Differential protective effects of Bcl-xL and Bcl-2 on apoptotic liver injury in transgenic mice

Alix de la Coste1, Monique Fabre, Nathalie McDonell1, Arlette Porteu1, Helène Gilgenkrantz1, Christine Perret1, Axel Kahn1, and Alexandre Mignon1

1 Institut National de la Sante et de la Recherche Medicale, U-129 Institut Cochin de Genetique Moleculaire, Université Paris V René Descartes, 75014 Paris; and Hôpital du Kremlin Bicêtre, 94275 Le Kremlin-Bicêtre, France


    ABSTRACT
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Fas ligand (CD95L) and tumor necrosis factor-alpha (TNF-alpha ) are pivotal inducers of hepatocyte apoptosis. Uncontrolled activation of these two systems is involved in several forms of liver injury. Although the broad antiapoptotic action of Bcl-2 and Bcl-xL has been clearly established in various apoptotic pathways, their ability to inhibit the Fas/CD95- and TNF-alpha -mediated apoptotic signal has remained controversial. We have demonstrated that the expression of BCL-2 in hepatocytes protects them against Fas-induced fulminant hepatitis in transgenic mice. The present study shows that transgenic mice overexpressing BCL-XL in hepatocytes are also protected from Fas-induced apoptosis in a dose-dependent manner. Bcl-xL and Bcl-2 were protective without any change in the level of endogenous Bcl-xL or Bax and inhibited hepatic caspase-3-like activity. In vivo injection of TNF-alpha caused massive apoptosis and death only when transcription was inhibited. Under these conditions, PK-BCL-XL mice were partially protected from liver injury and death but PK-BCL-2 mice were not. A similar differential protective effect of Bcl-xL and Bcl-2 transgenes was observed when Fas/CD95 was activated and transcription blocked. These results suggest that apoptosis triggered by activation of both Fas/CD95 and TNF-alpha receptors is to some extent counteracted by the transcription-dependent protective effects, which are essential for the antiapoptotic activity of Bcl-2 but not of Bcl-xL. Therefore, Bcl-xL and Bcl-2 appear to have different antiapoptotic effects in the liver whose characterization could facilitate their use to prevent the uncontrolled apoptosis of hepatocytes.

Fas/CD95; tumor necrosis factor-alpha ; apoptosis


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THE ROLE OF APOPTOSIS in normal liver biology and during liver diseases is well established (28). Hepatocyte apoptosis is involved in the removal of injured, infected, or transformed hepatocytes by the immune system as an adaptative and beneficial process. However, dysregulated hepatocyte apoptosis, mainly caused by death-domain receptor ligands such as Fas ligand (CD95L) and tumor necrosis factor-alpha (TNF-alpha ) is clearly implicated in several experimental and human liver diseases (see Ref. 16 for review).

Besides their structural and functional similarities, Fas/CD95 and TNF-alpha -receptor type 1 appear to recruit similar or at least partially overlapping pathways (3). Upon receptor trimerization, activated Fas and TNF-alpha -receptor type 1 initiate rapid protein interactions at the cell membrane, leading to the formation of a death inducing signaling complex responsible for downstream apoptotic events, which include the release of cytochrome c from mitochondria (29) and a proteolytic cascade of caspase activation (35).

The Fas/CD95 and TNF-alpha apoptosis signaling pathways appear to be differentially regulated in hepatocytes (16). Hepatocytes are very sensitive to Fas/CD95-mediated apoptosis. In vivo treatment with Fas-inducing agents leads rapidly to fulminant and lethal apoptotic hepatitis (27). This great sensitivity of hepatocytes to Fas/CD95-mediated apoptosis might be partly due to the small amounts of antiapoptotic proteins in these cells, in contrast to the constitutive synthesis of proapoptotic protein. Bcl-2 expression is indeed undetectable in hepatocytes (19); although low concentrations of Bcl-xL are present in the liver, they are lower here than in other tissues such as the brain or bone marrow (8, 17). In contrast to the Fas/CD95 pathway, hepatocytes are resistant to TNF-alpha -induced apoptosis in vivo. These cells proliferate when stimulated by TNF-alpha , a critical mechanism involved in liver regeneration (26). The resistance of many cell types, including hepatocytes, to TNF-alpha -induced apoptosis, was recently linked to the ability of TNF-alpha to stimulate the transcription of several protective genes, mainly those dependent on the nuclear factor (NF)-kappa B transcription factor (4). These observations are fully consistent with studies by Leist et al. (23), demonstrating that transcriptional inhibition was required to unveil the proapoptotic effects of TNF-alpha in hepatocytes.

