From the Department of Microbiology and Cell Biology,
The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, the
§ Department of Immunology, Graduate School of Medicine and
Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku,
Tokyo, 113-0033, Japan, the ¶ Department of Internal Medicine,
Self-Defense Forces Central Hospital, 1-2-24 Ikejiri, Setagaya-ku,
Tokyo, 154-0001, Japan, the
Department of Biochemistry, Kawasaki
Medical School, 577 Matsushima, Kurashiki, Okayama, 7.1-0192, Japan,
the ** Department of Molecular Cell Physiology, The Tokyo Metropolitan
Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo
113-8613, Japan, the
Department of
Microbiology and Immunology, Nippon Medical School, 1-1-5, Sendagi,
Bunkyo-ku, Tokyo 113-8602, Japan, the
§§ Department of Pathology, Tokyo Metropolitan
Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8677, Japan, and the ¶¶ Laboratory of Animal Science, The Tokyo
Metropolitan Institute of Medical Science, 3-18-22 Honkomagome,
Bunkyo-ku, Tokyo 113-8613, Japan
Received for publication, November 7, 2000, and in revised form, January 9, 2001
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ABSTRACT |
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Persistent hepatitis C virus (HCV)
infection often progresses to chronic hepatitis, cirrhosis, and
hepatocellular carcinoma. Numerous viruses have been reported to escape
from apoptotic mechanism to maintain persistent infection. In the
present study, we characterized the effect of HCV proteins on the Fas
signal using HCV transgenic mice, which expressed core, E1, E2, and NS2
proteins, regulated by the Cre/loxP switching system. The
transgene expression of HCV transgenic mice caused resistance to Fas
antibody stimulated lethality. Apoptotic cell death in the liver of HCV
protein expressing mice was significantly reduced compared with
nonexpressing mice. Histopathological analysis and DNA fragmentation
analysis revealed that the HCV proteins suppressed Fas-mediated
apoptotic cell death. To identify the target pathway of HCV proteins,
we characterized caspase activity. The activation of caspase-9 and -3/7
but not caspase-8 was inhibited by HCV proteins. Cytochrome
c release from mitochondria was inhibited in HCV protein
expressing mice. These results indicated that the expression of HCV
proteins may directly or indirectly inhibit Fas-mediated apoptosis and
death in mice by repressing the release of cytochrome c
from mitochondria, thereby suppressing caspase-9 and -3/7 activation.
These results suggest that HCV may cause persistent infection, as a
result of suppression of Fas-mediated cell death.
Hepatitis C virus (HCV)1
is a positive-strand RNA virus and major causative agent of
post-transfusion-associated and sporadic nonA nonB hepatitis.
Persistent HCV infection often progresses to chronic hepatitis,
cirrhosis, and hepatocellular carcinoma. The mechanism involved in the
development of persistent infection and the pathogenesis is still unclear.
A number of viruses have been reported to escape from the apoptotic
mechanism to maintain persistent infection. The expression of HCV
proteins is reported to influence apoptosis. HCV proteins have been
variously found to suppress or activate anti-Fas antibody and/or tumor
necrosis factor (TNF)- A transgenic mouse model using a stable expression system causes
immunotolerance to transgene products. Therefore, an HCV protein
switching expression system may be suitable for in vivo assay of HCV protein effects. Using the Cre/loxP system, we
developed a transgenic mouse model with efficient conditional transgene activation of HCV cDNA (core, E1, E2, and NS2) (8). HCV proteins were mainly detected in the liver of conditionally expressing transgenic mice. These methods allowed us to investigate the importance of HCV proteins in apoptotic signaling pathways by the conditional expression of HCV proteins in vivo.
Fas-mediated cell death appears to induce hepatic injury (9).
Fas-mediated liver injury is likely to play a critical role in some
forms of immune-mediated hepatitis. It has become evident that
activated T lymphocytes have the ability to kill Fas-bearing targets
through membrane expression of Fas ligand (Fas-L) (10). Experimental
liver injury models have demonstrated the important roles of this
molecule: intraperitoneal administration of agonistic anti-Fas
antibodies damages the liver by massive hepatocyte apoptosis, with (11)
or without lethality (12). The involvement of the Fas/Fas-L system has
been shown in many injury models (10). In addition, involvement of the
Fas/Fas-L system in liver homeostasis was suggested by a report that
Fas-deficient mice show substantial liver hyperplasia (13).
The Fas protein CD95 is a homotrimeric molecule and Fas-L causes
clustering of the Fas death domains. An adapter protein called FADD
then binds through its own death domain to the clustered receptor death
domains (14). Upon recruitment by FADD, caspase-8 is activated through
self-cleavage and oligomerization (15). Caspase-8 then activates
downstream effector caspases such as caspase-3/7 (16). Bcl-2 family
proteins play a pivotal role in controlling cell life and death, with
some members such as Bcl-2 and Bcl-XL, inhibiting
apoptosis, and others such as Bax, inducing cell death (17).
