From the
Department of Internal Medicine and
Therapeutics,
Department of Molecular
Therapeutics, ¶Department of Microbiology, and
||Department of General Medicine, Osaka University
Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Received for publication, October 13, 2002 , and in revised form, May 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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To understand the molecular mechanisms through which such biological
functions would be exhibited, the modulation effect of HCV core protein on
various cellular signaling pathways has been studied by several investigators.
Tsuchihara et al.
(14) showed that the HCV core
protein enhanced the mitogen-activated protein kinase pathway under epidermal
growth factor stimulation. It has also been demonstrated by Aoki et
al. (15) that the
activation of Raf-1 kinase, a member of the mitogen-activated protein kinase
kinase family, was exerted by binding of the HCV core protein to 14-3-3
protein. Zhu et al.
(13) reported that the HCV
core protein became bound to the tumor necrosis factor receptor 1, resulting
in enhanced tumor necrosis factor-
-induced apoptosis by modulating the
tumor necrosis factor-
-mediated signal transduction pathways.
Furthermore, it has recently been shown by Yoshida et al.
(16) that the HCV core protein
modifies the NF-
B-mediated signal transduction pathway.
The Janus kinase (JAK)-signal transducer and activator transcription factor
(STAT) signaling pathway is known to be a major cascade associated with the
signal transduction for many cytokines and growth factors. The interactions of
these cytokines with specific surface receptors trigger the activation of JAK
and then STAT. The activation of JAK and STAT depends on the phosphorylation
at the specific tyrosine residues. There are four JAK proteins (JAK13
and Tyk2) and seven STAT proteins (STAT14, -5a, -5b, and -6). After
phosphorylation of JAK and STAT, the STAT is dimerized, resulting in nuclear
translocation. Most STAT dimers recognize the DNA element with the consensus
sequence 5'-TT(N46)AA-3' and regulate
transcription of many STAT-responsive genes (reviewed in Refs.
1722).
For example, IL-6 stimulation leads to the phosphorylation of JAK1, JAK2, and
STAT3, followed by nuclear translocation of the STAT3 homodimer
(23,
24). In the case of
IFN-, JAK1, JAK2, and STAT1 phosphorylation occurs, followed by STAT1
homodimerization and its nuclear translocation
(25). As for
IFN-
/
, the stimulation results in binding of the interferon
stimulatory response element (ISRE) with the sequence
5'-AGTTTN3TTTCC-3', through the activation of JAK1,
Tyk2, STAT1, and STAT2 and the conformation of interferon-stimulated gene
factor 3 composed of STAT1, STAT2, and interferon regulatory factor 9
(26,
27). A few investigators have
so far studied the influence of HCV expression on the JAK-STAT signaling
pathway in the host cell. Naganuma et al.
(28) revealed the enhancement
of the IFN-
-inducible 2',5'-oligoadenylate synthetase gene
expression by HCV core protein. It has also been shown by Markus et
al. (29) that expression
of the full-length of HCV ORF suppressed the IFN-
-mediated STAT/DNA
binding activity. However, the detailed molecular mechanisms of HCV
core-mediated modification on the JAK-STAT signaling pathway have not been
fully elucidated.
