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
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ABSTRACT |
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Fas ligand (CD95L)
and tumor necrosis factor- (TNF-
) 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-
-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-
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-
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-; apoptosis
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INTRODUCTION |
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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- (TNF-
) 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--receptor type 1 appear to recruit similar or at least partially
overlapping pathways (3). Upon receptor trimerization, activated Fas
and TNF-
-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- 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-
-induced apoptosis in
vivo. These cells proliferate when stimulated by TNF-
, a critical
mechanism involved in liver regeneration (26). The resistance of many
cell types, including hepatocytes, to TNF-
-induced apoptosis, was
recently linked to the ability of TNF-
to stimulate the
transcription of several protective genes, mainly those dependent on
the nuclear factor (NF)-
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-
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--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-
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-
-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- 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-
pathways appear to be involved.
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MATERIALS AND METHODS |
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Materials.
Jo2 anti-Fas antibody in sterile 0.9% NaCl was purchased from
Pharmingen. Recombinant murine TNF- (rTNF-
) 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 -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- studies, rTNF-
was injected intraperitoneally at 20 µg/kg.
D-GalN (800 mg/kg) was injected
intraperitoneally 30 min before the rTNF-
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-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-
B probe. Competition assays used 10-50 ng of the sucrase
isomaltase factor or NF-
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 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.
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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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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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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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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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Activation of NF-B in hepatocytes following Fas/CD95
stimulation.
The activation of NF-
B on Fas/CD95 stimulation has to date not been
demonstrated in hepatocytes. We therefore determined whether NF-
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-
B
in nontransgenic or transgenic mice (lanes
2 and 6). By
contrast, a specific NF-
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-
B heterodimer, since competition with excess of
irrelevant probe (lane 10) did not
displace the complex, whereas a 50-fold excess of unlabeled NF-
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-
B activation was also detected in
PK-BCL-XL
transgenic mice (data not shown). As NF-
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-
B pathway.
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Partial protection of PK-BCL-XL mice, but
not PK-BCL-2, from TNF--induced liver apoptosis plus
D-GalN injection.
TNF-
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-
-mediated cell death by giving to
PK-BCL-XL,
PK-BCL-2, and nontransgenic mice a
lethal dose of murine rTNF-
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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Differential antiapoptotic effects of
Bcl-xL and Bcl-2 in the liver.
One major difficulty encountered when comparing the Fas/CD95 and the
TNF- pathways in the liver in vivo is that TNF-
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-
-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-
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-
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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DISCUSSION |
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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- 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-
B, which is known to prevent TNF-
-mediated apoptosis
(see Ref. 5 for review).
We show in this paper that activation of the Fas/CD95 receptor is
associated with NF-B activation, as it was already known on
activation of the TNF-
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-
, two conditions which are known to activate NF-
B (33).
Prevention of NF-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-
results in robust induction of antiapoptotic genes,
particularly through NF-
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-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 I
B kinase 2-deficient mice
(6, 25). 2) Murine hepatocytes
treated with NF-
B inhibitors also became apoptotic (7).
3) NF-
B downregulation is
required for TGF-
-induced apoptosis in hepatic cell lines (2).
4) Finally, blocking NF-
B
posthepatectomy results in increased TNF-
-mediated apoptosis and
liver dysfunction (20).
Numerous antiapoptotic targets of NF-B have now been described, in
particular the Bcl-2 homologue Bfl-1/A1, recently shown to be a direct
transcriptional target of NF-
B and shown to block TNF-
-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.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michel Raymondjean for helpful discussion and support
in performing NF-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.
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FOOTNOTES |
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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.
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