In the present paper, we establish an experimental system in which the
effects of HCV protein on Fas-mediated apoptosis can be examined. Some
of the potential target pathways of HCV proteins are also characterized.
Expression of HCV Transgene in Vivo--
HCV transgenic mice CN2
(BALB/c, 9-13 weeks old) were used in experiments. The CN2 transgenic
mouse is the HCV genotype 1b transgenic mouse regulated by the
Cre/loxP conditional switching system (8). AxCANCre and
AxCAw1 recombinant adenoviruses were replication-deficient, lacking the
E1A, E1B, and E3 regions (18). AxCANCre expressed recombinase Cre, and
AxCAw1, which was used as the control adenovirus, lacked the inserted
cre gene. AxCANCre or AxCAw1 (2 × 109 pfu)
were intravenously injected into transgenic or nontransgenic female
mice between 9 and 13 weeks of age. The CN2-8 mice were intravenously
injected with AxCANCre and AxCAw1 virus and killed at 1, 2, 3, 4, 5, 6, and 7 days after the injection. The liver tissue was stained with
anti-core polyclonal antibody RR8 using TSA direct method (PerkinElmer
Life Sciences). Four days after the inoculation of recombinant
adenovirus, mice were administered anti-Fas antibody (clone Jo2;
PharMingen) intravenously, at a dose of 0.14 µg/g. The animals (6-10
mice) were observed for mortality over 24 h, and sacrificed 1, 2, 3, 4, 5, 6, 8, 12, and 24 h after anti-Fas antibody administration
for liver histology and biochemical studies. Animal care was in
accordance with institutional guidelines.
Quantification of HCV Core Protein in Mouse Liver--
Mouse
liver was homogenized in 1 ml of RIPA buffer (1% SDS, 0.5% Nonidet
P-40, 10 mM Tris (pH 7.4), 0.15 M NaCl). HCV
core protein concentration of the liver lysates was quantified by
fluorescent enzyme immunoassay (FEIA) (19). The FEIA is based on two
high-affinity monoclonal antibodies directed against the HCV core
protein. These two monoclonal antibodies, 5E3 and 5F11, recognize amino
acids 21-40 and 41-60, respectively. These pretreated samples were
added to EIA tubes precoated with monoclonal antibody 5F11. The tube was incubated for 10 min at 37 °C. Monoclonal antibody 5E3
conjugated to Biochemical Analysis of Mouse Sera--
Blood samples were
collected from the supra orbital veins at 0, 1, 2, 3, 4, 5, and 6 h after Fas antibody administration or heart puncturing of sacrificed
mice, and centrifuged at 10,000 × g at 4 °C. Serum
alanine aminotransferase (ALT) levels were assayed by enzyme reaction
with Transnase Nissui kit (Nissui Pharmaceutical, Tokyo, Japan).
Measurement of Caspase Activity by Cleavage of Fluorogenic
Substrates--
The cytosolic fraction of liver tissues was isolated
as previously reported, with partial modification (20). Briefly, liver tissue was homogenized in a Dounce glass homogenizer (loose type) with
lysis buffer (0.3 M mannitol, 5 mM MOPS, 1 mM EGTA, 4 mM KH2PO4,
20 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride) and fractionated into
pellet, heavy membrane, light membrane, and cytosolic fractions. The
lysates were centrifuged at 600 × g for 10 min at
4 °C to remove debris or fiber. The supernatants were centrifuged at
10,000 × g for 15 min at 4 °C and the pellets
collected as the heavy membrane fraction. The supernatants were again
centrifuged at 100,000 × g for 45 min at 4 °C and
collected as the cytosolic fraction. The activities of caspase-8, -9, and -3/7 were measured using Ac-LETD-AFC, Ac-LQTD-AFC, Ac-LEHD-AFC, or
Ac-DEVD-AFC (Enzyme Systems Products) as substrates, respectively.
Substrates were preincubated for 10 min at 37 °C in reaction buffer
composed of 20 µM substrate, 100 mM Hepes (pH
7.5), 10% sucrose, 10 mM dithiothreitol (pH 7.5), and
0.1% CHAPS (21). After preincubation, 100 µg of lysate was added to
1.25 ml of reaction buffer and incubated for 60 min at 37 °C. The
fluorescence of released 7-amino-4-trifluoromethylcoumarin (AFC) was
detected by a fluorescence spectrometer (model F2000; Hitachi).
Excitation wavelength was 400 nm and emission wavelength was 505 nm.