Although many studies have clearly established the broad antiapoptotic activity of Bcl-2 and Bcl-xL in various apoptotic pathways, the ability of Bcl-2 or Bcl-xL to inhibit Fas/CD95- and TNF-alpha -mediated apoptotic pathways has remained controversial. The findings have varied with the experimental model used, the level of expression, and the cell type (1). We and others have demonstrated that Bcl-2 gene expression in the hepatocytes of transgenic mice protects them against Fas/CD95-induced liver apoptosis and death (22, 30). These results prompted us to study the protective effects of Bcl-xL gene overexpression on Fas/CD95-mediated liver injury. Because Fas/CD95 and TNF-alpha recruit similar or at least partially overlapping pathways, we also addressed the question of whether Bcl-2 and Bcl-xL could protect our transgenic mice from TNF-alpha -mediated liver injury. We therefore generated transgenic mice using the same L-PK liver-specific regulatory regions directing the expression of either human BCL-2 or BCL-XL cDNA in the liver.

We report here that Bcl-xL like Bcl-2 protected the liver of the mice against Fas/CD95-induced apoptosis. But Bcl-xL, unlike Bcl-2, partially prevented hepatic apoptosis in mice treated with TNF-alpha and D-galactosamine (D-GalN). Because these differing effects of Bcl-xL and Bcl-2 were observed when Fas/CD95-mediated apoptosis was associated with D-GalN administration, we assume that gene expression was required for the antiapoptotic effect of Bcl-2, but not for that of Bcl-xL. These results emphasize the different properties of these two related homologous proteins; they also suggest the potential prospects of increasing the antiapoptotic rheostat of hepatocytes in several liver diseases for which uncontrolled Fas/CD95 or TNF-alpha pathways appear to be involved.


    MATERIALS AND METHODS
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Materials. Jo2 anti-Fas antibody in sterile 0.9% NaCl was purchased from Pharmingen. Recombinant murine TNF-alpha (rTNF-alpha ) was purchased from Genzyme (Paris, France) and dissolved in pyrogen-free PBS. D-GalN dissolved in sterile 0.9% NaCl was purchased from Sigma Chemical (St. Louis, MO).

Transgenic mice. The expression of human BCL-XL cDNA was directed by the regulatory sequences of the rat L-type pyruvate kinase gene (L-PK). Most of the L-PK gene was replaced by the human BCL-XL cDNA kindly provided by C. B. Thompson (8), but the first exon with a mutated ATG, the first intron, and part of the second exon of the rat pyruvate kinase gene were retained. Transgene expression was restricted to the hepatocytes, enterocytes, and kidney cells. Full-length human BCL-XL cDNA was inserted into the KS-L-PK plasmid at the Spe I site with the 3' untranslated region and polyadenylation signal of the human beta -globin gene. Translation started at the human BCL-XL ATG, leading to a Bcl-xL protein of 29 kDa. Transgenic mice were identified by Southern blot analysis using the coding 0.9-kb BCL-XL sequence as a probe.

Experimental protocol and histological examination. In this study we used 8- to 16-wk-old nontransgenic littermates and transgenic mice (18-25 g) bred in a CBA/B6D2 background. All experiments were conducted in heterozygous mice, except for a few sets of experiments, such as survival analysis, where the use of homozygous mice is clearly stated. For the Fas/CD95 studies, mice were injected intravenously with either 0.5 or 0.15 mg/kg of Jo2 monoclonal anti-Fas antibody. For the TNF-alpha studies, rTNF-alpha was injected intraperitoneally at 20 µg/kg. D-GalN (800 mg/kg) was injected intraperitoneally 30 min before the rTNF-alpha and Jo2 injection. Mice were killed at various times, and liver samples were removed and fixed in 4% (vol/vol) paraformaldehyde in PBS and embedded in paraffin, and 3-µm sections were cut and stained with hematoxylin and eosin section and Masson trichrome. Sections from different lobes were assessed for the severity of apoptotis. Apoptotic cells were detected in liver sections of mice killed 2 h after Jo2 injection using an in situ cell death peroxidase detection kit (TUNEL kit; Boehringer, Meylan, France), according to the manufacturer's protocol.

