From the Department of Medical Biophysics, University
of Toronto, Toronto, Ontario M5G 2M9, the § Ontario Cancer
Institute, Princess Margaret Hospital, Toronto, Ontario M5G 2M9, and
¶ Amgen Research Institute,
Toronto, Ontario M5G 2C1, Canada
Received for publication, July 8, 2000, and in revised form, November 8, 2000
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ABSTRACT |
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The X protein from a chronic strain of hepatitis
B virus (HBx) was determined to inhibit Fas-mediated apoptosis and
promote cell survival. Fas-mediated apoptosis is the major cause of
hepatocyte damage during liver disease. Experiments demonstrated that
cell death caused by anti-Fas antibodies was blocked by the expression of HBx in human primary hepatocytes and mouse embryo fibroblasts. This
effect was also observed in mouse erythroleukemia cells that lacked
p53, indicating that protection against Fas-mediated apoptosis was
independent of p53. Components of the signal transduction pathways
involved in this protection were studied. The SAPK/JNK pathway has
previously been suggested to be a survival pathway for some cells
undergoing Fas-mediated apoptosis, and kinase assays showed that SAPK
activity was highly up-regulated in cells expressing the HBx protein.
Normal mouse fibroblasts expressing HBx were protected from death,
whereas identical fibroblasts lacking the SEK1 component
from the SAPK pathway succumbed to Fas-mediated apoptosis, whether HBx
was present or not. Assays showed that caspase 3 and 8 activities and
the release of cytochrome c from mitochondria were
inhibited, in the presence of HBx, following stimulation with anti-Fas
antibodies. Coprecipitation and confocal immunofluorescence microscopy
experiments demonstrated that HBx localizes with a cytoplasmic complex
containing MEKK1, SEK1, SAPK, and 14-3-3 proteins. Finally, mutational
analysis of HBx demonstrated that a potential binding region for 14-3-3 proteins was essential for induction of SAPK/JNK activity and
protection from Fas-mediated apoptosis.
Hepatitis B virus (HBV)1
is a leading cause of cirrhosis and hepatocellular carcinoma (reviewed
in Refs. 1-3), and currently more than 400 million people are
chronically infected with this virus worldwide. Multiple factors
including damage caused by inflammatory cytokines, mutations incurred
during liver regeneration, defects in DNA repair, integration of viral
DNA into the host cell genome, host genomic instability, activation of
cellular oncogenes, and induction of cell survival pathways have been
implicated as causes leading to liver cancer. However, the exact
molecular events progressing to liver carcinogenesis remain to be
elucidated. The genome of HBV consists of a partially double-stranded
circular DNA spanning 3200 nucleotides. Mammalian hepatitis viruses
(human, woodchuck, and ground squirrel) encode 4 overlapping reading
frames that code for surface glycoproteins (PreS1, PreS2, and S), core
proteins (C and PreC), polymerase (P), and the X protein (HBx). The
x gene encodes a 17-kDa (154 amino acid) protein that has
been attributed to a number of functions (reviewed in Refs. 2 and 4-8)
including transcriptional transactivation of viral and cellular genes,
binding to p53, inhibition of nucleotide excision DNA repair,
stimulation of signal transduction pathways, and interference with
proteosome activity.
There appears to be a close correlation between expression of HBx and
the development of chronic liver disease and hepatocellular carcinoma
(9-11). Removal of the x gene from woodchuck hepatitis viral DNA prevents the virus from establishing chronic infections and
tumors in experimental animals (12, 13). HBx is clearly a
multifunctional protein, but its mechanism of action has been controversial (2). It was initially described to be a "promiscuous transactivator" capable of stimulating a variety of viral and host
gene promoters indirectly through its interaction with transcription factors. These included the proto-oncogenes (c-myc,
N-myc, and c-jun), transcription factors (AP-1,
NF- Most DNA viruses such as simian virus 40, polyomavirus,
papillomaviruses, poxviruses, retroviruses, baculoviruses, and
herpesviruses contain gene products that block and reduce apoptosis
(reviewed in Refs. 21-23). These viral proteins can block cell death
in a variety of ways including interaction with p53, degradation of p53, subversion of components from the TNF or Fas pathways, inhibition of cytochrome c release from the mitochondria, or reduction
of caspase activity. Several laboratories (24-27) have demonstrated that HBx is able to interact with p53 and can interfere with
p53-mediated transcriptional activation of other genes. However, both
inhibition and activation of p53-mediated apoptosis by HBx has been
reported, causing many investigators (2, 28) to question the
significance of this interaction in vivo. In addition, many
of the viral anti-apoptotic proteins are multifunctional and can bind
p53 but are also able to block apoptosis in other ways. For example,
the simian virus 40 large T antigen and the adenovirus E1B/19K protein
were recently shown to protect cells from Fas-mediated apoptosis
(29-31). With these precedents, we were intrigued about the possible
effects of HBx upon apoptotic pathways, particularly Fas-mediated
apoptosis, within the infected cell.
Many viral oncogenes have been shown to up-regulate the SAPK/JNK signal
transduction pathway (reviewed in Ref. 32). These include the E1B/19K
protein of adenovirus (33), the TAT protein of human immunodeficiency
virus (34), the LMP1 protein of Epstein-Barr virus, the TAX protein of
HTLV-1, the angiogenic G protein receptor of Kaposi sarcoma virus, and
the HBx protein of HBV (35, 36). Cellular oncogenes such as
TPL2 (tumor progression locus 2 protein) and
BCR-ABL tyrosine kinase and the HER2/NEU gene product
associated with many breast tumors, epidermal growth factor-stimulated
proliferation, the RET oncoprotein, the MAS gene product, and the MET
protein increase SAPK levels (32). Clearly up-regulation of SAPK
activity, often in parallel with increased MAPK, appears to be
associated with cellular transformation. Higher levels of SAPK have
also been implicated in hepatocyte growth and regeneration (37, 38), and deletion of the upstream kinase, Sek1, has been shown to reduce SAPK activity and impair liver development in mice (39).
Recently HBx protein has been implicated in the activation of signal
transduction pathways that support the survival of the cell. Several
reports have demonstrated that HBx in liver and fibroblast cell lines
stimulates the receptor tyrosine kinase-Ras-Raf-MAPK pathway, activates
c-FOS/c-JUN-mediated transcription, favors G0/S cell
cycle transition, and protects cells from serum starvation (40-43). It
has been reported that Src kinases, but not Ras, are up-regulated in cells containing HBx and woodchuck hepatitis B x
protein and that HBV genome replication is stimulated by the enhanced
kinase activity (43, 44). Two other reports indicate that the SAPK/JNK
pathway is also up-regulated by HBx (35, 36). Still other findings
(45-48) suggest that HBx stimulates NF In the present study, we explored the ability of HBx protein to inhibit
apoptosis and dissected the signal transduction pathways involved in
this protection. We were primarily interested in Fas-mediated apoptosis
and cell death induced by serum starvation, since both Fas ligand and
growth factors play important roles in liver damage and the
regeneration of hepatocytes during hepatitis (51). We first observed
that HBx blocked Fas-mediated apoptosis in primary human hepatocytes
and that it supported their survival in culture. The same effects were
demonstrated in mouse erythroleukemia cells that lacked p53. Mouse
fibroblasts that lacked the SEK1 component of the stress kinase pathway
were also much more sensitive to Fas-mediated apoptosis than wild type
cells. Normal fibroblasts that contained HBx were protected against
cell death, whereas the SEK1-deficient cells underwent apoptosis
whether the viral protein was present or not. The SAPK/JNK pathway has
previously been suggested as a survival pathway for cells undergoing
Fas-mediated apoptosis, and assays show that SAPK is highly
up-regulated in cells containing HBx. Immunoprecipitation and
immunofluorescence confocal microscopy showed that HBx localizes with
the SAPK-JNK complex. Up-regulation of SAPK by HBx may help
rescue cells from Fas-mediated apoptosis.