Specific caspase-3/7 or -8 activity was determined by subtracting the
values obtained in the presence of 10 µM Ac-DEVD-CHO or
Ac-IETD-CHO (Peptide Institute, Osaka, Japan), inhibitors of caspase-3/7 or -8, respectively. One unit corresponds to the activity required to cleave 1 pmol of the substrate in 60 min.
Western Blotting of Caspase-8 and Bcl-2 Family Members--
The
liver was perfused with phosphate-buffered saline( Western Blotting of Cytochrome c--
Liver tissue was minced
and washed once in ice-cold homogenizing buffer composed of 0.1%
bovine serum albumin, 1 mM EGTA, 300 mM
sucrose, 5 mM MOPS, 5 mM
KH2PO4 (pH 7.4), and 1 × complete inhibitor mixture (Roche Molecular Biochemicals). The minced liver was
gently homogenized in the homogenate buffer in a Dounce glass homogenizer using five complete up and down cycles of a glass loose-type pestle (22). The homogenate was centrifuged at 600 × g for 15 min at 4 °C. The supernatant was recovered and
centrifuged at 10,000 × g for 15 min at 4 °C. The
pellet, representing the heavy membrane fraction, was washed once with
homogenization buffer and denatured with SDS sample buffer (67 mM Tris (pH 6.8), 2% SDS, 10% glycerol, 5%
2-mercaptoethanol). The protein concentration of the lysates was
measured using the Bradford method (Bio-Rad DC protein assay). Lysates
(30 µg) were electrophoresed by 15% SDS-polyacrylamide gel
electrophoresis using Tris Tricine Buffer (Daiichi Pure Chemicals),
transferred to polyvinylidene difluoride membrane (Amersham Pharmacia
Biotech), and blocked with 5% skim milk in TBST. The membrane was
washed with TBST for 5 min and incubated with anti-cytochrome
c rabbit polyclonal
antibody2 in 5% skim milk in
TBST for 16 h at 4 °C. The membrane was washed with TBST three
times and incubated with anti-rabbit IgG (Amersham Life Science) for
2 h at room temperature. The signal was detected by the ECL system
(Amersham Pharmacia Biotech).
Histology and Immunohistochemical
Staining--
Histopathological analysis of hematoxylin- and
eosin-stained tissue sections was undertaken. The liver tissues were
frozen with O.C.T. compound (Tissue Tech) for immunohistochemical
analysis. The sections were fixed with a 1:1 solution of
acetone:methanol at
For cytochrome c staining, the sections were incubated in
blocking buffer for 30 min at room temperature and incubated with rabbit anti-cytochrome c antibodies for 3 h at room
temperature. The sections were washed with phosphate-buffered
saline( FACS Analysis--
Cytofluorometry was performed by FACS Calibur
(Becton-Dickinson, San Jose, CA). Primary hepatocytes were stained with
an optimal dilution of anti-Fas-FITC antibody (06134D, PharMingen, San
Diego, CA), and examined by FACScan.
Statistical Analysis--
Data represent the mean ± S.D.
All statistical analysis was performed using Student's t
test. Statistical significance was established at p < 0.05.
HCV Protein Expression in the Liver of HCV Transgenic
Mice--
HCV proteins were expressed in the liver of HCV transgenic
mice. Expression of core protein in AxCANCre adenovirus-injected CN2-8
transgenic mice liver was confirmed by immunofluorescence staining.
Core protein was expressed in 50-60% of hepatocytes in the lobule of
the liver section at day 4, and 70-80% of hepatocytes at 7 days after
AxCANCre injection (Fig. 1A).
The genomic DNA was prepared from liver 4 or 5 days after the injection
of adenovirus and analyzed by Southern blot analysis. Transgene
recombination occurred in the livers of the transgenic mice (Data not
shown). In contrast, core protein was not expressed in liver sections from transgenic mice without AxCANCre injection or in AxCAw1-injected CN2 mice (Fig. 1A). The core protein levels in hepatocytes
were measured by FEIA. The mean level of core protein was 5.7 ng/mg total protein in the HCV transgenic mice liver 4 days after the administration of AxCANCre adenovirus (Fig. 1B). The
expression of core, E1 and E2 proteins was detected in
AxCANCre-injected mouse liver by Western blot analysis (data not
shown).
The livers of CN2 mice, 5 days after administration of AxCANCre and
AxCAw1, did not show infiltration of mononuclear cells or an increase
in serum ALT levels (Fig. 1C). A significant liver injury
was not evoked until day 5. Therefore, 4 days after the administration
of AxCANCre or AxCAw1 was a suitable time point to characterize the
effect of HCV proteins on Fas-mediated cell death.