In the present study, we investigated the influence of the HCV core protein
on the JAK-STAT signaling pathway under IL-6 and IFN- stimuli using
murine cells constitutively expressing HCV core protein. The results of this
study demonstrated that the expression of HCV core protein has different
effects on the JAK-STAT signaling pathway; it causes inhibition in the case of
IL-6 treatment or enhancement in the case of IFN-
treatment of JAK-STAT
activation. We also showed that such HCV core-mediated modulation of JAK-STAT
signaling pathway was exerted by the two mechanisms; its binding to the JAK
protein and the up-regulation of the IFN-
receptor 2 (IFNGR2)
expression.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and TransfectionA murine normal liver cell
line, BNL CL. 2 (CL2) (33),
was maintained in the Dulbecco's modified Eagle's medium (Sigma, Tokyo, Japan)
supplemented with 10% fetal calf serum, 100 µg/ml streptomycin sulfate, 100
units/ml penicillin G sodium, and 0.25 µg/ml of amphotericin B in 5%
CO2 at 37 °C. For the establishment of cells constitutively
expressing HCV core protein, CL2 cells were transfected with pc/3EFNCTH
using Lipofectin (Invitrogen)
(34). Next, the cells were
cultured in the presence of 800 ng/ml G418 (Invitrogen). After 14 days,
G418-resistant colonies were isolated. Finally, three clones of HCV
core-expressing cells were established (designated CL2 core-I, -II, and -III).
In the present study, the CL2 core-I cell was mainly used for subsequent
experiments. In some experiments, the results were confirmed in CL2 core-II,
and -III cells. As a negative control, CL2 cells were transfected with
pc/3EFpro and cultured in the presence of 400 ng/ml G418 without the colony
isolation (designated CL2 mock). In this study, a human hepatoma cell line,
HepG2 (35), was also
maintained in the same condition as CL2 cells. For the transient transfection
study, 2 x 106 cells were seeded on a 10-cm culture dish and
transfected with pc/3EF
NCTH using Lipofectin. Next, the cells were
incubated in serum-free medium (Opti-MEM, Invitrogen), containing the
DNA-Lipofectin complex. Twenty-four hours after transfection, the medium was
removed, followed by further culture in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum for 24 h. Finally, the cells were lysed and
subjected to the subsequent experiments. Cells transfected with pc/3EFpro were
also used as a negative control.
Reporter Gene AssayFor the reporter gene assay using cells
constitutively expressing HCV core protein, 8.0 x 104 CL2
mock or CL2 core-I cells were seeded in the 6-well culture dishes and
transfected with 1.5 µg of the reporter plasmid (pAPRELuci for IL-6
stimulation, pGASLuci for IFN- stimulation, or pISRELuci for
IFN-
/
stimulation) and 0.1 µg of pRLtk using Lipofectin. Next,
the cells were incubated in Opti-MEM (Invitrogen) containing the
DNA-Lipofectin complex. Twelve hours after transfection, the medium was
removed, followed by further culture in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum for 24 h. For the last 6 h, the cells were
stimulated with 10 or 100 ng/ml of murine IL-6 (Pepro Tech, London, UK), 20 or
200 units/ml of murine IFN-
(Pepro Tech), 50 or 500 units/ml of murine
IFN-
/
(Sigma), or left unstimulated. In the cotransfection
experiment using CL2 and HepG2 cells, 8.0 x 104 CL2 or 2.0
x 105 HepG2 cells were seeded in the 6-well culture dishes
and cotransfected of 0.75 µg of the effector plasmid (pCore
(1191)-V5, pCoreMut-V5, pCoreDel-V5, pCore (1173)-V5, pCore
(1122)-V5, or pcDNA3.1/V5-HisA), with 0.75 µg of the reporter
plasmid (pAPRELuci or pGASLuci) and 0.1 µg of pRLtK. For the last 6 h, the
cells were stimulated with 100 ng/ml murine or human IL-6 (Pepro Tech), 200
units/ml of murine or human IFN-
(Pepro Tech), or left unstimulated.
Finally, the cells were lysed and subjected to the dual luciferase assay (Toyo
Ink Co., Ltd., Tokyo, Japan). Both firefly and seapansy luciferase activities
were measured with a luminometer (Lumat LB9507, EG&G Berthold, Bad
Wildbad, Germany), and the firefly luciferase activity was normalized for
transfection efficiency based on the seapansy luciferase activity. The
relative light unit of the unstimulated sample was regarded as 1, and the fold
activity of each sample was calculated. All assays were done in
triplicate.