Cell Lines--
Chang liver cells were purchased from the
American Type Culture Collection (Manassas, VA). Human primary
hepatocytes were supplied by Clonetics Corp. (San Diego, CA) and were
grown in hepatocyte culture medium provided by the company. DP-16-1
mouse erythroleukemia cells were provided by Dr. S. Benchimol (Ontario Cancer Institute, Toronto, Canada), and the cells were propagated in
minimum Eagle's medium- Plasmids--
The DNA coding regions of HBx and HBx deletion
mutant flanked by engineered restriction enzyme sites were generated by
polymerase chain reactions from the plasmid pAM6 (45020D)
(American Type Culture Collection) harboring the wild type HBV and HBx
open reading frame (GenBankTM accession number X51970). The
expression vector pRBK-HBx was constructed by inserting HBx DNA
fragments downstream of the Rous sarcoma virus promoter between the
NheI and XhoI cloning sites of the pRBK vector
(Invitrogen, Carlsbad, CA). Expression vectors containing N-terminal
deletions in HBx were also made using the same insert sites. The
bicistronic expression vector pIRES-EGFP-HBx was constructed by
ligation of the HBx DNA fragments between the EcoRV-EcoRI cloning sites of vector pIRES-EGFP
(CLONTECH, Palo Alto, CA). Through use of the
cytomegalovirus promoter, a bicistronic mRNA was transcribed that
directs the synthesis of the HBx protein from its own AUG and enhanced
green fluorescent protein (EGFP) under control of the internal ribosome
entry site (IRES). The pBMN retroviral expression vector was obtained
from G. Nolan (Stanford University) and modified to contain an IRES-GFP
element (J. Ruland, Amgen Institute, Toronto, Canada). Mutations in the
HBx gene were generated with the QuickChangeTM XL
site-directed mutagenesis kit (Stratagene, La Jolla, CA).
Transfections and Generation of HBx Cell
Lines--
Erythroleukeima cells and Chang liver cells at 50-70%
confluency were transfected or cotransfected with plasmids, using
LipofectAMINE (Life Technologies, Inc.). SuperFect (Qiagen, Valencia,
CA) was used in transfections of the primary human hepatocytes and
mouse fibroblast cell lines. DNA transfections were performed with
protocols supplied with the reagents. Transfection efficiencies ranged
from 2 to 4% for primary hepatocytes and 60% for Chang liver cells but were as high as 80% for mouse fibroblast lines. Stable cell lines
were selected by culturing DP-16 erythroleukemia cells, Chang liver
cells, or mouse fibroblasts containing pRBK-HBx in the presence of
hygromycin (270 µg/ml). Expression of HBx in the various cell lines
was verified by RT-PCR, immunoprecipitation followed by immunoblot
analysis, and immunofluorescence microscopy.
Antibodies--
Monoclonal antibodies directed against HBx
peptide (amino acids 50-88) were supplied by Chemicon International
Inc. (Temecula, CA). Polyclonal antiserum directed against HBx protein
was produced in our laboratory by immunizing rabbits with a
maltose-binding protein-HBx fusion generated in Escherichia
coli. Protein was purified by affinity chromatography. Antibodies
from this antiserum were purified using affinity chromatography with
HBx-(His6) coupled to nickel-Sepharose. Rabbit
polyclonal antisera directed against HBx peptide (amino acids 2-21)
coupled to keyhole limpet hemocyanin was also generated in our
laboratory. The antibodies specific for this HBx peptide were purified
from these antisera by affinity chromatography using peptide-conjugated
to Affi-Gel 10 (Bio-Rad). Other antibodies came from commercial sources
as follows: rabbit polyclonal antibody against 14-3-3 Immunofluorescence Microscopy--
Chang liver cells were grown
to confluency in 8-well chamber slides (Nalge Nunc International).
Medium was removed, and the cells were washed once with PBS prior to
fixation. Cells were fixed with 4% paraformaldehyde in PBS for 15 min
and washed once with PBS. Subsequently the cells were permeabilized
with 0.2% Triton X-100 in PBS at room temperature for 10 min.
Nonspecific protein binding to the cells was blocked by incubating the
cells with blocking solution (1% bovine serum albumin and 0.1% Triton X-100 in PBS) at 37 °C for 30 min. Primary antibody was diluted 1:50
in blocking solution, incubated with fixed cells for 3 h, and
washed 3 times with PBS at room temperature. Cells were subsequently incubated with secondary antibody consisting of either goat anti-mouse TRITC (Sigma) or goat anti-rabbit FITC (Sigma) that had been diluted 1:50 in blocking buffer. Incubations were performed at
37 oC for 1 h and washed 4 times with PBS. Finally,
the chambers were removed, and the slides were mounted with coverslips
using Fluorescent Mounting Solution (Dako, Hiroshima, Japan).
Fluorescently labeled cells were viewed with a Zeiss LSM510 confocal
microscope, and the images were analyzed by the LSM510 image browser software.
Analysis of DNA Ladders Due to Apoptosis Using Gel
Electrophoresis--
Following induction of apoptosis, 104
cells were lysed in Tris-EDTA buffer containing 1% Triton X-100 and
RNase (10 µg/ml) for 30 min at 37 °C. Lysates were subsequently
treated with proteinase K (0.5 mg/ml) at 37 °C for about 8 h.
The entire lysate was loaded onto a 2% agarose gel and subjected to
electrophoresis for 2 h at 65 V. The DNA ladder was visualized by
staining DNA fragments with ethidium bromide and examining the gel
under UV light.
Cell Viability and Apoptosis Analysis by Fluorescence Cytometry
(FACS)--
Cells were plated and grown overnight until they were 80%
confluent when they were treated as indicated with Fas antibodies. Subsequently, cell media containing detached cells were collected, and
the remaining adherent cells were released by trypsinization (1 min)
and combined with the detached cells. Collected cells were centrifuged
and washed twice with cold PBS and resuspended in 100 µl of binding
buffer (PharMingen). Subsequently, 5 µl of Annexin V-PE and 5 µl of
7-AAD (PharMingen) were added to the cell suspension and mixed gently.
The cells were stained at room temperature in the dark for 15 min and
analyzed by on a Becton Dickinson fluorescence cytometer using
CellQuest software (52).
SAPKs/JNKs Kinase Assay--
A nonradioactive method of
measuring SAPK/JNK activity was employed based on the SAPK/JNK Assay
Kit provided by New England Biolabs (Beverly, MA). Briefly, 5 × 106 cells were lysed, and 250 µg of total protein was
used in each reaction. An N-terminal c-JUN fusion protein bound to
Sepharose beads was used to pull down SAPK/JNK from cell lysates. The
kinase reaction was carried out in the presence of ATP, and c-JUN
phosphorylation was selectively measured using a phospho-c-JUN antibody
and immunoblot analysis. Proteins associated with the c-JUN beads were
also detected on the immunoblots using specific antibodies. For the
radioactive kinase assay, specific antibodies to SAPK/JNK (New England
Biolabs) were used to immunoprecipitate selectively SAPK/JNK from cell lysates overnight at 4 °C. Protein G was used to bind the
immunocomplexes. The resulting immunoprecipitate was then incubated
with c-JUN-(1-89) fusion protein (New England Biolabs) in the presence
of 1 mM cold ATP and 10 mCi of [ Caspase Assays--
6-Well microtiter plates containing 1 × 106 cells per chamber were grown to 80% confluency and
treated with Fas antibodies. Cells were suspended by trypsin treatment
and collected by centrifugation at 400 × g for 10 min.
Pellets were resuspended in 100 µl of cold cell lysis buffer provided
in ApoAlert caspase fluorescent assay kits
(CLONTECH, Palo Alto, CA). Cell lysates were
centrifuged at 18,000 × g for 3 min at 4 °C, and 50 µl of the supernatants were transferred to 96-well microtiter plates
for detection of caspase 3 or caspase 8 activity. The remaining
portions of the supernatants were assayed for protein concentration.
Caspase 3 and caspase 8 activities were measured using fluorescent
peptide substrates (DEVD-AFC and IETD-AFC, respectively) using a
Wallac fluorimeter according to the ApoAlert kit instructions.
Cytochrome c Release Assay--
Adherant cells were suspended by
treatment with trypsin (Life Technologies, Inc.). 5 × 106 cells were suspended in 0.5 ml of serum-free medium and
incubated with 5 µg/ml of protein G (Sigma) and either 5 µg/ml of
hamster antibodies directed against Fas or hamster control IgG. The
cells were gently shaken at 37 °C for 4 h and collected by
centrifugation at 600 × g for 2 min. Mitochondria and
cytosol fractions were prepared as described previously (53) with the
following modifications. Cells were suspended in cold buffer consisting
of 250 mM mannitol, 0.5 mM EGTA, 0.1%(w/v)
bovine serum albumin, leupeptin (1 µg/ml), pepstatin A (1 µg/ml),
antipain (50 µg/ml), phenylmethylsulfonyl fluoride (0.1 mM), 5 mM HEPES, pH 7.2, and disrupted using a
Dounce homogenizer. Unbroken cells and nuclei were sedimented and
removed by centrifugation at 600 × g for 5 min at
4 °C. The supernatants were further centrifuged at 12,000 × g for 10 min at 4 °C to sediment the mitochondria.