Inhibition of Fas-mediated Death by HCV Proteins--
Mice not
expressing HCV were treated with 0.14 µg/g mice body weight of
anti-Fas antibody, which killed 50% of the mice over the initial
24 h (n = 6). In contrast, all of the HCV protein expressing transgenic mice were resistant to Fas stimulated lethality over the initial 24 h (Fig.
2A). Another transgenic mice
strain, CN2-29 (8) was also analyzed to better understand the effects of the transgene integration site. CN2-29 mice expressed an average of
0.54 µg/mg core protein on day 4 after AxCANCre administration. Ten
of the AxCAw1 adenovirus-injected CN2-29 mice were administered 0.14 µg/g anti-Fas antibody and observed for 24 h (Fig.
2B). Half of the AxCAw1 adenovirus-infected CN2-29 mice
died. In contrast, 80% of the AxCANCre adenovirus-injected CN2-29 mice
survived after the administration of anti-Fas antibody
(n = 10). To investigate the effects of adenovirus,
AxCANCre and AxCAw1 adenovirus-injected nontransgenic BALB/c mice were
administered 0.14 µg/g anti-Fas antibody. Within 24 h of
administration, 50% AxCAw1 and 50% AxCANCre injected nontransgenic
BALB/c mice died (n = 10) (Fig. 2C).
Expression of HCV proteins appeared to inhibit the progression of death
after the injection of anti-Fas antibody.
Inhibition of Hepatic Injury in Transgenic Mice with HCV Protein
Expression--
Serum ALT levels dramatically increased within 3 h of injection of the anti-Fas antibody in AxCAw1-injected CN2-8 mice
(Fig. 3A). In contrast,
significantly lower ALT levels were observed in HCV protein expressing
CN2-8 transgenic mice 3 and 6 h after injection of the anti-Fas
antibody (p = 0.0352, and p = 0.0266 respectively).
Liver tissue of anti-Fas antibody-treated mice was sampled from 0 to
4 h after the intravenous injection for histological examination
(Fig. 3B). Four h after the anti-Fas antibody injection, hepatic injury was observed predominantly in the liver of mice not
expressing HCV proteins (Fig. 3B) and fragmented nuclei were observed in over 80% of the hepatocytes. In contrast, histological analysis of livers from HCV protein expressing transgenic mice revealed
that liver injury was suppressed and cell death was far less prevalent
than in the liver of mice not expressing HCV proteins (Fig.
3B). The nuclear shrinkage and fragmentation observed in hepatic cell death resembled apoptotic cell death. Therefore, DNA
fragmentation was assessed in the livers of anti-Fas antibody injected
mice by genomic DNA laddering. Ladder formation was much more moderate
in the HCV protein expressing transgenic hepatocytes than in the
hepatocytes in the HCV-nonexpressing mice at 5, 6, 8, and 12 h
after the injection of anti-Fas antibody (Fig.
4). These findings indicate that
expression of HCV proteins inhibited the progress of apoptotic
hepatocyte death caused by anti-Fas antibody injection.
Expression of HCV Proteins Inhibited Activation of Caspase-3/7 and
Caspase-9--
To clarify the mechanism of Fas-mediated apoptosis
inhibition by HCV proteins, expression of Fas antigen on hepatocytes
and activation of caspase-8, -9, and -3/7 proteases were examined in
liver samples. Four h after the administration of anti-Fas antibody,
Fas protein levels in hepatocytes and expression on the cell surface
did not differ significantly between HCV-positive and HCV-negative mice
(Fig. 5, A and
B).
Caspase-8 was similarly activated within 1 h in both HCV-negative
and HCV-positive transgenic mice treated with anti-Fas antibody (Fig.
6). The active form of the caspase-8
subunit (p18) appeared within 1 h after anti-Fas antibody
injection (Fig. 6A). No significant difference was observed
in p18 patterns between HCV protein expressing and nonexpressing CN2-8
mice (Fig. 6A). To determine the activity of caspase-8, -9, and -3/7, the fluorogenic substrates with the cleavage site by each
caspase were used. The results were consistent with the results from
Western blot analysis (Fig. 6A). Caspase-8 was similarly
activated in the liver tissue of HCV protein expressing and
nonexpressing mice within 1 h after anti-Fas antibody injection (Fig. 6B). HCV proteins expression did not repress substrate
cleavage activity by caspase-8 as shown by the substrate cleavage assay using both Ac-LETD-AFC and Ac-LQTD-AFC, which are the known cleavage sites of Bid (22). After 3 and 4 h of Fas antibody administration, caspase-8 activity decreased in liver tissues from mice not expressing HCV proteins (Fig. 6B). The results suggested that the
greater damage to liver tissue observed in mice not expressing HCV
proteins contributed to the decrease in caspase-8 activity itself in
HCV-negative livers. In contrast, activation of caspase-3/7 and -9 was
suppressed in HCV protein expressing liver tissue, 3 and 4 h after
antibody administration (Fig. 6B).