Northern and Western blot analyses. Total liver mRNAs were extracted from homogenized tissues using the single-step guanidium thiocyanate procedure (14). Total RNA (15 mg) was fractionated in 2% (wt/vol) agarose-formaldehyde gels, transferred to Hybond-N-nylon membranes, and hybridized with the 900-bp fragment containing the full-length BCL-XL coding sequence. For protein analysis, liver tissues were mixed 1:1 with 2× Laemmli sample buffer, boiled for 3 min, and centrifuged for 10 min at 10,000 g. Supernatants were electrophoresed on 12% (wt/vol) SDS-polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose filters. Membranes were blocked with 5% (wt/vol) skimmed milk powder in Tris-buffered saline with 0.05% (vol/vol) Tween 20 (Sigma) and incubated with a goat polyclonal anti-Bcl-2 antibody (Santa-Cruz, SC-492G), a rabbit polyclonal anti-Bcl-xL antibody (Santa Cruz, SC-634), and a rabbit polyclonal anti-Bax antibody (Santa Cruz, SC-493). Immunostaining was performed with peroxidase-coupled anti-goat IgG (Dako) or anti-rabbit IgG (Amersham, NA-9340), respectively, and visualized by enhanced chemiluminescence (Amersham).

Hepatic caspase-3-like activity. Liver lysates were prepared by homogenization (Dounce homogenizer) in hypotonic buffer (25 mM HEPES, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml leupeptin and aprotinin). Homogenates were centrifuged at 15,000 rpm for 15 min. The protein concentrations of the supernatants were determined by the Bradford method. The extracted proteins (50 µg) were tested in duplicate experiments with the Promega Caspace kit by incubation with 1 mM fluorescent substrate for CPP32/caspase-3 (Ac-Asp-Glu-Val-Asp-aminomethyl coumarin, DEVD-AMC) in 0.1 ml ICE standard buffer assay at 30°C for 1 h. The specific contribution of caspase-3-like enzyme activities was assessed by running assays in the presence or absence of a selective inhibitor DEVD-CHO, for CPP32/caspase-3. Levels of AMC released by enzymatic reaction were measured spectrofluorometrically with excitation wavelength of 360 nm and an emission wavelength of 460 nm. The difference between the substrate cleavage activity levels in the presence and absence of caspase inhibitor reflected the contribution of CPP32-like enzyme activity. Caspase activity (pmol per min per mg protein) was measured for five animals per group from 30 to 120 min after anti-Fas Jo2 injection. The caspase-3 activity of PK-BCL-XL transgenic mice was measured only on liver extracts of mice whose histological and serum transaminases clearly indicated almost full inhibition of Fas-mediated apoptosis. Five nontransgenic littermates were injected with sterile saline, killed 120 min later, and used as controls.

Electrophoretic mobility shift assay. Nuclear proteins were harvested as described previously (10). The sequence of NF-kappa B oligonucleotide was 5'-TCGATTACAAAGGGACTTTCCCAT-3'. Electrophoretic mobility shift assay (EMSA) were performed at 4°C in binding buffer containing 5 µg liver nuclear extracts, 2 µg poly(dI-dC) plus 0.1-0.5 ng kinase-treated 5' end-labeled double-stranded NF-kappa B probe. Competition assays used 10-50 ng of the sucrase isomaltase factor or NF-kappa B-unlabeled probes as unspecific and specific competitors. Anti-p50 (kindly provided by A. Israel, Paris, France) and anti-p65 antibodies (Santa Cruz, C20-G) were included in the binding reactions for supershift experiments. Protein-DNA complexes were separated by electrophoresis through 6% nondenaturating polyacrylamide gels. Gels were dried under vacuum and exposed to Kodak films.

Serum analysis. The biochemical parameters of the serum (aspartate aminotransferase and alanine aminotransferase) were measured using a standard clinical automatic analyzer (Hitachi, type 7150).