Proteins in the supernatants were denatured with 4× SDS sample buffer,
and mitochondrial pellets were solubilized in 1× SDS sample buffer.
Samples were subjected to electrophoresis on 10-20%
acrylamide/Tricine-buffered gels; proteins were transferred to
nitrocellulose membranes, and immunoblot analysis was performed with
antibodies directed against cytochrome c.
Generation of Recombinant Retroviruses Expressing HBx--
HBx
was inserted between the EcoRI and XhoI of the
retroviral expression vector, pBMN-IRES-GFP. Recombinant retrovirus was generated by introducing 10 µg of pBMN-HBx-IRES-GFP into 5 × 106 cells of the ecotropic packaging Phoenix cell line
using the calcium phosphate transfection method (54). Generation of
high titer, helper-free retroviruses occurred following the transient transfection. Recombinant retrovirus was harvested at 48 h
post-transfection. Mouse embryo fibroblasts and DP-16 mouse
erythroleukemia cells were infected at a multiplicity of infection of
10 in 6-well microtiter plates (5 × 105
cells/chamber). Cells were incubated for 24 h prior to conducting assays.
Immunoprecipitation and Western Blotting--
Cell lysates were
prepared by using a Dounce homogenizer and ice-cold buffer containing 1 mM EGTA, 5 mM MgCl2, 142.5 mM KCl, 10 mM HEPES, pH 7.5, with 0.5% (w/v)
Nonidet P-40 supplemented with the protease inhibitors of leupeptin (1 µg/ml), pepstatin A (1 µg/ml), antipain (50 µg/ml), and
phenylmethylsulfonyl fluoride (0.1 mM). The lysates were
further disrupted by sonication at a maximum frequency output for
6 s. Unbroken cells and nuclei were sedimented and removed by
centrifugation at 600 × g for 5 min. Cell lysates were
further centrifuged at 10,000 × g for 15 min at
4 °C, and the pellets were discarded. For immunoprecipitation studies, antibodies were added to the cell lysates (10 µg/ml), and
incubations were performed at 4 °C overnight. Protein G beads (Amersham Pharmacia Biotech) were then added to the reaction for additional 3 h of incubation. The beads were washed at least three times using the cell lysis buffer. Proteins associated with the beads
were solubilized in electrophoresis sample buffer and resolved by
SDS-PAGE, transferred to polyvinylidene difluoride membranes (Roche
Molecular Biochemicals), incubated with 5% skim milk and 0.05%
Tween 20 in PBS, and probed with specific primary antibodies. Bound antibodies were detected with goat anti-rabbit or anti-mouse antibodies that had been conjugated to horseradish peroxidase and ECL
(Roche Molecular Biochemicals).
Expression of HBx Protein in Human Primary Hepatocytes Protects
Cells from Fas-mediated Apoptosis--
To study the effects of HBx in
human liver, we transfected expression vectors containing the
x gene into primary human hepatocytes (Fig.
1). The hepatocytes were cotransfected
with two plasmids containing the genes for HBx and EGFP, respectively.
EGFP expression verified the efficiency of transfection with the HBx
gene. Recombinant HBx protein expression was also checked by
immunoprecipitation followed by immunoblot (data not shown). These
cells were subsequently treated with Fas antibodies at 48 h
post-transfection, and apoptosis was monitored by fluorescence
microscopy. Transfection efficiencies for primary hepatocytes are
typically low (2-5%), but over 80% of these cells expressed both HBx
and EGFP and survived over 2 days following this treatment (Fig.
1A). Those cells expressing control plasmid and EGFP died
within 16 h of adding the Fas antibodies (Fig. 1B). In
the course of observing the transfected cells under the microscope for
26 days, we also found that the primary hepatocytes transfected with
HBx and EGFP (Fig. 1C) survived much longer than control
cells cotransfected with EGFP alone (Fig. 1D). Similar results were obtained with the p(HBx)IRES-GFP duo-expression vector, but the fluorescent signal was less intense (data not shown). Chang
liver cells were also transfected with an expression vector pRBK-HBx,
and 6 stable cell lines were isolated by hygromycin selection. These
cell lines were also resistant to cell death induced by Fas stimulation
or serum starvation and were also partially protected against
TNF Mouse Embyro Fibroblasts Infected with an HBx Recombinant
Retrovirus Are Viable and Are Protected Against Fas-mediated
Apoptosis--
To increase the efficiency of transfection, the HBx
gene was cloned into a mouse mammary tumor virus vector that contained the HBx and EGFP reporter genes under control of a cytomegalovirus promoter and IRES element, respectively. Greater than 90% of mouse embryo fibroblasts (MEFs) and mouse erythroleukemia cells (DP-16) could
be infected with recombinant virus that expressed both HBx and EGFP, as
shown by immunoblot analysis and fluorescence microscopy. The DP-16
cells are a mouse erythroblastoid cell line from mice infected with
Friend leukemia virus that was previously reported to be deficient in
p53 production (55). Synthesis of HBx in MEFs and DP-16 cells had no
adverse effects, and the cells remained viable in the presence of HBx
and EGFP (Fig. 2A). Cell
viability was quantitated by annexin V/7-AAD staining of the
transiently infected mouse cells. In addition, expression of HBx
protected both MEFs and DP-16 cells against Fas-mediated apoptosis.
Cell death was monitored by flow cytometry (Fig. 2B) and
fluorescence microscopy (Fig. 3,
G and H). Expression of HBx was verified by immunoblot using a specific monoclonal antibody (Fig. 2C).
In these experiments, apoptosis was inhibited by at least 75% of the
levels found in cells infected with control retrovirus that lacked the
HBx gene. Similar results were found with mouse 3T3 fibroblasts and in
experiments using a transient expression plasmid, pHBx-IRES-EGFP. FACS
analysis also indicated that the level of Fas on the cell surface was
unaffected by HBx expression (data not shown). Although a few reports
recently suggested that HBx could induce cell death when expressed at
high levels (28, 56-59), we found that the HBx of virus derived from a
chronic carrier had no toxic effects and instead protected cells from
Fas-mediated apoptosis. Most other laboratories support the role of HBx
as a survival and growth-stimulating factor.
HBx Prevents Apoptosis in Cells Lacking p53--
Several
laboratories have demonstrated that HBx is able to interact with p53,
and it subsequently interferes with the p53-mediated transcriptional
activation of other genes (24-27). However, both inhibition and
activation of p53-mediated apoptosis by HBx-p53 interaction have been
reported, causing many investigators to question the significance of
this interaction in vivo (28). To test whether the presence
of p53 is a factor during the inhibition of Fas-mediated apoptosis by
HBx protein, we used a murine erythroleukemia cell line, DP-16, which
does not synthesize this endogenous tumor suppressor (55). Stable cell
lines expressing HBx were generated by transfecting pRBK-HBx into DP-16
cells and culturing them in the presence of hygromycin. Expression of
HBx was verified by RT-PCR (Fig.
4B) and immunoprecipitation
followed by immunoblot analysis. These cell lines expressed HBx at
relatively low levels that were comparable to the amounts of viral
protein that are found in the livers of patients with hepatitis. We
subsequently tested the effect of HBx on Fas-mediated apoptosis in the
DP-16-HBx cell lines. Cells were treated with antibody directed against the Fas receptor, which mimics the effect of Fas ligand (FasL). Viability of the treated cells and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assay, flow cytometry, and DNA fragmentation analysis. In Fig.
4A, DNA fragmentation analysis showed that Fas-mediated
apoptosis was blocked in stable DP-16 cell lines that expressed HBx.
Analysis of Fas surface expression by flow cytometry (FACS) again
indicated that HBx did not alter the levels of Fas on the cell surface
(data not shown). A cell line derived from the DP-16 parental cells, ts5.207.3, which overexpresses a temperature-sensitive form of p53
(tsp53Val-135) that is active at 33 °C, was also used to
test the effect of HBx on Fas-mediated apoptosis. Regardless of whether
p53 was present or not in the DP-16 cell line, they were protected from
Fas-mediated apoptosis (data not shown). Therefore, in the
erythroleukemia cell lines, HBx appears to block apoptosis induced by
Fas antibodies, irrespective of whether p53 is present in the
cells.
HBx Prevents Fas-mediated Apoptosis in Normal Mouse Fibroblasts but
Not in the Same Cells Lacking SEK1 Expression--
NF HBx Inhibits Caspase 8 and Caspase 3 Activities That Are Induced
during Fas-mediated Apoptosis--
Caspase 8 and caspase 3 are the
effectors of Fas-mediated apoptosis, and their activities arise
following the cleavage of inactive proenzymes after Fas is stimulated.