Immunoblot Analysis of Bcl-2 Family Proteins in Hepatocytes of Mice
Injected with Anti-Fas Antibody--
The inhibitory mechanism of
Fas-mediated cell death by HCV genome expression was analyzed further.
We examined expression levels of the Bcl-2 protein family, which
localize in the mitochondria and regulate the progression of apoptotic
cell death. Bcl-2 family protein level, including Bid,
Bcl-XL, Bcl-2, Bad, and Bax was analyzed by immunoblot
(Fig. 7, A and B).
Caspase-8 activity of Bid cleavage was not inhibited by HCV proteins.
Translocation of the C-terminal of Bid protein into mitochondria was
not repressed by HCV proteins 4 h after the administration of
anti-Fas antibody in HCV transgenic mice (Fig. 7A). Bcl-2
protein increased gradually after the administration of anti-Fas
antibody in liver tissue of both HCV protein expressing and
nonexpressing mice (Fig. 7B). Bcl-XL protein was
consistently expressed in liver tissue of HCV protein expressing mice
after 3 h (Fig. 7B). In contrast, the Bcl-XL protein decreased to the basal level over 3 h
in nonexpressing mouse liver tissue (Fig. 7B). HCV protein
did not contribute to the degradation of Bad protein after anti-Fas
antibody-mediated cell death. Bax was consistently expressed even after
the administration of anti-Fas antibody in both HCV protein expressing
and nonexpressing mice.
Bax protein level was analyzed by Western blot in four fractions,
including the soluble, light membrane, heavy membrane (mitochondria rich), and low speed pellet fractions in liver lysates taken 0, 1, 2, 3, and 4 h after the administration of anti-Fas antibody. Bax
protein translocated similarly from the cytosolic fraction to the
mitochondria-rich fraction within 1 h of the administration of
anti-Fas antibody in both HCV nonexpressing and expressing mice (Fig.
7C). This translocation of Bax has also been reported in
some cell lines (23).
Inhibition of Cytochrome c Release by Expression of HCV
Proteins--
To clarify HCV mediated-inhibitory mechanisms on the
Apaf-1/caspase-9 amplification loop, the distribution pattern of
cytochrome c was examined by immunohistochemistry. The
originally prepared anti-cytochrome c
antibody3 strongly detected
cytochrome c in cytoplasm released from mitochondria, and
weakly detected cytochrome c in mitochondria in a spotty
pattern. Liver sections of pretreated mice showed a spotty staining
pattern in the cytoplasm (Fig.
8A). Immunoreactivity of
cytoplasmic cytochrome c increased in HCV-negative
hepatocytes after 4 h of Fas antibody administration (Fig.
8A), reflecting the release of cytochrome c from
mitochondria. In contrast, staining of cytochrome c was much
weaker in HCV protein expressing hepatocytes (Fig. 8A).
Therefore, HCV protein inhibited the release of cytochrome c
from mitochondria.
To further examine the localization of cytochrome c, the
liver lysates were fractionated by centrifugation into supernatant, heavy membrane, and nuclear fractions. The heavy membrane fraction was
a mitochondria-rich fraction that contained cytochrome c, as
indicated by immunoblot analysis with anti-cytochrome c
antibody (Fig. 8B). The mitochondrial fraction of liver
tissue was collected 0, 1, 3, and 4 h after anti-Fas antibody
administration from both HCV protein expressing and nonexpressing mice.
The quantity of cytochrome c in the heavy membrane fraction
of HCV protein expressing mice liver after 4 h of Fas antibody administration was not significantly reduced compared with HCV nonexpressing mice (Fig. 8B). Hence, the cytoplasmic
fraction after both 3 and 4 h in HCV protein expressing mice liver
had 3-4-fold decrease of cytochrome c than that of
hepatocytes in HCV nonexpressing mice (Fig. 8C). These
results indicated that the release of cytochrome c from
mitochondria to cytoplasm in hepatocytes was suppressed by HCV protein expression.
A part of the defense mechanism against virus infection is induced
to initiate apoptotic cell death by signals delivered from CTL. On the
other hand, numerous viruses have been reported to escape from
apoptotic mechanism to maintain persistent infection (24-28). In this
study, we investigated that HCV might cause persistent infection, as a
result of suppression of Fas-mediated cell death and inhibition of
HCV-infected hepatocyte rejection in the liver (Fig.
9).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mediated cell death (1-4). In addition, HCV
reportedly activates nuclear factor
B (NF-
B) and represses Fas
and TNF-
-mediated cell death (5-7). The effects of cell death by
HCV proteins are not fully understood because several discrepancies
have been observed in the activation and repression of Fas or TNF-
related cell death.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactosidase was added after washing
with buffers, and the mixture was incubated for 9 min at 37 °C.