Data analysis. We used the chi 2 test for survival, Kruskal and Wallis nonparametric ANOVA for biochemical and histological parameters, and one-way ANOVA followed by the Bonferroni t-test for hepatic caspase activity. P < 0.05 was considered to be significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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PK-BCL-XL transgenic mice. The expression of human BCL-XL cDNA was directed in hepatocytes by the regulatory sequences of the rat L-type pyruvate kinase gene (Fig. 1A). Five transgenic PK-BCL-XL lines were characterized. Lines 27 and 45 expressed the highest level of Bcl-xL (Fig. 1, B and C). Human PK-BCL-XL mRNA (1 kb) and murine Bcl-xL mRNA (3 kb) were detected in their livers (Fig. 1B). Both murine and human Bcl-xL proteins were detected as a single 29-kDa band (Fig. 1C), contrasting with a lower constitutive level of Bcl-xL expression in the liver. Overexpression resulted in a Bcl-xL protein concentration two- to five-fold higher than the endogenous one; this varied with the lines and was generally higher in line 27 than in line 45 but also between individual animals in a given line. Like PK-BCL-2 mice (22), the transgenic mice had a normal phenotype and did not develop spontaneous hepatocarcinomas, even in old mice whose life span appears to be normal.


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Fig. 1.   PK-BCL-XL transgenic mice. A: schematic representation of the L-PK human-BCL-XL construct. B: Northern blot. C: Western blot of nontransgenic (non Tg) littermates and two heterozygous PK-BCL-XL transgenic lines (L.45 and L.27). Murine Bcl-xL mRNA (3 kb) and protein were detected in nontransgenic littermate. Unspliced (1.5 kb) and expected (1 kb) PK-BCL-XL transgenic RNAs were detected in transgenic lines, whereas endogenous murine Bcl-xL mRNA was still observed. Overexpression of Bcl-xL was detected in liver of transgenic mice at same 29-kDa size, because anti-Bcl-xL antibody used cross-reacts with human and mouse Bcl-xL.

Expression of endogenous Bcl-2, Bcl-xL, and Bax is not changed in PK-BCL-2 and PK-BCL-XL transgenic mice. The endogenous expression of murine Bcl-2, Bcl-xL, and Bax was unaffected, both at the mRNA (data not shown) and the protein levels (Fig. 2) in PK-BCL-2 and heterozygous PK-BCL-XL transgenic mice from all lines. The Bcl-2-to-Bax and Bcl-xL-to-Bax ratios were therefore increased in the livers of the transgenic mice, suggesting increased antiapoptotic rheostat in transgenic hepatocytes.


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Fig. 2.   Bcl-2 family proteins expression in PK-BCL-2 and PK-BCL-XL transgenic mice. Western blot analysis of Bcl-2, Bcl-xL, and Bax expression in PK-BCL-2 and PK-BCL-XL transgenic mice. Bax expression remained unchanged in transgenic mice. Lane 1: uterus sample, positive control for expression of all 3 Bcl-2 family proteins, Bcl-2, Bcl-xL, and Bax. Lane 2: nontransgenic liver, synthesizing Bax, small amount of Bcl-xL, and undetectable Bcl-2. Lane 3: PK-BCL-2 transgenic mouse, with abundant Bcl-2. Lanes 4 and 5: increased expression of Bcl-xL in liver of heterozygous PK-BCL-XL transgenic mice from lines 45 and 27. Western blot was standardized by annexin V used as internal control.

PK-BCL-XL mice are protected from Fas/CD95-mediated apoptosis. PK-BCL-XL transgenic mice and their nontransgenic littermates were injected intravenously with a lethal dose of Jo2 anti-Fas antibody (0.5 mg/kg). The PK-BCL-XL mice were significantly protected against massive lethal liver injury, as were PK-BCL-2 transgenic mice (Table 1). Apoptosis was greatly inhibited (TUNEL assays) in PK-BCL-XL and PK-BCL-2 livers, compared with nontransgenic mice (Fig. 3). Of 26 heterozygous PK-BCL-XL mice, 15 (57%) survived Jo2 injection. This protection was not as pronounced as for PK-BCL-2 mice, which were almost fully protected (93%). The effect of Bcl-xL appeared to be dose dependent, inasmuch as 11 of 13 (85%) homozygous mice survived the lethal challenge.

