Bak KO mice (bak−/−baxflox/flox), which served as control littermates of this mating, were injected with Jo2 and analyzed 3 hours later. We confirmed the hepatocyte-specific defects of Bax protein in Bak/Bax DKO mice by way of western blot analysis (Fig. 3A). The serum ALT levels of Bak/Bax DKO mice were in the normal range and were significantly lower than those of Bak KO mice (Fig. 3B). Liver histology and TUNEL staining did not show evidence of hepatocyte apoptosis in Bak/Bax DKO livers, in contrast to Bak KO livers (Fig. 3C,D). Taken together, these results indicate that Bak and Bax are basically redundant molecules for execution of hepatocellular apoptosis induced by Fas activation, although the former appears to be clearly required for full-blown apoptosis in vivo.

Figure 3.

Bak/Bax DKO mice are fully resistant to Fas-induced hepatocellular apoptosis in early phase. (A) Western blot analysis of the indicated fraction of the liver for the expressions of Bax. (B-D) Bak/Bax DKO mice (Bak−/− Baxfl/fl Alb-Cre) or control Bak KO littermates (Bak−/− Baxfl/fl) were analyzed 3 hours after intraperitoneal injection of Jo2 anti-Fas antibody (1.5 mg/kg). (B) Serum ALT levels (n = 10 per group). (C) Hematoxylin and eosin (HE) and TUNEL staining of the liver sections. (D) Number of TUNEL-positive cells (n = 9 per group).

Fas Stimulation Leads to Late-Onset Hepatocellular Death Even in Bak/Bax Deficiency with Moderate Caspase-3/7 Activation Without Mitochondrial Disruption.

To examine whether the inhibition of Fas-induced rapid liver injury in Bak/Bax deficiency is a durable effect, we analyzed the survival rate after Jo2 injection. The survival rate of Bak/Bax DKO mice was significantly higher than that of Bak KO mice, but approximately half of the Bak/Bax DKO mice died within 12 hours (Fig. 4A). To examine the cause of this late-onset lethality, we analyzed the serum ALT levels and liver tissue 6 hours after Jo2 injection. Unexpectedly, the serum ALT levels were highly elevated in Bak/Bax DKO mice (Fig. 4B). Liver histology revealed many hepatocytes with cellular shrinkage and scattered regions of sinusoidal hemorrhage (Fig. 4C), indicating that Bak/Bax DKO mice still developed severe liver injury at this time point. TUNEL staining revealed many TUNEL-positive hepatocytes in the liver sections. Of importance, electron microscopic analysis revealed mitochondrial alterations (such as disruption of the membrane and herniation of the matrix) in hepatocytes of Bak KO mice but not in hepatocytes of Bak/Bax DKO mice with chromatin condensation (Fig. 4E). Because some reports showed that hepatocytes act like type I cells with a high dose of Jo2 anti-Fas antibody and that anti-Fas antibody does not always reliably mimic the action of the physiological Fas ligand,23, 24 we also injected 0.5 mg/kg Jo2 or recombinant Fas ligand into Bak/Bax DKO mice. Similarly, both injected mice showed severe elevation of serum ALT levels and severe hepatitis with many TUNEL-positive cells at 6 hours (Supporting Figs. 1 and 2).

Figure 4.

Bak/Bax DKO mice develop late-onset severe liver injury upon Fas stimulation. Bak/Bax DKO mice (Bak−/− Baxfl/fl Alb-Cre) or control Bak KO littermates (Bak−/− Baxfl/fl) were intraperitoneally injected with 1.5 mg/kg Jo2 anti-Fas antibody. (A) Survival rate after Jo2 injection (n = 11 per group). (B) Serum ALT levels of Bak/Bax DKO mice. (C, D) Hematoxylin and eosin (C) and TUNEL (D) staining of the liver sections of Bak/Bax DKO mice 6 hours after Jo2 injection. Representative photomicrographs are shown. (E) Representative electron microscopy photomicrographs of the livers of Bak/Bax DKO mice before and 6 hours after Jo2 anti-Fas injection (1.5 mg/kg) and control Bak KO mice 2 hours after Jo2 anti-Fas injection (1.5 mg/kg). Right panels are enlarged images of the square area of each left panel. N, nucleus.