By using fluorescent peptide substrates, we were able to show caspase 3 and 8 activities were inhibited by at least 80% in sek1+/+
MEFs that stably expressed the HBx protein (Fig.
6). Inhibition of the caspases 8 and 3 was much less in sek1 HBx Inhibits the Release of Cytochrome c from Mitochondria
following Induction of Fas-mediated Apoptosis--
The release of
cytochrome c from mitochondria is an intermediate event in
one arm of the Fas-mediated apoptotic pathway that promotes the
activation of apoptotic caspases (70). Interaction of cytochrome
c with APAF1 activates procaspase-9 which in turn cleaves and activates the precursors of caspases 3, 6, and 7. However,
an alternative Fas-mediated pathway bypasses the mitochondrion in many
cell types and is mediated by the activation of procaspase 8 by FADD,
which in turn stimulates caspase 3 activity directly (70, 71). The Fas
stimulus is actually amplified by the mitochondrial pathway through
action of BID on the mitochondrion, which triggers the release of
cytochrome c into the cytosol. We measured the effect of HBx
on cytochrome c release from mitochondria in MEFs (Fig.
7A) and DP-16 cells (Fig.
7B) following stimulation of the Fas pathway. Our results
indicated that expression of HBx in sek1+/+ and DP-16 cells
prevented the release of cytochrome c during Fas-mediated apoptosis but had no effect in sek1 HBx Protein Up-regulates SAPK/JNK Activity in Mouse Fibroblast and
Erythroleukemia Cell Lines--
Previous studies have shown that HBx
protein induces a 20-25-fold increase in phosphorylation of the N
terminus of c-JUN due to up-regulation of SAPK/JNK (35, 36). To
determine whether HBx protein can modify the SAPK/JNK function in DP-16
erythroleukemia cells and MEFs, the specific protein kinase activity
was measured using either a nonradioactive solid phase method or
32P incorporation assay (Fig.
8). The substrate for SAPK/JNK consisted of the N terminus of c-JUN (amino acids 1-89) fused to GST, which was
in turn linked to glutathione-Sepharose beads. In the nonradioactive solid phase method for measuring kinase activity, an antibody specific
for phosphorylated c-JUN was used to probe immunoblots prepared from
cells containing HBx. Our results showed that the presence of HBx
protein did correlate with increased SAPK/JNK activity in the mouse
fibroblasts following stimulation of the cells with Fas antibody (Fig.
8A, lanes 2) or TNF HBx Protein Is Associated with the MEKK1-SEK1-SAPK Kinase
Complex--
Based upon our preceding results, we asked whether HBx
protein could interact directly with components of the SAPK-JNK
signaling complex and activate this pathway. A c-JUN-(1-89)-GST fusion
protein conjugated to Sepharose beads was used as bait to precipitate associated kinase complexes. This situation is somewhat artificial since c-JUN is normally found in the nucleus. However, both
activated and nonactivated forms of SAPK/JNK can interact with c-JUN
beads. The precipitates from Chang liver cells (Fig.
9, A and B) and MEFs (Fig. 9C) were subsequently examined by immunoblot
analysis to determine whether HBx protein was associated with c-JUN
beads, SAPK/JNK, or SEK1. Results from both types of cells showed that MEKK1, SEK1, SAPK/JNK, 14-3-3, and HBx were associated with c-JUN beads, suggesting that HBx protein might be physically associated with
the SAPK/JNK kinase complex. A deleted form of HBx, which lacked the
N-terminal 50 amino acids, did not associate with this complex (Fig.
9B, lane 3). GST-Sepharose beads that were not linked to the
c-JUN amino acids did not precipitate HBx (data not shown). In addition
the amount of HBx that associated with c-JUN in the sek1( Confocal Immunofluorescence Microscopy Confirms That HBx
Colocalizes with SEK1, SAPK, and 14-3-3 Proteins in the Cytoplasm of
Chang Liver Cells--
To ascertain the cellular location of HBx and
also check whether the protein actually colocalized with components of
the SAPK pathway, Chang liver cell lines that stably expressed HBx were fixed and stained with either TRITC-conjugated rabbit polyclonal or
mouse monoclonal antibodies directed against HBx. When viewed by
immunofluorescence confocal microscopy, cells containing HBx exhibited
a punctate cytoplasmic labeling with an increased intensity surrounding
the nucleus (Fig. 10). The staining is
somewhat similar to that found previously by another group (17) who
concluded that HBx was localized to the proteosomes. Another laboratory indicated that HBx is associated with mitochondria and causes them to
aggregate (72). Polyclonal or monoclonal antibodies directed against
HBx gave similar results, and the background staining of Chang control
cells was negligible (data not shown). FITC-conjugated antibodies
directed against SAPK (Fig. 10B) and SEK1 (Fig.
10C) colocalized exactly with the TRITC-conjugated
antibodies that recognized HBx. Overlapping staining is represented by
a yellow color. We concluded that HBx did indeed
colocalize as a complex with several components of the SAPK pathway. As
one would expect in the intact cell, HBx did not associate with c-JUN
transcription factor, which is found predominantly in the nucleus (Fig.
10E). Some members of the 14-3-3 scaffolding protein family
have been shown previously to colocalize with MEKK1 (73). However, HBx did not colocalize as strongly with MEKK1 in our immunofluorescence experiments compared with what we would have predicted from our coimmunoprecipitation experiments (Fig. 10D). This could be
due to nonspecificity of the MEKK1 antibody staining or a weaker
interaction with HBx when compared with SEK1 and SAPK/JNK. HBx did
appear to interact with some members of the 14-3-3 family of proteins (Fig. 10A). The significance of the interaction of protein
kinases with 14-3-3 proteins is being studied in a number of
laboratories (74). It is interesting to note that HBx contains an
RXRXXpS phosphorylation motif (amino acids
26-33; where pS is phosphoserine) which is found in many
phosphoproteins that bind to these scaffolding proteins (75, 76). The
association of HBx with this complex of stress kinases may up-regulate
the SAPK/JNK activity and help alleviate the apoptotic effects of Fas
antibodies on the four different types of cells that were tested in our
laboratory.
Mutation of the 26RXRXXS Motif of HBx Confirms
That This Region Is Essential for SAPK Up-regulation and Inhibition of
Fas-mediated Apoptosis--
To identify regions critical for enhancing
SAPK activity and maintaining the ability of the viral protein to
suppress Fas-mediated apoptosis, point mutations and deletions in HBx
were generated (Fig. 11A).
Since we discovered that there was an interaction between HBx, MEKK1,
SEK1, SAPK, and 14-3-3 proteins (Fig. 9), we focused on the role of
14-3-3 protein binding motif in up-regulating SAPK activity and
inhibiting Fas-mediated apoptosis. Human liver cell lines (Huh7 and
Chang cells) were transfected with mutated versions of HBx inserted
into pRBK or pIRES-EGFP expression vectors. Mouse fibroblast cells were
also infected with retroviral vectors expressing mutated HBx.
Immunoprecipitation with an HBx-specific antibody followed by Western
blot analysis with an antibody specific for the phosphorylated 14-3-3 binding motif (New England Biolabs) revealed that the
26RXRXXS was indeed phosphorylated
and constituted a 14-3-3 recognition domain. Mutation of the serine
residue at position 31 to an alanine, and deletion of the
26RXRXXS,
25XRXRXXSX, or
amino acids 2-50, abolished the interaction of this antibody with the
phosphorylated serine in the 14-3-3 binding motif (Fig.
11B). The effect of specific mutations and deletions on SAPK
activity was also evaluated in liver cells and mouse fibroblasts following stimulation of the SEK1/SAPK pathway with Fas antibodies, heat shock, or anisomycin treatment. Anisomycin treatment was previously shown to stimulate strongly the SEK1-dependent
pathway (65). HBx was shown to enhance dramatically SAPK/JNK activity and the phosphorylation of c-JUN following stimulation with anisomycin (Fig. 11C). Longer ECL exposures of the immunoblots showed
that SAPK/JNK was also stimulated by anisomycin in the control cells containing vector alone but not nearly to the degree as when HBx was
present. On the other hand, the point mutation HBx31S-A or deletion of
the 26RXRXXS motif greatly reduced or
abolished the enhanced SAPK activity due to anisomycin in cells
containing the mutated HBx (Fig. 11C). The effect of these
HBx mutations on Fas-mediated apoptosis was further assessed in mouse
embryonic fibroblasts infected with retroviral expression vectors.