                              
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Table 1.   Sensitivity of nontransgenic and PK-BCL-2 and PK-BCL-XL transgenic mice to fulminant lethal apoptotic hepatic injury induced by systemic injection of agonist anti-Fas Jo2 antibody (0.5 mg/kg)



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Fig. 3.   TUNEL assays on liver sections of nontransgenic, PK-BCL-2, and PK-BCL-XL transgenic lines 120 min after systemic injection of anti-Fas antibody Jo2. Extensive labeling of apoptotic hepatocytes in nontransgenic mouse liver (A) but few scattered apoptotic hepatocytes in PK-BCL-2 and heterozygous PK-BCL-XL transgenic mice (B and C) are shown.

Bcl-xL and Bcl-2 block caspase-3 activity. We examined the molecular mechanisms by which Bcl-xL and Bcl-2 inhibit Fas/CD95-induced liver injury by measuring the caspase-3 activity in liver extracts of either nontransgenic, heterozygous PK-BCL-XL, or PK-BCL-2 transgenic mice 30-120 min after Jo2 injection (n = 5 per group). Sustained increases in caspase-3 (CPP32) and caspase-3-like activity have been reported after anti-Fas treatment in vitro (31) and in vivo in mice. Bcl-xL and Bcl-2 overexpression fully inhibited the dramatic increase in caspase-3 activity, which was, in contrast, detected in nontransgenic mice after Jo2 injection (Fig. 4).


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Fig. 4.   Hepatic caspase-3-like activity in nontransgenic, PK-BCL-2, and PK-BCL-XL transgenic lines 120 min after systemic injection of anti-Fas antibody Jo2 (0.5 mg/kg). AMC, aminomethyl coumarin. There was a large increase in caspase-3-like activity in the liver of nontransgenic mice (open bars). By contrast, this activity was almost fully inhibited in PK-BCL-2 (hatched bars) and heterozygous PK-BCL-XL (cross-hatched bars) transgenic mice. Basal hepatic caspase-3-like activity is shown for nontransgenic mice treated with sterile saline (solid bar). Measurements were performed on 5 animals per group. * P < 0.05.

Activation of NF-kappa B in hepatocytes following Fas/CD95 stimulation. The activation of NF-kappa B on Fas/CD95 stimulation has to date not been demonstrated in hepatocytes. We therefore determined whether NF-kappa B was activated in vivo in the liver after stimulation of the Fas/CD95 pathway and/or selectively by Bcl-xL or Bcl-2 on Fas/CD95 receptor activation. Liver nuclear extracts were prepared 1 h after various systemic injections and tested in EMSA (Fig. 5). Saline alone did not activate NF-kappa B in nontransgenic or transgenic mice (lanes 2 and 6). By contrast, a specific NF-kappa B band shift was detected in both the control and PK-BCL-XL transgenic mice challenged with Jo2 anti-Fas antibody (lanes 4 and 8). This band was specific for the p50/p65 NF-kappa B heterodimer, since competition with excess of irrelevant probe (lane 10) did not displace the complex, whereas a 50-fold excess of unlabeled NF-kappa B probe did (lane 9). Supershift experiments performed with specific anti-p50 and p65 (lanes 11 and 12) clearly demonstrated that the Fas/CD95 signaling pathway in the liver activated the p50/p65 heterodimer. Similar NF-kappa B activation was also detected in PK-BCL-XL transgenic mice (data not shown). As NF-kappa B was activated in the liver of both nontransgenic and transgenic mice challenged with anti-Fas antibody, the antiapoptotic effects of Bcl-xL or Bcl-2 would not only be explained by an effect on the NF-kappa B pathway.