Western Blot AnalysisFor the detection of HCV core protein, CL2 mock and core-I cells at a confluent state were lysed in low salt buffer (10 mM HEPES (pH 7.8), 10 mM KCl, 0.1 mM EDTA, 0.1% Nonidet P-40, 1 mM sodium vanadate, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 2 mM aprotinin, leupeptin, and pepstatin) at 4 °C for 5 min. After centrifugation for 5 min at 5,000 rpm, the supernatant was used as the cytoplasmic fraction for subsequent experiments. On the other hand, the pellet was suspended in high salt buffer (50 mM HEPES (pH 7.8), 420 mM KCl, 0.1 mM EDTA, 0.1% Nonidet P-40, 1 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 2 mM aprotinin, leupeptin, and pepstatin, 5 mM MgCl2, and 2% glycerol) at 4 °C for 30 min. Next, the sample was centrifuged at 4 °C for 15 min at 15,000 rpm. The supernatant was collected as a nuclear function. Both cytoplasmic and nuclear fractions were used for the Western blot analysis with mouse monoclonal HCV core antibody (East Coast Biologics, Inc., North Berwick, MN).
For the detection of JAK1, JAK2, STAT1, STAT3, tyrosine phosphorylated JAK1
(pY1022,1023JAK1; pJAK1), JAK2 (pY1007,1008JAK2; pJAK2),
STAT1 (pY701STAT1; pSTAT1), and STAT3 (pY705STAT3;
pSTAT3), the CL2 mock and core-I cells were stimulated with 100 ng/ml murine
IL-6 or 200 units/ml of murine IFN- and then lysed with buffer
containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1
mM EGTA, 50 mM NaF, 5 mM
Na2P2O7, 0.1% 2-mercaptoethanol, 1% Triton
X-100, 10 mM
-glycerophosphate, 0.5 mM sodium
vanadate, 1 mM aprotinin, leupeptin, pepstatin, and
phenylmethylsulfonyl fluoride. Antibodies against JAK1, JAK2, and pSTAT1 were
purchased from Upstate Biotechnology (Lake Placid, NY), and antibodies against
pJAK1 and pJAK2 were from BioSource (Camarillo, CA). Antibodies against pSTAT3
were obtained from Cell Signaling Technology (Beverly, MA), and antibodies
against STAT1 and STAT3 were from Santa Cruz Biotechnology (Santa Cruz, CA).
As for the Western blot analysis, the sample was mixed with 5x sample
buffer containing 15% 2-mercaptoethanol, 15% sodium dodecyl sulfate (SDS),
1.5% bromphenol blue, and 50% glycerol, separated with SDS-polyacrylamide gel
electrophoresis (PAGE), and blotted onto polyvinylidene difluoride membrane
(Hybond P; Amersham Biosciences, Buckinghamshire, UK). After blocking with
milk, the membrane was incubated with the first antibody, followed by
incubation with horse-radish peroxidase-labeled immunoglobulin as a second
antibody. Finally, the immune complex was detected by an enhanced
chemiluminescent assay (Super Signal, Pierce).
Protein-Protein Interaction AnalysisTo examine the binding
of the JAK protein to HCV core protein, the immunoprecipitation/Western blot
analysis was applied in this study. From CL2 mock, CL2 core-I, and transiently
transfected HepG2 cells, the cellular protein was extracted by the method
described above. The lysates were precleared by the incubation with protein
A-Sepharose beads (Amersham Biosciences, Uppsala, Sweden) at 4 °C for 1 h.
Next, the sample was incubated with the beads coupled to the JAK1, JAK2,
STAT1, or STAT3 antibody, or the rabbit nonspecific -globulin (Dako,
Glostrup, Denmark) for 18 h. The immune complex were eluted by being boiled
for 5 min. Finally, the supernatant was used for Western blot to detect the
HCV core protein as described above.