Infected cells that expressed both the mutated version of HBx and GFP
were sorted by fluorescence flow cytometry. GFP expression correlated
with levels of HBx in the cells. Both the HBx31S-A and HBx A role for HBx in the generation of hepatocellular carcinoma is
well documented, but the mechanism of action of this protein has
remained elusive. In this study, we investigated the effect of HBx
protein on Fas-mediated apoptosis in hepatocytes, erythroleukemia cells
lacking p53, normal fibroblasts, and fibroblasts deficient in SEK1. Our
results indicate that HBx protein from the virus of a chronic carrier
is a strong survival factor that is able to protect cells from death
induced by anti-Fas antibody, both in transient and constitutive
expression systems. Unlike some previous studies, the anti-apoptotic
action of HBx is independent of p53. Our data provide the first
evidence that the SEK1-dependent SAPKs/JNKs pathway is
required for the inhibitory effect of HBx protein on Fas-mediated
apoptosis. We also showed that HBx either directly or indirectly
inhibits caspase 8, caspase 3, and the release of cytochrome
c from the mitochondria. Subsequent studies showed that HBx
associated with a protein kinase complex in the cytoplasm that
contained MEKK1, SEK1, SAPK, along with the c-JUN-Sepharose beads and
that the SAPK activity in these cells was up-regulated 30-fold. These
experiments suggest that the presence of HBx increases the kinase
activity associated with this complex without affecting the levels of
SAPK or SEK1 mRNA or proteins in the cell.
Our investigation indicates that overexpression of the HBx protein of
virus isolated from the blood of a chronic carrier inhibits Fas-mediated apoptosis. We have also confirmed that HBx favors survival
of the cell under low serum conditions, but it does not appear to
protect the cell from chemical apoptotic stimuli. Our data are in good
agreement with other reports that HBx favors cell cycle progression
(41) and inhibits apoptosis during serum starvation (19, 26, 77). HBx
is known to stimulate NF Up-regulation of the SAPK pathway is associated with a variety of
effects that are largely determined by the cell type and situation. The
effects of stress-activated kinases can range from induction of
apoptosis in neurons, increased survival in other cell types,
transformation of cells expressing oncogenes, stimulation of
angiogenesis, activation of T cells, proliferation of B cells, production of cytokines, inflammation, liver regeneration, or responses
to cardiovascular and renal damage (32). Deletion of MEKK1 can also
prevent activation of SAPK and favors apoptosis in embryonic stem cells
(90). The deletion of c-JUN and SEK1 has severe consequences on
hepatogenesis and liver development in mice (39, 66, 91, 92).
Interestingly, the Ras/Rac1/Cdc42/SEK/JNK/c-JUN signaling pathway is
important in the early proliferative response of hepatocytes after
partial hepatectomy in vivo and in the stimulation of DNA
synthesis in primary cultures of rat hepatocytes (37). Cellular
oncogenes such as BCR-ABL, TPL2, MET, HER2/NEU,
RET, and MAS can activate SAPK (32). In many
cases both the MAPK/extracellular signal-regulated kinase and SAPK
pathways are up-regulated, but inhibition of SAPK activity is usually
associated with loss of the transformed phenotype. In addition,
mitogens and growth factors such as epidermal growth factor also
activate the SAPK pathway. Many viruses act to up-regulate SAPK
activity to increase their survival (32). For example, adenovirus
activates SAPK activity and c-JUN transcription via its E1B19K protein
(33). The LMP1 protein of Epstein-Barr virus also activates SAPK, which
may contribute to the transforming properties of this viral protein.
Human immunodeficiency virus type 1 TAT protein, HTLV-1 TAX protein,
and Kaposi sarcoma virus also activate the SAPK pathway. Therefore, it
is most likely that activation of SAPK/JNK by HBx protein provides a
survival or anti-apoptotic signal for nontransformed hepatocytes and
contributes at least in part to the transformation of hepatocytes and
the development of hepatocellular carcinoma.
The molecular mechanism by which HBx stimulates the SAPK pathway and
inhibits Fas-mediated apoptosis and cell death through serum starvation
is unknown. However, the situation bears some resemblance to the
inhibition of apoptosis and SAPK activation associated with the E1B19K
protein of adenovirus (33). This viral protein up-regulates SAPK
activity but also inhibits Fas-mediated apoptosis by binding to
BAX, APAF1, and FADD/caspase 8 in the signal transduction
pathway (31, 93). It is not known whether HBx also interacts directly
with components in the Fas pathway, with mitochondrial membrane
proteins, or indirectly influences the apoptotic signal transduction
pathway by activating SAPK. Inhibition of apoptosis by HBx may involve
"cross-talk" between the Fas apoptosis and SAPK pathway, but a
simpler, less elegant explanation might be that the MAPK and SAPK
survival/proliferative pathways just overpower the death pathways due
to Fas activation or serum starvation.
Our results indicate that MEKK1, SEK1, SAPK, 14-3-3 protein, and HBx
form a complex in the cytoplasm. It was recently demonstrated that
14-3-3 proteins interact with MEKK1, -2, and -3 but not MEKK4 (73). The
14-3-3 proteins associate with a number of different signaling proteins
through phosphoserine and have been proposed to be important in
controlling mitogenic signaling pathways and inhibiting apoptosis (74,
94). 14-3-3 proteins also interact with Raf-1, polyoma middle tumor
antigen, PKB, ASK1, and the Bcl family member BAD. It has been
suggested that 14-3-3 proteins behave as a "scaffolds" or
"anchors" to localize protein kinase activity. It is known that
proteins that bind to 14-3-3 usually contain
RSXpSXP or RXRXXpS domains.
Analysis of the HBx protein sequence reveals that the HBx protein has a
potential 14-3-3 binding domain
(26RXRXXS), and preliminary findings
indicate that 14-3-3 is also in the SAPK complex. It is also
interesting to note that 14-3-3 proteins have a punctate cytoplasmic
distribution that colocalizes with MEKK1. By using confocal microscopy,
we observed that the majority of HBx in the cell is also present in the
cytoplasm as punctate structures that colocalize with 14-3-3 protein.
However, we have not yet determined which of the 7 isoforms of 14-3-3 is present in the SAPK complex.
Although there is no unifying hypothesis as to how HBx initiates
hepatocarcinogenesis, numerous publications seem to indicate that this
protein is multifunctional. Many viral oncogenes (SV40 T antigen,
adenovirus E1B19K, adenovirus E1B55K, HTLV
tax, and lmp1 of Epstein-Barr virus) have similar
functions in the process of cellular transformation. Our results show
for the first time that HBx inhibits some apoptotic processes that are
independent of the effects of p53. We also confirm that up-regulation
of SAPK activity and the presence of the SEK1 upstream kinase are
associated and required for protection from Fas-mediated apoptosis. HBx
may stimulate SAPK activity through its presence in a complex
consisting of MEKK1, SEK1, SAPK, and 14-3-3 proteins. Further
dissection of this interaction is in progress. It has previously been
observed that the so-called transcription activation domains of HBx
(amino acids 67-69 and 110-139) are neither required nor sufficient
for cell transformation (95). Point mutations in these two regions have
no effect upon the transformation process. Instead, the first 50 amino
acids of HBx have been implicated in dimerization, repression of
transcriptional activation, and transformation (8, 95). The
significance of the 14-3-3 protein binding motif requires further
investigation, and the kinase that phosphorylates this region remains
to be identified. Naturally occurring mutations in HBx from fulminant,
chronic, and hepatocellular carcinoma strains of HBV also affect this
region. Our studies suggest that the 14-3-3 binding motif may play a
very important role in transformation process. Since HBx has been shown
to interact with and stimulate other kinases such as PKC (96),
JAK1 (49), Src-like kinases (43), I
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, ATF/CREB, ERCC3, and RPB5 of RNA polymerase), HBV enhancers,
and the human immunodeficiency virus long terminal repeat (see Refs.
6-8 and 14 and references therein). The X gene product has also been
suggested to be a protease inhibitor due to the presence of serpin-like
protease domains in its sequence (15). To support this hypothesis, HBx
has been found to associate with proteosomes (16-18). More recently it
has been proposed that HBx inhibits caspase 3 activity (19). However, the role of HBx as a protease inhibitor is still controversial since
the existence of true serpin-like domains in the protein has been
disputed (2). Another intriguing property of HBx is its association
with the DNA repair protein DDBP1/XPE, which could account for an
accumulation of mutations in the liver over the long course of chronic
hepatitis (20).