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Fig. 5.   Nuclear factor (NF)-kappa B-specific electromobility shift assay of liver nuclear extracts performed in nontransgenic littermate and heterozygous PK-BCL-XL transgenic mice 1 h after systemic injection of anti-Fas antibody Jo2 (0.5 mg/kg). Probe was a double-stranded synthetic radiolabeled oligonucleotide corresponding to NF-kappa B binding site. Lane 1: free-labeled probe. Lanes 2 and 3: liver nuclear extract from nontransgenic littermate taken 1 h after injection of sterile saline or D-galactosamine (D-GalN), respectively, demonstrating undetectable NF-kappa B binding activity. Lane 4: liver nuclear extract from nontransgenic mouse injected with a lethal dose of anti-Fas antibody Jo2; specific NF-kappa B binding activity was detected. Lane 5: liver nuclear extract from nontransgenic mouse injected with lethal dose of anti-Fas antibody Jo2 plus D-GalN; specific NF-kappa B binding activity was detected. Lanes 6 and 7: liver nuclear extract from heterozygous PK-BCL-XL transgenic mice 1 h after injection with sterile saline or D-GalN, respectively, demonstrating undetectable NF-kappa B binding activity. Lane 8: liver nuclear extract from heterozygous PK-BCL-XL transgenic injected with lethal dose of anti-Fas antibody Jo2; specific NF-kappa B binding activity was detected. Lane 9: competition experiment with same nuclear extract and 50-fold excess of unlabeled NF-kappa B oligonucleotide, which fully displaced NF-kappa B- specific retarded band. Lane 10: experiment with same nuclear extract and 50-fold excess of unlabeled irrelevant oligonucleotide sequence (sucrase isomaltase factor, SIF); NF-kappa B-specific retarded band was not displaced. Lanes 11 and 12: supershift experiments with same extract and anti-p50 (lane 11) or anti-p65 (lane 12) antibodies, demonstrating specificity of p50/p65 heterodimer activated by Fas/CD95 stimulation of liver in vivo. Lane 13: liver nuclear extract from heterozygous PK-BCL-XL transgenic mice injected with lethal dose of anti-Fas antibody Jo2 plus D-GalN; specific NF-kappa B activity was detected. Similar patterns were obtained with extracts from PK-BCL-2 mice (not shown).

Partial protection of PK-BCL-XL mice, but not PK-BCL-2, from TNF-alpha -induced liver apoptosis plus D-GalN injection. TNF-alpha induces apoptosis in hepatocytes, provided that gene transcription is blocked with transcription inhibitors such as D-GalN. D-GalN selectively blocks transcription in hepatocytes because this amino sugar exclusively metabolized in hepatocytes causes the selective depletion of uridine nucleotides so that transcription stops 0.5 h after injection and persists for ~3 h (24). We tested the resistance of the transgenic mice to TNF-alpha -mediated cell death by giving to PK-BCL-XL, PK-BCL-2, and nontransgenic mice a lethal dose of murine rTNF-alpha plus D-GalN. The PK-BCL-2 mice died from massive hepatocyte apoptosis within 8 h, but 12 of 34 (35%) heterozygous PK-BCL-XL mice survived the lethal challenge (Table 2). Surprisingly, there was no increased survival of homozygous PK-BCL-XL mice as with the anti-Fas challenge. Because D-GalN blocks transcription in hepatocytes, we first determined whether D-GalN also affected the amounts of antiapoptotic transgenic mRNAs and proteins. Northern and Western blot analyses were therefore performed in transgenic mice killed 3, 6, or 9 h after D-GalN injection. The amount of Bcl-2 and Bcl-xL mRNAs (data not shown) and proteins was not decreased (Fig. 6).

                              
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Table 2.   Protection by Bcl-xL against acute lethal apoptotic hepatic injury induced by systemic injection of recombinant murine tumor necrosis factor-alpha (20 mg/kg) and D-galactosamine (800 mg/kg)



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Fig. 6.   Effects of D-GalN on BCL-2 and BCL-XL transgene expression. Bcl-2 (A) and Bcl-xL (B) expression was analyzed by Western blot experiments in liver of transgenic mice 3, 6, and 9 h after systemic injection of D-GalN. A: lane 1, basal Bcl-2 protein level in liver of PK-BCL-2 transgenic mice; lanes 2-7, Bcl-2 protein concentration remained unchanged in PK-BCL-2 transgenic mice 3, 6, and 9 h after D-GalN treatment. Two representative animals per time point are shown. B: lane 1, basal Bcl-xL protein level in liver of heterozygous PK-BCL-XL transgenic mice; lanes 2-7: Bcl-xL protein level remained unchanged in heterozygous PK-BCL-XL transgenic mice 3, 6, and 9 h after D-GalN treatment; lanes 2, 4, and 6, transgenic mice from line 27; lanes 3, 5, and 7 transgenic mice from line 45. Note great variability in Bcl-xL protein between animals of same line as mentioned in text.