4-Methylumbelliferyl
-D-galactopyranoside was used as a
substrate. After incubation for 9 min at 37 °C, the relative
fluorescence intensity was determined using a fluorometer after
addition of 0.1 M glycine-NaOH (pH 10.3). HCV core protein
concentration in the liver samples was divided by the total protein
concentration and expressed as nanograms/mg of total protein.
) at 37 °C
through the portal vein to remove monocytes, sectioned, and rapidly
frozen in liquid nitrogen. Liver tissue was homogenized in a Dounce
glass homogenizer using five complete up and down cycles of a glass
loose-type pestle in a homogenate buffer composed of 1% SDS, 0.5%
Nonidet P-40, 0.15 M NaCl, 10 mM Tris (pH 7.4), and 1 × complete protease inhibitor mixture (Roche Molecular
Biochemicals). The lysates were centrifuged at 10,000 × g for 15 min at 4 °C. The supernatant was collected and
used as liver lysate. Lysates (30 or 50 µg) were separated via 15%
SDS-polyacrylamide gel electrophoresis using Tris Tricine buffer
(Daiichi Pure Chemicals) and transferred onto polyvinylidene difluoride
membrane (Amersham Pharmacia Biotech). The membrane was incubated in
blocking buffer composed of 5% skim milk in TBST (20 mM
Tris (pH 7.4), 137 mM NaCl, 0.5% Tween 20) followed by
primary antibody; anti-Caspase-8 (T-16, Santa Cruz Biotechnology),
anti-Bcl-XL (S-18, Santa Cruz Biotechnology), Bid (R&D
Systems), Bax (N-20, Santa Cruz Biotechnology), Bcl-2 (4C-11, Santa
Cruz Biotechnology), and Bad (New England Biolabs, Inc.).
20 °C for 10 min and then washed with
phosphate-buffered saline(
). Subsequently, the sections were
incubated with the IgG fraction of an anti-HCV core rabbit polyclonal
antibody (RR8) (8) in blocking buffer for 16 h at 4 °C. The
sections were incubated with secondary antibody, horseradish
peroxidase-conjugated anti-rabbit IgG, for 2 h at room
temperature. Immunohistochemical staining was completed using tyramide
signal amplification (PerkinElmer Life Sciences).
). Primary antibodies were detected by Texas Red-conjugated
anti-rabbit IgG (CAPPEL). Fluorescently labeled sections were stained
with 0.5 µg/ml Hoechst 33342 dye (Molecular Probes) for 1 min at room
temperature before coverslipping to stain cell nuclei. Fluorescence was
observed under a fluorescence microscope (Carl Zeiss).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Immunofluorescence analysis of HCV core
protein in liver. A, liver sections of CN2-8
mice were fixed and stained using rabbit anti-core polyclonal
antibodies as described under "Experimental Procedures." The CN2
mice (n = 3) were sacrificed 0, 1, 2, 3, 4, 5, 6, and 7 days after AxCANCre injection (HCV expressed). The control
adenovirus AxCAw1-injected livers were examined on day 4 (HCV not
expressed). The immunohistochemistry of HCV protein expressing
hepatocytes was assayed by staining with anti-core antibody.
B, core protein levels in hepatocytes of AxCANCre-injected
CN2-8 mice were quantified by FEIA. The quantity of core protein shown
represents the mean and S.D. of three individual experiments.
C, the serum ALT level after injection of AxCANCre
adenovirus in CN2-8 mice (mean and S.D. of three individual
experiments).
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Fig. 2.
Survival rates of HCV transgenic mice after
administration of anti-Fas antibody. Survival rate of CN2-8
(A), CN2-29 (B), or BALB/c nontransgenic mice (-)
injected with AxCANCre (closed circle) or AxCAw1 (open
circle) in response to an intravenous injection of 0.14 µg/g
anti-Fas antibody.
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Fig. 3.
Serum ALT level and histological analysis of
liver after Fas antibody administration. A, serum ALT
levels of Fas antibody injected HCV nonexpressing (open
circles) and expressing (closed circle) mice. ALT
levels represent the mean and S.D. of three individual experiments.
B, hematoxylin and eosin staining of liver sections from
transgenic mice, pretreatment and 4 h after anti-Fas antibody
injection.
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Fig. 4.
Genomic DNA fragmentation in HCV-negative
(A) or HCV-protein expressing (B)
hepatocytes. AxCAw1-injected (A) and AxCANCre-injected
(B) CN2-8 mice were administered anti-Fas antibody on day 4 after adenovirus injection. Genomic DNA was prepared from livers at 0, 1, 2, 3, 4, 5, 6, 8, 12, and 24 h after anti-Fas antibody
injection as described under "Experimental Procedures".