B signal transduction
pathways, and another group (49) suggests it activates JAK1-STAT
signaling, and more recently HBx has been shown (50) to activate the
phosphatidylinositol 3-kinase pathway. Clearly HBx can modulate a
number of signal pathways in the cytoplasm.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Life Technologies, Inc.) supplemented with
10% fetal calf serum. sek1
/
and sek1+/+
mouse fibroblasts came from Dr. J. Woodgett, and the cells were
cultured in Dulbecco's modified Eagle's medium containing 10% fetal
calf serum.
protein (Santa
Cruz Biotechnology), rabbit polyclonal antibody against MKK4/SEK1
(Upstate Biotechnology, Inc.), monoclonal antibody against MEKK1 (Santa
Cruz Biotechnology), rabbit polyclonal antibody against SAPK1/JNK
(Upstate Biotechnology, Inc.), monoclonal antibody against the
phospho-14-3-3 binding motif (New England Biolabs), anti-mouse Fas
(PharMingen, San Diego), goat anti-mouse TRITC (Sigma), goat
anti-rabbit FITC (Sigma), and anti-hamster Ig (PharMingen).
-32P]ATP
and supplemented with kinase buffer as used for the nonradioactive kinase assay; immunoprecipitated active SAPK phosphorylated c-JUN at
30 °C for 30 min. Phosphorylated c-JUN was separated by SDS-PAGE and
detected and quantitated by PhosphorImager analysis. The c-JUN bands
were also cut out from the gel and quantitated by liquid scintillation assays.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mediated apoptosis. The presence of HBx in the cell also
rendered the primary hepatocytes and liver cell lines resistant to
serum starvation but, on the other hand, appeared to make cells more
sensitive to chemical apoptotic stimuli such as actinomycin D,
anisomycin, cisplatin, cycloheximide, dexamethasone, doxorubicin,
mitomycin C, okadaic acid, staurosporine, sorbitol, G418, and
wortmannin. The analysis of stable cell lines expressing HBx was
approached with caution, since additional cell mutations could
cooperate with the viral oncoprotein during the course of transformation and hepatocarcinogenesis. Transient expression of HBx in
primary cell lines could more closely reflect the situation occurring
within the cell during early stages of HBV infection. However, in our
experiments transient and stable expression of HBx yielded similar
findings. We concluded that HBx could prevent Fas-mediated apoptosis in
liver cells and also promote the survival of primary hepatocytes in
culture in the absence of antibiotic selection.
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Fig. 1.
HBx protein expression in primary human
hepatocytes protects cells from Fas-mediated apoptosis and promotes
cell survival. Cells were cotransfected with coding sequences for
HBx and EGFP (A and C) or EGFP alone
(B and D) using Superfect transfection reagent.
After 48 h, the cells were incubated with anti-Fas antibodies (5 µg/ml) at 37 °C, and the cells were examined by fluorescent
microscopy at 0, 16, and 24 h following stimulation of the Fas
signal transduction pathway (A and B). Other
cells in which HBx was present (C) or absent (D)
were incubated for 26 days and were viewed by fluorescent microscopy at
1, 14, 26 days. HBx+ indicates that the coding region for
HBx was present in the transfected cells, and HBx
indicates that it was absent.
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Fig. 2.
Quantitation of viability and apoptosis in
MEFs and DP-16 mouse erythroleukemia cells treated with Fas antibodies
in the presence and absence of HBx expression. MEFs and DP-16
cells were infected with recombinant mouse retroviruses expressing HBx
and EGFP. Viability was measured with an annexin V/7-AAD assay, and
cells infected with the retrovirus vector alone
(Retro-vector) were compared with cells infected with
retrovirus expressing HBx (Retro-HBx) (A).
Apoptosis induced by treatment with Fas antibodies was quantitated by
annexin V/7-AAD assays, and death of cells expressing HBX
(Retro-HBx) was compared with control cells infected with
the retrovirus vector alone (Retro-vector) (B).
Expression of HBx expression with the recombinant retrovirus vector
(Retro-HBx) was confirmed by Western immunoblot using monoclonal
antibodies directed against HBx followed by ECL detection. Control
cells infected with vector alone (Retro-vector) did not contain HBx
(C).
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Fig. 3.
Photomicrographs of MEFs infected with
recombinant retrovirus vectors expressing HBx and EGFP. At 24 h post-infection, cells were treated with Fas antibodies (10 µg/ml)
for a further 24 h. Cells were viewed by illuminating the cells
with polarized light (A, C, E, and G) or by
UV-induced fluorescence (B, D, F, and H).
Untreated controls were infected with recombinant retrovirus that
expressed just EGFP alone (A and B) or retrovirus
that expressed both HBx and EGFP (E and F). MEFs
that were treated with Fas antibodies in the absence of HBx
(C and D) and the presence of HBx (G
and H) are shown. HBx expression showed no cytotoxicity
(E and F) and protected cells from Fas-mediated
apoptosis (G and H). Cells were photographed
at × 250 magnification.
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Fig. 4.
HBx protects p53( ) mouse erythroleukemia
cells (DP-16) from Fas-mediated apoptosis. A, cell
lines were generated by transfecting DP-16 cells with the expression
plasmid pRBK-HBx or the empty vector pRBK, and three cellular clones
with HBx and one clone without HBx were selected in the presence of
hygromycin B. DP-16 cells (105) were treated with anti-Fas
(5 µg/ml) for 0, 3/4, 4, and 6 h. Total DNA was isolated
from these cells, and DNA fragments produced by apoptosis were resolved
on 1.8% agarose gels. B, the expression of HBx mRNA in
DP-16 cells (lanes 1-3) was verified by RT-PCR and compared
with cell lines (lanes 4-6) transfected with the expression
vector alone (DPpRBK). RT-PCR analysis of actin mRNA was
performed as an internal control. PCR analysis of the HBx vector was
included as a positive control (lane 7).
B, SAPK, and
PI3K/PKB kinase pathways help to overcome the apoptotic signal (4, 39,
60-64) associated with TNF
signaling. A survival pathway that
rescues the cell from Fas-mediated apoptosis has yet to be described
with certainty. However, it was recently shown that the SAPK/JNK
signaling pathway may contribute to protection against Fas-mediated
apoptosis and stimulating regeneration of the liver following damage
(65, 66). Other laboratories (37, 67-69) generally support this
observation. Still others (39) have shown that SEK1/MKK4 provides a
growth signal for hepatocytes during organogenesis. The protein kinase
Sek1 (JNKK/MKK4) is a direct upstream activator of SAPK. Thymocytes
from sek1
/
mice are significantly more
susceptible to Fas-mediated apoptosis than similar cells from
sek1+/+ mice, although the expression of Fas on the surface
of sek1
/
and sek1+/+ cells is the same (65). We found this observation was also true for mouse fibroblasts derived
from sek1
/
mouse embryos (Fig.
5A). To test whether HBx
protein can inhibit Fas-mediated apoptosis through activation of the
SAPK/JNK pathway, sek1
/
and sek1+/+
fibroblast cell lines were transfected with pRBK-HBx, and stable cell
lines were generated. Mouse fibroblasts, in which HBx protein was
either present or absent, were incubated with Fas antibodies and
assayed for apoptosis using annexin V/7-AAD staining and flow
cytometry. We observed that HBx-sek1
/
cells succumbed to
Fas antibody treatment, whereas the HBx-sek1+/+ became
resistant to the Fas-mediated apoptosis (Fig. 5B). These
results were in good agreement with those using mouse fibroblasts that
were transiently infected with recombinant retrovirus expressing HBx.
We concluded that the SEK1/SAPK pathway cooperates with HBx to protect
cells from Fas-mediated apoptosis.
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Fig. 5.
Mouse fibroblasts deficient in SEK1 are much
more sensitive to Fas-mediated apoptosis, and cell death cannot be
reversed by the presence of HBx. Embryonic fibroblasts were
derived from SEK1-deficient mice (sek1 /
). The
susceptibility to Fas-mediated apoptosis in
sek1
/
-deficient fibroblasts was compared with those from
wild type mice (sek+/+) in A. Cell lines that
expressed HBx were prepared with the expression vector pRBK-HBx, and
control cells were made with the empty vector pRBK. Normal fibroblasts
were protected from cell death by HBx, whereas SEK1-deficient
fibroblasts (sek1
/
) were equally sensitive to Fas
antibodies in the presence or absence of HBx (B). Apoptosis
was evaluated by annexin V/7-AAD assays and flow cytometry. DP-16 cells
with and without HBx served as a positive control in our experiments,
and HBx inhibited apoptosis as shown in Fig. 4.