To examine the kinetics of caspase activation and apoptosis in the liver after Jo2 administration, we performed western blot analysis for caspase activation and agarose gel electrophoresis for DNA laddering. All signals for cleaved forms of caspase-3, caspase-7, and PARP in the liver were clearly detected at 6 hours in Bak/Bax DKO mice, although they were weaker than those at 3 hours in control Bak KO littermates (Fig. 5A). Regarding the cleaved form of caspase-9, two bands were detected at 3 hours in Bak KO liver, but not in Bak/Bax DKO liver. Previous research established that procaspase-9 has two sites for cleavage upon activation: both Asp353 and Asp368 sites are autoprocessed by caspase-9 activation after cytochrome c release, whereas the Asp368 site is preferentially processed over the Asp358 site by caspase-3.25 In our western blot analysis, the slow migrating species corresponding to the fragment cleaved at Asp368, but not the rapid migrating species corresponding to that at Asp353, was weakly detected at 6 hours in Bak/Bax DKO liver. This indicated that caspase-3–mediated cleavage of procaspase-9 takes place without evidence of cytochrome c–induced autoprocessing of procaspase-9. Agarose gel electrophoresis clearly detected oligonucleosomal DNA laddering at 6 hours in Bak/Bax DKO livers, similar to our observation at 3 hours in control Bak KO livers (Fig. 5B). Collectively, these morphological and biochemical data support the idea that hepatocellular death occurring at 6 hours in the Bak/Bax DKO liver seems to involve apoptosis.

Figure 5.

Fas-mediated hepatocellular death in Bak/Bax DKO mice is associated with caspase-3/7 activation and oligonucleosomal DNA breaks. Bak/Bax DKO mice (Bak−/− Baxfl/fl Alb-Cre) or control Bak KO littermates (Bak−/− Baxfl/fl) were intraperitoneally injected with Jo2 anti-Fas antibody (1.5 mg/kg). (A) Western blot analysis for expression of cleaved caspase-8, -9, -3, -7, and PARP. (B) DNA laddering of the liver. (C) Western blot analysis for expression of IAP family proteins.

To examine the underlying mechanisms by which caspase-3/7 was increasingly activated from 3 to 6 hours in Bak/Bax DKO mice, we analyzed the expression of inhibition of apoptosis proteins (IAPs), which can block cleavage of procaspase-3, -7, and -9.26 The expression levels of cIAP1 and cIAP2 were not changed in the liver after Jo2 injection (Fig. 5C, Supporting Fig. 3). In contrast, the expression levels of XIAP were up-regulated in the livers of both Bak KO and Bak/Bax DKO mice at 3 hours after Jo2 injection, as in WT mice (Fig. 5C, Supporting Fig. 3), which is consistent with previous reports.27 However, this up-regulation disappeared from the livers of Bak/Bax DKO mice at 6 hours. Repression of XIAP overexpression might explain why weak activation of capsase-3/7 gradually increased from 3 to 6 hours in the Bak/Bax DKO liver.

Cell Death with Bak/Bax Deficiency Is Not Dependent on a Necrotic Pathway.

Fas activation was reported to induce not only caspase-dependent apoptosis but also caspase-independent necrosis, which is required for receptor-interacting protein (RIP) kinase.28 To exclude the possibility of this necrotic cell death in the Bak/Bax DKO liver, we first examined the effect of necrostatin-1, which specifically inhibits RIP kinase to protect against necrotic cell death caused by death-domain receptor stimulation.2, 29 Bak/Bax DKO mice were injected with 2 mg/kg necrostatin-1 at 2 hours after or 1 hour before Jo2 injection. The ALT levels at 6 hours after Fas stimulation were clearly elevated without a significant difference between the necrostatin-1 injection group and the vehicle injection group (Fig. 6A and Supporting Fig. 4). We next examined the effect of CypD, which is a key molecule of mitochondrial permeability transition generated by Ca2+ overload and/or oxidative stress leading to necrotic cell death.14, 30 We injected Jo2 into CypD−/− mice with a Bak/Bax-deficient background (cypd−/−bak−/−baxflox/floxAlb-Cre) or control CypD+/+ or +/− littermates (cypd+/+ or +/−bak−/−baxflox/floxAlb-Cre). The ALT levels of CypD/Bak/Bax triple KO mice upon Fas stimulation were the same as those of control mice (Fig. 6B). These results indicate that liver injury in Bak/Bax deficiency induced by Fas stimulation was not dependent on the necrotic pathway, at least that mediated by RIP kinase and/or CypD.

Figure 6.

Fas-induced hepatocellular death in Bak/Bax DKO mice is independent of RIP kinase and/or CypD. (A) Bak/Bax DKO mice (Bak−/− Baxfl/fl Cre) were intraperitoneally injected with 2 mg/kg necrostatin-1 in vehicle containing 0.2% dimethylsulfoxide or vehicle alone at 2 hours after injection of 1.5 mg/kg Jo2 anti-Fas antibody. Serum ALT levels at 6 hours after Jo2 injection are shown (n = 8 per group). (B) CypD+/+ or +/− mice in a Bak/Bax-deficient background (CypD+/+ or +/− Bak−/− Baxfl/fl Alb-Cre) or control CypD−/− littermates (CypD−/− Bak−/− Baxfl/fl Alb-Cre) were intraperitoneally injected with 1.5 mg/kg Jo2 anti-Fas antibody. Serum ALT levels at 6 hours after injection are shown (n = 7 per group or 8 per group, respectively).