Differential antiapoptotic effects of Bcl-xL and Bcl-2 in the liver. One major difficulty encountered when comparing the Fas/CD95 and the TNF-alpha pathways in the liver in vivo is that TNF-alpha pathways require that transcription be blocked by D-GalN. The range of effects of these drugs remains unclear. We therefore compared the antiapoptotic action of BCL-XL and BCL-2 on Fas/CD95- and TNF-alpha -mediated liver apoptosis using the same experimental conditions. We injected heterozygous PK-BCL-XL and PK-BCL-2 transgenic mice with D-GalN plus a lethal dose (0.5 mg/kg) of anti-Fas antibody. Eight of nine PK-BCL-2 (89%) and 16 of 19 (85%) PK-BCL-XL mice died (Table 3). D-GalN was not toxic by itself (Table 3), nor did it activate NF-kappa B alone (Fig. 5, lanes 2 and 7). On the other hand, injection of D-GalN plus Jo2 antibody did not inhibit the rapid NF-kappa B activation in the liver as with Jo2 treatment alone (Fig. 5, lanes 5 and 13). This result suggests that Bcl-xL and Bcl-2, besides blocking the activation of downstream caspases, require gene transcription to exert their antiapoptotic effects. To elucidate whether gene transcription is necessary for their protective effect, we treated heterozygous PK-BCL-XL, PK-BCL-2, and nontransgenic mice with D-GalN and a nonlethal dose of the anti-Fas Jo2 agonistic antibody (0.15 mg/kg). Seven of eight (88%) nontransgenic mice, which otherwise survived this low dose of the Fas-activating agent, succumbed to the combined effect of Jo2 and D-GalN. Interestingly, 8 of 10 (80%) PK-BCL-2 transgenic mice also died, whereas only 2 of 10 (20%) PK-BCL-XL transgenic mice suffered lethal liver failure (Table 3). This indicates that transcription inhibition sensitizes hepatocytes to apoptosis in vivo as in vitro and inhibits the antiapoptotic effect of Bcl-2 but not of Bcl-xL.

                              
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Table 3.   Effects of Bcl-xL and Bcl-2 in apoptotic hepatic experimental protocol induced by injecting anti-Fas antibody Jo2 and D-GalN


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We show here that PK-BCL-XL transgenic mice are significantly protected from liver failure induced by the Fas/CD95 pathway. These results occur with a relatively modest BCL-XL overexpression (2- to 5-fold). Greater BCL-XL overexpression, produced by gene transfer or pharmacological intervention, would probably provide full protection. Accordingly, hepatic growth factor was shown recently to significantly block Fas/CD95-induced liver injury in vivo. This protection by hepatic growth factor was probably due to its ability to cause great overexpression of Bcl-xL in hepatocytes (21).

As expected, and in agreement with many in vitro studies (9, 13, 36), Bcl-xL and Bcl-2 act upstream of caspase-3 in vivo, because they completely inhibited the significant increase in caspase-3 activity in response to Fas/CD95 triggering. In fact, Bcl-xL and Bcl-2 probably act at the mitochondrial step of the apoptotic cascade (18), in agreement with the properties of mitochondria isolated from our PK-BCL-2 transgenic mice (32).

We find that Bcl-xL and Bcl-2 block apoptotic liver injury in transgenic mice differentially. Whereas both PK-BCL-XL and PK-BCL-2 mice are protected from Fas/CD95-mediated apoptosis, only PK-BCL-XL mice appear to be protected, at least partially, from liver injury caused by TNF-alpha plus D-GalN. Many studies have clearly demonstrated that Bcl-2 and Bcl-xL play a central role in regulating programmed cell death (1). The question has been raised as to whether each molecule has an entirely distinct function or whether they similarly regulate common apoptotic pathways. Several hypothesis could be proposed to explain the differential ability of Bcl-2 and Bcl-xL to inhibit apoptosis, at least in the liver. 1) Different BH1 and BH2 regions of Bcl-xL and Bcl-2 could lead to differential interaction with Bax (12). 2) Bcl-2 and Bcl-xL could directly interact as substrates with different subsets of caspases. These interactions with and the cleavage specificity of particular subsets of caspases for Bcl-2 or Bcl-xL might be regulated differently (11, 15). 3) On going or induced transcription could be essential for the antiapoptotic action of Bcl-2, whereas a certain Bcl-xL action could continue in the presence of a transcription inhibitor. The factor responsible for stimulating antiapoptotic partners of Bcl-2 could be NF-kappa B, which is known to prevent TNF-alpha -mediated apoptosis (see Ref. 5 for review).