/
MEFs, with only 30-35%
reduction in activity, when HBx was present. This result was consistent
with the inability of HBx to block Fas-mediated apoptosis in
sek1
/
cells. However, the general effect of HBx on
apoptosis probably arises not from the inhibition of caspase activity
directly but by the stimulation of survival signal transduction
pathways that override the effects of the death pathway. Inhibition of
caspase 8 activity was also greatly inhibited in DP-16 erythroleukemia
cells in the presence of HBx (Fig. 6A). Interestingly,
caspase 3 activity could not be detected even in control DP-16 cells,
indicating that other caspases were probably involved in executing cell
death in these cells. Inhibition of caspase 3 activity by HBx was
previously reported (19). This group suggested that cleavage of
procaspase 3 was not inhibited but that the downstream effects of
caspase 3 on poly(ADP-ribose) polymerase and lamin degradation
were blocked by HBx. Inhibition of caspase 3 did not appear to be due
to direct interaction of HBx with the protease. However, this effect of HBx on caspase 3 activity requires further documentation. Since caspase
3 lies near the end of the apoptotic cascade, its inhibition confirms
that HBx inhibits Fas-mediated apoptosis.
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Fig. 6.
Caspase activity is inhibited in mouse
fibroblasts and DP-16 cells containing HBx. Mouse embryo
fibroblasts, with and without the sek1 gene, and DP-16
erythroleukemia cells were transfected with expression vector alone
(pRBK) or vector expressing HBx (pRBK-X). Cells
were treated with Fas antibodies (10 µg/ml) for 2 h to induce
apoptosis and were quickly lysed. Cytosolic fractions were obtained by
centrifuging the lysates at high speed, and caspase 8 (A)
and caspase 3 (B) activities were measured using fluorescent
substrates that were specific for each protease as specified in the
ApoAlert kit from CLONTECH. Relative fluorescence
due to caspase activity was measured with a fluorimeter.
/
cells (Fig.
7A). A modified form of HBx which lacked its first 50 amino
acids (X
2-50) was ineffective in blocking the release of cytochrome
c (Fig. 7A). Thus, inhibition of cytochrome
c release correlates with diminished caspase 8 and caspase 3 activities that we previously observed. It remains to be determined
whether HBx interacts directly with the mitochondrion to prevent
cytochrome c release or whether HBx interferes with the
activation of caspase 8 or cleavage of BID. However, preliminary data
in our laboratory indicate that HBx does not interact directly with
procaspase 8, FADD, or BID. The presence of HBx in MEFs and DP-16 cells
correlates with reduced cytochrome c release, which again
supports the role of this viral protein in inhibiting Fas-mediated
apoptosis.
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Fig. 7.
Expression of HBx in mouse embryo fibroblasts
and DP-16 cells inhibits the release of cytochrome c
from mitochondria. sek1+/+ and
sek /
fibroblasts (A) and DP-16 cells
(B) were transfected with an HBx expression vector
(pRBK-HBx), an HBx deletion mutant (pRBK-X
2-50), or the vector alone, were stimulated with
Fas antibodies (10 µg/ml) for 4 h. Nonspecific IgG1 was used in
place of Fas antibodies in the controls. Cytochrome c
released into the cytosol was measured by SDS-polyacrylamide gel
electrophoresis followed by immunoblot analysis.
(Fig. 8A, lanes 3).
Activation of SAPK/JNK was also observed in Chang liver cell lines
expressing HBx. These results were confirmed with a quantitative assay
that measured incorporation 32P onto the same substrate.
Expression of HBx correlated with a 30-fold increase in SAPK activity
in DP-16 erythroleukemia cells and mouse fibroblasts following
activation of the Fas signal transduction pathway (Fig. 8, B
and C). Cells without HBx only had a 6-fold increase
following stimulation. The presence of HBx in Chang liver cells also
increased the activity of SAPK 20-fold following stimulation of the
stress kinase pathway with either anisomycin or heat shock (data not
shown). Northern blot analysis indicated that the amounts of SEK1 and
SAPK mRNA were not increased due to the presence of HBx, suggesting
that either the viral protein increased transcription of upstream
kinases (e.g. MLK1-4) or acted directly on SEK1 or SAPK. We
concluded that the presence of HBx correlated with increased SAPK
activity in a variety of different cell types.
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Fig. 8.
HBx expression is associated with increased
SAPK/JNK activity. A, MEFs expressing HBx were prepared
using the expression plasmid pRBK-HBx, whereas control cells were made
with the empty vector pRBK. MEFs were stimulated with anti-Fas
(lane 2) and TNF (lane 3) and lysed according
to instructions for the SAPK assay from New England Biolabs. The cell
lysates were incubated with c-JUN coupled to Sepharose beads in the
presence of ATP. Proteins associated with the beads were resolved by
SDS-PAGE, transferred to nitrocellulose, and detected by ECL. In these
experiments, antibodies specific for phosphorylated c-JUN
(anti-P-c-JUN) or antibodies that recognized the nonphosphorylated form
of c-JUN (anti-c-JUN) were used to monitor phosphorylation. The
intensity of the P-c-JUN band was determined, and the increases of
SAPK/JNK activity over unstimulated background (lane 1) were
noted. B and C, quantitative determination of
increased SAPK/JNK activity in DP-16 cells (DP) and mouse
fibroblasts (MEF) in the presence (solid lines) or absence
(dashed lines) of HBx. Cell lines were prepared using the
expression plasmid pRBK-HBx (solid lines) or the empty
vector, pRBK, as a control (dashed lines). Specific
antibodies to SAPK/JNK were used to recognize the kinase in cell
lysates. Protein G coupled to Sepharose was used to precipitate the
SAPK/JNK that was subsequently incubated with c-JUN in the presence of
[
-32P]ATP. Phosphorylated c-JUN was resolved by
SDS-PAGE, detected by PhosphorImage analysis, and quantitated by liquid
scintillation counting. Samples of SAPK were isolated, and activity was
quantitated at the indicated times following anti-Fas
stimulation.
/
) fibroblasts was almost negligible (Fig.
9C) suggesting that an intact SEK1-SAPK complex was required
for HBx interaction. In addition, antibodies directed against HBx
precipitated 14-3-3 proteins (30 kDa), but SEK1 and SAPK protein bands
were obscured by the heavy chain of IgG (Fig. 9D). Further
studies to dissect the role of HBx and its role in the kinase complex
are underway in our laboratory, but it is intriguing to speculate that
this viral polypeptide may function as an adaptor that up-regulates kinase activity.
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Fig. 9.
HBx coprecipitates with MEKK1, SAPK, SEK1,
and 14-3-3 as a complex using c-JUN-Sepharose beads as bait.
A, Chang liver cells in which HBx was absent (lanes
1) or present (lanes 2) were lysed and incubated with
c-JUN-Sepharose beads. The beads were washed at least 3 times, and
associated proteins were solubilized in electrophoresis sample buffer
and resolved by SDS-PAGE along with samples of the initial cell
lysates. The proteins on the gels were transferred to nitrocellulose
membranes and probed with primary antibodies directed against MEKK1,
SAPK, SEK1, HBx, and 14-3-3. Detection was performed with secondary
antibodies and ECL. B, Chang liver cells expressing
full-length HBx (lane 2), N-terminally deleted HBx
(lane 3), or empty pRBK expression vector (lane
1) were lysed and incubated with c-JUN-Sepharose beads. Proteins
were resolved by SDS-PAGE and probed with primary antibodies directed
against SAPK and poly clonal antibodies directed against HBx. Detection was performed
with ECL. C, SEK1-deficient mouse fibroblasts
(sek1 /
) and normal fibroblasts (sek1+/+) in
which HBx was either present (lane 1) or absent (lane
2) were lysed and incubated with c-JUN-Sepharose beads. Protein
complexes were disrupted in sample buffer, resolved by SDS-PAGE, and
subjected to immunoblot analysis with primary antibodies directed
against MEKK1, SAPK, SEK1, 14-3-3, and HBx. In all experiments,
non-c-JUN-conjugated Sepharose beads did not form complexes with HBx or
components of the stress kinase pathway. D, normal Chang
liver cells (lane 1) and Chang liver cells containing HBx
(lane 2) were lysed and incubated with polyclonal antibodies
directed against HBx. Immunoprecipitated proteins were resolved on
immunoblots and probed with antibodies against 14-3-3 and HBx. The
secondary antibody also detects the rabbit IgG heavy chain and obscures
SAPK and SEK1 protein bands.