Late-Onset Cell Death in Bak/Bax Deficiency Is Completely Dependent on Caspase.

Although cell death observed in Bak/Bax DKO mice appears to be apoptosis, the question arose of whether relatively weak caspase-3/7 activity compared with that observed in Bak KO mice is sufficient for inducing liver injury 6 hours after Fas simulation. To this end, Bak/Bax DKO mice were given 40 mg/kg Q-VD-Oph, a potent broad spectrum caspase inhibitor,31 2 hours after injection of Jo2. Western blot analysis revealed the existence of truncated Bid and cleaved caspase-8 in the liver 2 hours after Jo2 injection, demonstrating that caspase-8 had already been activated by this point (Fig. 7A). Administration of the caspase inhibitor at 2 hours completely blocked the elevation of serum ALT levels and hepatocellular apoptosis, as evidenced by liver histology and TUNEL staining 6 hours after Jo2 injection (Fig. 7B-D). Finally, we tried to analyze the survival rate of Bak/Bax DKO mice and control Bak KO mice when therapeutically injected with the caspase inhibitor 2 hours after Jo2 injection. None of the Bak/Bax DKO mice showed lethal liver injury upon Jo2 injection, whereas half of the Bak KO mice died from severe liver injury (Fig. 7E). These findings suggest that Fas-induced liver injury in Bak/Bax deficiency was dependent on caspase activity, which could be fully negated by the caspase inhibitor. On the other hand, caspase activation in Bak KO mice was too high to be negated by the same dose of the caspase inhibitor.

Figure 7.

Hepatocellular death in Bak/Bax DKO mice is dependent on caspase activation. (A) Bak/Bax DKO mice were analyzed before and 2 hours after intraperitoneal injection of Jo2 anti-Fas antibody (1.5 mg/kg). Western blot analysis of the liver for the expression of cleaved caspase-8 and truncated Bid (tBid). (B-D) Bak/Bax DKO mice were intraperitoneally administered 40 mg/kg Q-VD-Oph in 10 mL/kg dimethylsulfoxide (DMSO) or DMSO alone, as a vehicle, 2 hours after injection of 1.5 mg/kg Jo2 anti-Fas antibody and analyzed at 6 hours. (B) Serum ALT levels (n = 6 or 7 per group, respectively). (C) Hematoxylin and eosin (HE) and TUNEL staining of the liver sections. (D) Number of TUNEL-positive cells (n = 6 or 7 per group, respectively). Because intraperitoneal injection of DMSO leads to injury at the surface layer of the liver, TUNEL positivity close to the surface layer was excluded from the cell count. (E) Bak/Bax DKO mice (Bak−/− Baxfl/fl Alb-Cre) or control Bak KO littermates (Bak−/− Baxfl/fl) were given 40 mg/kg Q-VD-Oph intraperitoneally in 10 mL/kg DMSO or DMSO alone at 2 hours after injection of 1.5 mg/kg anti-Fas antibody. The disease-free survival rate of lethal liver injury after Jo2 injection is shown (n = 8 per group).

Discussion

In the present study, we demonstrate that Bak KO, but not Bax KO, provides partial resistance to Fas-induced hepatocellular apoptosis in vivo. We demonstrated previously that Bak KO mice, but not Bax KO mice, showed resistance to apoptosis induced by Bcl-xL deficiency, which depended mainly on Bid activation.16 Research has shown that Fas induces apoptosis in hepatocytes through the Bid pathway,10, 11 and the present study also demonstrates that Bid becomes truncated in the liver upon anti-Fas injection. Therefore, truncated Bid may preferentially activate Bak rather than Bax in the liver. However, the present study also reveals that, in the absence of Bak, Bax plays an essential role in mediating the early onset of hepatocellular apoptosis. The most important finding of this study is that Bak/Bax deficiency failed to protect against the late onset of liver injury after Jo2 anti-Fas injection as well as Fas agonist injection. Wei et al.,32 in their historical paper establishing the importance of Bak and Bax in the mitochondrial pathway of apoptosis, reported that hepatocytes were protected from Jo2-induced apoptosis in traditional Bak/Bax DKO mice (bak−/−bax−/−). Because perinatal lethality occurs with most traditional Bak/Bax DKO mice, they could only analyze three animals, which did not enable detailed analysis of cell death due to Jo2 stimulation. The present study is the first to (1) thoroughly examine the impact of Bak and Bax in the liver using conditional KO mice and (2) demonstrate that Bak/Bax deficiency can protect against Fas-induced severe injury in the early phase but not in the late phase.