To confirm the binding site of HCV core protein to JAK proteins, HCV core proteins possessing the wild-type or the mutated amino acid sequences were synthesized from the plasmid pCore (1191)-V5, pCoreMut-V5, or pCoreDel-V5 by the in vitro translation method. The reaction was performed with the TNT T7 coupled reticulocyte lysate system (Promega) and labeled with L-[35S]methionine (Amersham Biosciences). The cellular lysates from CL2 and HepG2 cells were extracted and mixed with the in vitro synthesized various types of the HCV core proteins. Then, the mixture was subjected to the immunoprecipitation using antibodies against JAK1/2 as above. Finally, the immunoprecipitate was separated with SDS-PAGE, and the gel was dried and autoradiographed.
Northern Blot AnalysisThe expression levels of interferon
regulatory factor (IRF)-1, fibrinogen -chain, and IFNGR2 mRNAs were
analyzed by Northern blot analysis. The total cellular RNA was extracted from
CL2 mock and core-I cells using an Isogen kit (Nippon Gene Co., Toyama, Japan)
based on the guanidine-isothiocyanate method. The RNA sample were denatured
and electrophoresed, followed by the transfer to the nylon membrane (Hybond N;
Amersham Biosciences). The membrane was then hybridized with the
32P-labeled cDNA probe at 65 °C for 24 h, washed, and
autoradiographed. The band intensity was quantitated with an image analyzer
(BAS 2500, Fuji Film, Tokyo, Japan).
Flow CytometryCL2 mock and core-I cells were suspended in
100 µl of phosphate-buffered saline supplemented with 2% bovine serum
albumin and 0.1% sodium azide and incubated in the presence or absence (for
negative control) of the specific antibody at 4 °C for 1 h. Antibodies
against mouse gp-80, gp-130, IFN- receptor 1 (IFNGR1), and IFNGR2 were
purchased from Santa Cruz. Then, cells were washed and resuspended in 100
µl of phosphate-buffered saline with 10 µl of fluorescein
isothiocyanate-labeled anti-rabbit IgG antibody (Santa Cruz, CA). Finally,
samples were fixed with 1% parafolmaldehyde and subjected to the flow
cytometry. The expression level of the proteins on the cell surface was
quantitated by calculating the mean fluorescein intensities.
Statistical AnalysisStatistical analysis was performed using the non-paired t test as appropriate. p values less than 0.05 were considered to be statistically significant.
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RESULTS |
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Modulation of STAT-mediated Transcriptional Activity under IL-6 and IFN
Stimuli by HCV Core ProteinTo examine whether the expression of
HCV core protein would affect the activation of JAK-STAT signaling cascade
under various types of cytokines stimulation, we first performed the reporter
gene assay by transient transfection of the plasmids pAPRELuci (for IL-6
stimulation), pGASLuci (for IFN- stimulation), or pISRELuci (for
IFN
/
stimulation). The induction level of APRE-dependent
transcription activity under 100 ng/ml of IL-6 was 2.6-fold lower in CL2
core-I cells than in the mock cells (Fig.
2A). On the other hand, 200 units/ml of IFN-
stimulation led to 2.2-fold higher induction of GAS-dependent transcription
activity in the CL2 core-I cells than in the mock cells
(Fig. 2B). In the case
of IFN-
/
stimulation, no significant differences in the induction
of ISRE-dependent transcription activity between CL2 mock and core-I cells
(Fig. 2C). CL2 core-II
and -III cells displayed the same tendency for the luciferase activities as
CL2 core-I cells (data not shown). Next, the expression levels of the
fibrinogen
-chain and the IRF-1 mRNAs were studied as
IL-6/APRE-responsive and IFN-
/GAS-responsive genes, respectively. As
shown in Fig. 2D, the
expression of fibrinogen
-chain was almost absent in both cells before
IL-6 stimulation. But its induction level was
2.5-fold lower in the CL2
core-I cells than in the mock cells. As for expression of IRF-1 mRNA, the
induction level after IFN-
stimulation was 2.1-fold higher in the CL2
core-I cells than in the mock cells (Fig.