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Fig. 5.
A, expression of Fas protein after
administration of anti-Fas antibody in the presence or absence of HCV
protein was analyzed by Western blotting. B, FACS analysis
of Fas expression on the surface of primary hepatocytes in the presence
or absence of HCV, before anti-Fas antibody administration.
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Fig. 6.
A, analysis of caspase-8 in hepatocytes
by Western blotting after injection of anti-Fas antibody. Migration
positions of full-length (Procaspase-8, 57 kDa), cleaved intermediate
(p44 and p46), and the activated subunit (p18) of caspase-8 are
indicated. B, the time course of caspase activity of
hepatocytes in AxCANCre- or AxCAw1-injected CN2-8 mice after treatment
with anti-Fas antibody. Caspase activity was measured in cytosolic
fractions prepared from hepatocytes 0, 1, 2, 3, and 4 h after
injection of anti-Fas antibody (HCV not expressed, open
bars; HCV expressed, black bars). Caspase and Bid
cleavage activities were measured by release of AFC from peptide
substrates. Units represent the mean and S.D. of three individual
experiments.
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Fig. 7.
A, Bid was analyzed by Western blotting
of heavy membrane and cytosolic fractions of transgenic mice livers at
0, 1, 3, and 4 h after injection of anti-Fas antibody.
B, Western blot analysis of Bcl-2 family proteins (Bcl-2,
Bad, Bcl-XL, and Bax) in hepatocytes of anti-Fas antibody
injected mice. C, subcellular distribution of Bax protein in
the liver after anti-Fas antibody injection. Bax was analyzed by
immunoblot of cytosol (soluble fraction, S), plasma membrane
(light membrane fraction, LM), mitochondria-rich (heavy
membrane fraction, HM), and nuclear, (contained nuclei and
some mitochondria; low speed pellet, P1) fractions of livers
from transgenic mice, pretreatment and after anti-Fas antibody
injection.
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Fig. 8.
A, liver sections from HCV protein
expressing and nonexpressing mice at 0 and 4 h after anti-Fas
antibody injection were stained with anti-cytochrome c
antibody. B, cytochrome c (Cyt. c) was
analyzed by Western blotting of the heavy membrane fractions
(HM) from transgenic mice livers. C, cytochrome
c was analyzed by Western blotting of the cytosolic
fractions (Cytosol) from transgenic mice livers. The
quantification of detected cytochrome c by Western blotting
with chemiluminescence assay was performed with a Chemi Doc (Bio-Rad)
and relative intensity of each band was indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
The model of the HCV protein target in the
signaling pathway of Fas-mediated apoptosis. Two discrete
apoptotic signaling pathways, a mitochondrial pathway dependent on
cytochrome c/Apaf-1/caspase-9, and another signaling pathway
involving direct activation of caspase-3, -6, and -7 (30, 35) are
shown. Expression of HCV proteins may directly or indirectly suppress
the release of cytochrome c from mitochondrial
intermembrane.
Some reports indicated that the HCV core protein has antiapoptotic (1,
2, 5-7) and proapoptotic (3, 4) functions. The reason for the
discrepancy among these reports is still unclear. Moreover, NF-B has
an antiapoptotic function against anti-Fas antibody and TNF-
-induced
cell death. It was recently shown that HCV core protein activated
NF-
B-associated signal (5-7). It may be that the core has
multifunctional roles in the apoptotic signaling. This discrepancy may
be explained by the possibility that it was caused by using established
cell lines and clonically selected permanent transfectant cells in the
previous studies. To avoid the artificial effects in cell lines and
transgenic mouse with persistent expression of HCV protein, we examined
the transgenic mouse using conditional transgene expression system (8).
These hepatocytes of transgenic mouse retain original sensitivity to the apoptosis mediated by Fas signal. The conditional transgene expressed mouse makes it possible to examine native effect of HCV
protein to Fas-mediated apoptosis.
The results of the present study revealed that Fas-mediated apoptotic cell death was suppressed by the expression of HCV proteins (core, E1, E2, and NS2) in vivo. In the presence of HCV proteins, lethality after the administration of anti-Fas antibody significantly decreased (Fig. 2). The survival rate correlated to HCV protein expression levels in transgenic mice (CN2-8 and 2-29) (Fig. 2, A and B).