The late onset of liver injury observed in Bak/Bax DKO appeared to be apoptosis based on biochemical and morphological observations, including caspase activation, oligonucleosomal DNA breaks and, most importantly, identification of cell death with caspase dependency. In addition, the well-established necrotic pathway mediated by RIP kinase and/or CypD was not involved. However, the difference from apoptosis observed in Bak KO mice was the absence of mitochondrial alteration or cytochrome c–dependent caspase-9 processing in Bak/Bax DKO mice. We also confirmed that Bak/Bax-deficient mitochondria were not capable of releasing cytochrome c in the presence of truncated Bid (Supporting Fig. 5). These data support the idea that activation of the mitochondrial pathway of apoptosis is fully dependent on either Bak or Bax even in the late phase, indicating at the same time that late onset of apoptosis takes place through an extrinsic pathway rather than the mitochondrial pathway.

Although hepatocytes are generally considered to be type II cells, recent work has shown that the requirement of the mitochondrial pathway may be overcome through changes induced by in vitro culture conditions33, 34 or the strength of Fas stimulation.23 Schüngel et al.23 demonstrated that hepatocytes act as type II cells with a low-dose Jo2 injection (0.5 mg/kg) and act as type I cells with an extremely high-dose Jo2 injection (5 mg/kg). This agrees with the generally accepted idea that type I cells exhibit strong activation of DISC and caspase-8, which itself is sufficient to induce apoptosis, whereas type II cells exhibit weak activation and therefore require amplification of the apoptosis signal through the mitochondrial loop. In the present study, we used 1.5 mg/kg or 0.5 mg/kg Jo2 antibody, which could be considered relatively low doses, and found that hepatocytes act like type II cells in WT mice or Bak/Bax single KO mice but act like type I cells in Bak/Bax DKO mice. The present study therefore demonstrates that hepatocytes can act as type I cells in the absence of Bak and Bax independent of the strength of DISC formation or signals from microenvironments.

The question arises of why hepatocytes can act as type I cells where the levels of DISC formation or caspase-8 activation may be insufficient to induce activation of downstream caspases. Recently, Jost et al.27 reported a discriminating role of XIAP between type I and type II cells; in type II cells, the levels of XIAP expression increased after Fas stimulation but decreased in type I cells. In agreement with this report, XIAP expression was up-regulated at 3 hours in both Bak KO and Bak/Bax DKO livers. Interestingly, this XIAP up-regulation disappeared at 6 hours after Jo2 injection in Bak/Bax DKO mice. Because XIAP is a potent inactivator of caspase-3, -7, and -9 processing, repression of XIAP may be one reason why hepatocytes can act as type I cells at this time point.

Previous studies have reported that liver endothelial cells express Fas receptor and have suggested that apoptosis of these cells may participate in the liver damage in mice receiving Jo2 antibody, especially in the case of high-dose administration.35 However, we did not find liver injury in the sinusoidal hemorrhage in Bak/Bax DKO mice at 3 hours after Jo2 injection, which is the time point when Bak KO mice developed it (Fig. 3C). Together with the fact that Bax, but not Bak, was active in liver nonparenchymal cells in our Bak/Bax DKO mice, as was the case in Bak KO mice (Fig. 3A), we speculate that Bak-deficient sinusoidal cells could not contribute much to liver injury at 3 hours after Jo2 injection (1.5 or 0.5 mg/kg).

Recently, a pan-caspase inhibitor was reported to reduce hepatic damage in liver transplant recipients and patients with chronic hepatitis C in clinical trials.36, 37 For treatment of fulminant liver injury, caspase inhibitors seem to be attractive drugs. However, the present study demonstrates that Fas-induced apoptotic signals could be efficiently amplified through the mitochondrial pathway, leading to high lethality even if caspase inhibitor was administered 2 hours after Jo2 injection. In contrast, administration of the same dose of the caspase inhibitor was able to fully block hepatocyte apoptosis and lethality in Bak/Bax DKO mice. From a clinical point of view, when using caspase inhibitors to prevent fulminant liver failure, concomitant inactivation of the mitochondrial amplification loop may be required.

In conclusion, the extrinsic pathway of apoptosis exists in hepatocytes and causes late onset of lethal liver failure in the absence of Bak and Bax independent of the strength of Fas ligation. This pathway could be therapeutically intervened through the use of caspase inhibitors, presumably due to low levels of DISC formation and subsequent weak activation of effector caspases in hepatocytes. The present study unveils the entire framework of the Fas-mediated signaling pathway in hepatocytes, placing the mitochondrial pathway of apoptosis as a potent loop for amplifying activation of the caspase cascade to execute complete and rapid cell death in hepatocytes.

Acknowledgements

We thank Xiao-Ming Yin (Department of Pathology and Laboratory Medicine, Indiana University School of Medicine) for providing the anti-mouse Bid antibody.

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