We show in this paper that activation of the Fas/CD95 receptor is associated with NF-kappa B activation, as it was already known on activation of the TNF-alpha receptor (4). This result is in agreement with recent reports indicating that animals could be protected against Fas/CD95-mediated apoptosis by partial hepatectomy or the injection of rTNF-alpha , two conditions which are known to activate NF-kappa B (33).

Prevention of NF-kappa B activation would explain the sensitization by D-GalN to Fas/CD95-mediated apoptosis, even in PK-BCL-2 mice, while the PK-BCL-XL transgene mice still have some protective effect. We propose that exogenous TNF-alpha results in robust induction of antiapoptotic genes, particularly through NF-kappa B activation, that is sufficient itself to prevent hepatocytes apoptosis in vivo. In contrast, the antiapoptotic gene response to Fas/CD95 activation alone is insufficient to prevent apoptosis but could act synergistically with Bcl-2. Although this antiapoptotic transcriptional response may be permissive for the Bcl-2 action, Bcl-xL could still have some antiapoptotic activity when transcription is blocked.

The major antiapoptotic role of NF-kappa B in the liver has now been extended to numerous models (4, 34). 1) Embryonic lethal apoptotic liver destruction has been described in p65 and Ikappa B kinase 2-deficient mice (6, 25). 2) Murine hepatocytes treated with NF-kappa B inhibitors also became apoptotic (7). 3) NF-kappa B downregulation is required for TGF-beta -induced apoptosis in hepatic cell lines (2). 4) Finally, blocking NF-kappa B posthepatectomy results in increased TNF-alpha -mediated apoptosis and liver dysfunction (20).

Numerous antiapoptotic targets of NF-kappa B have now been described, in particular the Bcl-2 homologue Bfl-1/A1, recently shown to be a direct transcriptional target of NF-kappa B and shown to block TNF-alpha -induced apoptosis (37). This suggests that combined with the transgenic expression of Bcl-2 or Bcl-xL other inducible antiapoptotic factors can increase the hepatocyte "apopstat" and confer resistance to Fas-mediated hepatocyte cell death.

In conclusion, we have provided evidence that Bcl-xL and Bcl-2 can protect from Fas/CD95-mediated fulminant hepatitis liver in vivo. However Bcl-xL and Bcl-2 have slightly different actions, at least in the liver. The antiapoptotic effect of Bcl-2 but not that of Bcl-xL is absolutely dependent on active transcription. The very frequent involvement of apoptosis in liver diseases (28) suggests that increasing Bcl-xL or Bcl-2 synthesis in the liver, by gene transfer or by pharmacological agents, could provide a promising field of therapeutic research.


    ACKNOWLEDGEMENTS

We thank Dr. Michel Raymondjean for helpful discussion and support in performing NF-kappa B EMSA, Dr. Craig B. Thompson for providing the hBcl-xL cDNA, Drs. Olivier Soubrane and Virginie Joulin for helpful discussion and critical reviews, Dr. Robert Palau for technical assistance, and Nathalie Bâ, Arlette Dell'amico, Isabelle Lagoutte, and Hervé Gendrot for technical assistance.


    FOOTNOTES

This work was supported by the Ligue Nationale contre le Cancer, Association de Recherche contre le Cancer, and the European Union BIO4CT 960052.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. Kahn, INSERM U-129 ICGM, Université Paris V René Descartes, 24, Rue du Faulourg Saint-Jacques, 75014 Paris, France (E-mail: kahn{at}icgm.cochin.inserm.fr).

Received 2 November 1998; accepted in final form 27 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastroint Liver Physiol 277(3):G702-G708
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