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Fig. 10.
Confocal immunofluorescent
microscopy confirms that HBx colocalizes with SEK1, SAPK, and
14-3-3 in Chang liver cells. Cells were
fixed with paraformaldehyde and permeabilized with Triton X-100
detergent. A mouse monoclonal antibody specific for HBx and rabbit
polyclonal antibodies specific for 14-3-3.
, SAPK, and SEK1 were
incubated with the liver cells. Binding of primary antibodies was
detected with goat anti-mouse antibodies conjugated to TRITC
(red) or goat anti-rabbit conjugated to FITC
(green). Fluorescently labeled cells were viewed with a
Zeiss LSM510 confocal microscope (× 800 magnification), and the images
were analyzed with LSM510 image browser software. Colocalization of the
two fluorescent dyes produces a yellow color. Cells
containing HBx (Chang-pRBK-HBx) and cells without HBx (Chang-pRBK) were
also analyzed.
26-31
mutants were sensitive to Fas-mediated apoptosis, whereas MEFs infected
with normal HBx were protected from cell death (Fig. 11D).
Thus, the 26RXRXXS motif appears to
play an important role in the protective effect of HBx against
Fas-mediated apoptosis and the up-regulation of SAPK. The effect of
these mutations on the overall structure of HBx will require further
investigation.
View larger version (24K):
[in a new window]
Fig. 11.
The 14-3-3 binding motif of HBx protein is
required for induction of SAPK/JNK activity and the suppression of
Fas-mediated apoptosis. The open reading frame of HBx
(GenBankTM accession number X51970) was used to
generate truncated, deletion, and mutant constructs (A).
Mutant HBx proteins were expressed in human liver cells (Huh7) and
MEFs. B, cells were transfected or infected with expression
vectors containing mutant HBx-coding sequences. At 48 h
post-transfection cells were stimulated with anisomycin (10 ng/ml) for
30 min. Cell lysates were prepared, and HBx was immunoprecipitated with
specific polyclonal antibodies, and proteins were resolved by PAGE and
transferred to immunoblots and probed with an antibody specific for the
phosphorylated 14-3-3-binding motif (New England Biolabs) in the
upper panels or a monoclonal antibody directed against HBx
in the lower panels. Huh7 cells were transfected with pRBK vector (lane 1), pRBK-HBx
(lane 2), pRBK-HBx31S-A (lane
3), pRBK-HBx 2-50 (lane 4),
pRBK-HBx
104-153 (lane 5), pRBK-HBx
114-153
(lane 6), pRBK-HBx
124-153 (lane
7) and stimulated with anisomycin. MEFs were infected with
retrovirus vector (lanes 1* and 1),
retrovirus-HBx (lanes 2* and 2),
retrovirus-HBx31S-A (lanes 3* and 3),
retrovirus-HBx
26-31 (lanes 4* and
4), and retrovirus-HBx
25-32 (lanes
5* and 5). The * indicates treatment with
anisomycin. C, cells were transfected with vectors
expressing wild type and mutant forms of HBx. At 48 h
post-transfection, cells were stimulated with anisomycin (10 ng/ml) for
30 min; cell lysates were prepared, and SAPK/JNK activity was measured
as described in Fig. 8A. Phosphorylated c-JUN was detected
with a specific monoclonal antibody. On longer ECL exposures, SAPK/JNK
activity could be detected in cells containing the vectors alone. Huh7
cells were transfected with pRBK vector alone (lane 1),
pRBK-HBx (lane 2), pRBK-HBx31S-A (lane 3),
pRBK-HBx
2-50 (lane 4), pRBK-HBx
104-153 (lane
5), pRBK-HBx
114-153 (lane 6), and pRBK-HBx
124-153
(lane 7). MEFs were infected with retrovirus vector
(lane 1), retrovirus-HBx
26-31 (lane 2),
retrovirus-HBx31S-A (lane 3), or retrovirus-HBx (lane
4). D, MEF cells were infected with recombinant mouse
retroviruses expressing HBx, HBx31S-A, and HBx
26-31. Cells were
sorted by flow cytometry for the GFP and the corresponding mutant HBx
expression. The cells were then treated with Fas antibodies for 24 h, and apoptosis was measured by annexin V/7-AAD analysis. The percent
of cell death was measured.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (45-48), SAPK (35, 36), and PI3K/PKB cell
survival pathways (50). However, there are some reports that HBx
overexpression in G418-selected cells or induction of the protein with
cre-lox, tetracycline, or dexamethasone controlled promoters
sensitized cells to apoptosis due to chemical stimuli (56, 58, 78-80). Over-stimulation of stress kinases could account for this effect since
these molecules have been shown to favor either cell survival or cell
death depending upon the cell type and apoptotic stimulus (32). Other
investigators have shown that HBx, either by itself or in the presence
of E1A, Ha-Ras, and v-Myc, could inhibit transformed cell focus
formation in the presence of G418, (56, 59, 80, 81). These observations
could result from increased sensitivity of cells to the chemical agent
G418 used in the selection of foci. Other results show that HBx induces
transformation of murine hepatocytes (81-84), murine fibroblasts (82),
and rat fibroblasts (19). Transgenic mice that express high levels of
HBx under control of the viral promoter in their livers (85-87) often
go on to develop hepatocellular carcinoma. Other laboratories (88) were
not able to reproduce this effect with different promoters. However,
low levels of expression of HBx in the presence of c-MYC favors
transformation in immortalized cell lines and transgenic mice (89). All
in all, it appears that expression of HBx alone cannot lead to cancer; other changes within the cell must also occur. HBx expression levels,
effects on other signal transduction pathways, the specific apoptotic
stimulus, and the type of cells assayed could account for these
discrepancies. We have also observed that the sequences of individual X
proteins differ from acute fulminant, chronic, and hepatocellular
carcinoma patients, and HBx sequences can be grouped into these
categories. The protein sequence of HBx used in our studies aligns best
with homologous proteins from virus isolated from the blood of chronic
and hepatocellular carcinoma patients. Our laboratory is currently
looking at the effects of these sequence differences on HBx function.
BK (46, 48), PI3K (50),
and PKB/AKT,2 it is
interesting to speculate that HBx might act as an adaptor or kinase
activator that enhances the phosphorylation of HBx-associated proteins.
By analogy, other investigators (97, 98) have shown that the TAX
protein of HTLV-1 increases SAPK/JNK activity. TAX protein has recently
been shown to bind to and up-regulate MEKK1, accounting for both the
up-regulation of SAPK and and NF
B activity (99). HBx has also been
reported to up-regulate NF
B activity, and it may do so through a
similar mechanism. In addition, whether HBx and SAPK interact directly
with the mitochondria and other components of the Fas pathway, as was
recently suggested by two laboratories (72, 100), is also being
explored. Clearly HBx is a multifunctional protein that has the
capacity to interact with components of several signal transduction
pathways within the cell, resulting in deregulation of growth and
proliferation. Its effects appear to be a first step in the process of hepatocarcinogenesis.
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ACKNOWLEDGEMENTS |
---|
We thank Marees Harris-Brandts and Suda Arya for assistance and preparation of polyclonal and monoclonal antibodies directed against the X protein of hepatitis B virus. We also thank Dr. Garry Nolan (Stanford University) for making the mouse mammary tumor virus expression vector pBMN and the Phoenix packaging cell line available to us.
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FOOTNOTES |
---|
* This work was supported by Medical Research Council of Canada Operating Grant MT-10638 and an Ontario Graduate Scholarship (to J. D.).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: Amgen Research
Institute, 620 University Ave., Suite 706, Toronto, Ontario M5G 2C1,
Canada. Tel.: 416-204-2280; Fax: 416-204-2278; E-mail:
crichard@amgen.com.
Published, JBC Papers in Press, November 30, 2000, DOI 10.1074/jbc.M006026200
2 J. Diao, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: HBV, hepatitis B virus; HBx, hepatitis B virus; TNF, tumor necrosis factor; SAPK, stress-activated protein kinase; JNK, c-JUN N-terminal kinase; EGFP, enhanced green fluorescent protein; MAPK, mitogen-activated protein kinase; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; IRES, internal ribosome entry site; GFP, green fluorescent protein; RT-PCR, reverse transcriptase-polymerase chain reaction; TRITC, tetramethylrhodamine B isothiocyanate; HTLV, human T cell lymphotrophic virus; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis; MEFs, mouse embryo fibroblasts; FACS, fluorescence-activated cell sorter; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; 7-AAD, 7-amino-actinomycin D; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; GST, glutathione S-transferase.
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