These results revealed that apoptosis was inhibited by HCV protein expression, as shown by DNA ladder examination of mouse liver (Fig. 4). Expression of Fas on the surface of hepatocytes did not differ significantly among AxCANCre- or AxCAw1-injected mice and untreated mice (Fig. 5). Therefore, the downstream target of Fas signaling pathway was characterized. Many caspases were involved in the downstream of Fas signaling pathway (Fig. 9). Each caspase has a specific function, for example, as an initiator or effector. The initial caspase-8 cascade was activated in both HCV protein expressing mice and nonexpressing mice (Fig. 6, A and B). Caspase-9 and -3/7 activities were down-regulated in liver tissue of HCV protein expressing mice (Fig. 6). Apoptosis of hepatocytes requires effector caspases such as caspase-3/7 (16, 29). These results suggest that caspase-9 and -3/7 activation were inhibited by HCV proteins and/or that the mitochondria amplification loop of the Fas-mediated signaling pathway is inhibited by HCV proteins (Fig. 9).
Bcl-2 family proteins are involved in progression of apoptotic cell death in response to Fas signal (30). Bid is likely the activator of the mitochondrial pathway through cleavage by caspase-8 and translocation into the mitochondria. Bid then interacts with Bcl-2 and/or Bax protein causing the release of cytochrome c from mitochondria into the cytoplasm (31, 32). In the present study, distribution of Bid protein in the cytosol and heavy membrane fraction did not differ significantly between HCV protein expressing and nonexpressing mice (Fig. 7A). Therefore, the Bid pathway was not influenced by HCV protein. Bcl-2, Bad, and Bax protein quantities did not differ between HCV protein expressing and nonexpressing mice (Fig. 7, A and B). On the other hand, in the presence of HCV protein, Bcl-XL protein did not decrease 3 and 4 h after the administration of Fas antibody. This may have resulted from the down-regulation of caspase-3/7 activity (Fig. 7A) (16, 29). Therefore, the Bcl-2 family may not be significantly influenced by HCV proteins.
Immunohistochemical analysis of affected liver regions confirmed the
release of cytochrome c from the mitochondria of HCV nonexpressing mice and expressing mice by anti-Fas antibody
administration. The release of cytochrome c from the
mitochondria to cytoplasm was suppressed in the HCV protein expressing
mice liver compared with the HCV nonexpressing mice liver (Fig.
8A). Consistent with this result, cytochrome c
released 5.2-6.6-fold increase from heavy membrane fraction to
cytoplasmic fraction in the HCV nonexpressing liver tissue after 3 and
4 h of Fas antibody administration (Fig. 8, B and
C). In contrast, cytochrome c was mainly present
in the heavy membrane fraction, and the release of cytochrome
c from the mitochondria into the cytoplasm was suppressed in
the presence of HCV after 3 and 4 h of Fas antibody administration
(Fig. 8, B and C). These results indicated that
the release of cytochrome c from mitochondria to cytoplasm
was inhibited by the expression of HCV proteins in transgenic mice. The
inhibition of the activation of effector caspase-3/7 resulted from the
suppression of the cytochrome c/Apaf-1/caspase-9
amplification loop by HCV and decreased lethality after Fas antibody
administration. Further experiments are needed to determine whether the
inhibition of cytochrome c from mitochondoria is due to the
direct interaction of particular HCV proteins with a cellular protein.
Or, it is possible that the interaction of expression of HCV E1 and E2
glycoproteins may induce a more general cellular response, stimulating
chaperones and delaying the entry into apoptosis (33, 34).
Further elucidation of the apoptotic signaling functions of HCV will
not only advance the understanding of molecular mechanisms of HCV
pathogenesis, but also shed light on the basic mechanism of apoptosis.
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ACKNOWLEDGEMENTS |
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We express our gratitude to Tadatsugu Taniguchi for support of this study and Kazuaki Inoue and Kentaro Tomita for advice on histology. We thank Masao Miwa, Otoya Ueda, and Mitugu Takahashi for breeding the transgenic mice.
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FOOTNOTES |
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* This work was supported in part by a research fellowship from the Japan Society for the Promotion of Science, a grant-in-aid for Specially Promoted Research on Viral Diseases from the Tokyo Metropolitan Government, a grant from the Ministry of Education, Science, and Culture of Japan, and a grant from the Ministry of Health and Welfare of Japan.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. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Microbiology and Cell Biology, The Tokyo Metropolitan Institute of
Medical Science, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. Tel.: 81-3-3823-2101; Fax: 81-3-3828-8945; E-mail:
mkohara@rinshoken.or.jp.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M010137200
2 F. Shibasaki, manuscript in preparation.
3 F. Shibasaki et al., manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
HCV, hepatitis C
virus;
ALT, alanine aminotransferase;
FEIA, fluorescent enzyme
immunoassay;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonic acid;
TSA, tyramide signal amplification;
Ac-DEVD-AFC, Ac-Asp-Glu-Val-Asp-7-amino-4-(trifluoromethyl)-coumaride;
NF-B, nuclear factor-
B;
MOPS, 4-morpholinepropanesulfonic acid;
FACS, fluorescence-activated cell sorter;
TNF-
, tumor necrosis
factor-
.
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