2E). Taken together, the activation of JAK-STAT signaling
cascades was altered significantly by HCV core protein under stimulation with
IL-6 and IFN-
, but not with IFN-
/
.
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Influence of HCV Core Protein on Tyrosine Phosphorylation of JAK and
STAT Proteins by IL-6 StimulationThe reporter gene assay and
Northern blot analysis revealed that the expression of HCV core protein
modulated the JAK-STAT signaling cascade differently under stimulation with
IL-6 or IFN-. We thus examined the change in the level of
tyrosine-phosphorylated JAK and STAT proteins before and after IL-6
stimulation in CL2 mock and core-I cells. As shown in
Fig. 3A (upper
panel), the degree of JAK1 phosphorylation after the stimulation was
reduced in the CL2 core-I cells, compared with the mock cells. The induction
level of JAK2 phosphorylation after IL-6 stimulation was also lower in the CL2
core-I cells than in the mock cells (data not shown). As for the
phosphorylation of STAT3, it was also lower in the CL2 core-I cells than in
the mock cells (Fig.
3A, lower panel). This tendency of
phosphorylation status of JAK and STAT proteins under IL-6 stimulation was
also seen in CL2 core-II and -III cells (data not shown). When the expression
levels of the whole JAK1, JAK2, and STAT3 proteins were compared between CL2
mock and core-I cells (Fig.
3B), their levels were the same irrespective of IL-6
stimulation in both cells. Also, the expression of HCV core protein did not
considerably affect the expression levels of JAK1, JAK2 and STAT3. We also
studied the STAT3/DNA binding activity in the nuclear extracts from CL2 mock
and core-I cells after IL-6 stimulation using the double-stranded DNA probe
corresponding to the APRE. The STAT3/DNA binding activity was considerably
reduced in the CL2 core-I cells, compared with the CL2 mock cells at both 1
and 6 h after IL-6 stimulation (data not shown).
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Influence of HCV Core Protein on Tyrosine Phosphorylation of JAK and
STAT Proteins by IFN- StimulationThe changes in
the pJAK1, pJAK2, and pSTAT1 levels before and after the IFN-
stimulation were further examined in both CL2 mock and core-I cells. As shown
in Fig. 4A, the
induction levels of the JAK1 (upper panel) and JAK2 (middle
panel) phosphorylation after IFN-
stimulation were stronger in the
CL2 core-I cells than in the mock cells. As for changes in the STAT1
phosphorylation, its induction level was higher in the CL2 core-I cells than
in the mock cells (Fig.
4A, lower panel). CL2 core-II and -III cells
displayed the same tendency for the phosphorylation status of JAK and STAT
proteins under IFN-
stimulation as CL2 core-I cells (data not shown).
We also investigated the expression levels of the whole JAK1, JAK2, and STAT1
proteins in CL2 mock and core-I cells (Fig.
4B), resulting in the same expression levels of these
proteins irrespective of IFN-
stimulation. As for the influence of HCV
core on the expression levels of these proteins, it did not substantially
affect their expression levels. In the nuclear extracts from CL2 mock and
core-I cells after IFN-
stimulation, the EMSA was also performed to
examine the STAT1/DNA binding activity using the double-stranded DNA probe
corresponding to the GAS. The STAT1/DNA binding activity was considerably
enhanced in the CL2 core-I cells, compared with the CL2 mock cells at both 1
and 6 h after IFN-
stimulation (data not shown).
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Binding of HCV Core Protein to JAK ProteinsOur results
showed that the constitutive expression of HCV core protein caused inhibition
(in the case of IL-6 treatment) or enhancement (in the case of IFN-
treatment) of tyrosine phosphorylation of JAK and STAT proteins, resulting in
modification of the STAT-mediated transcription. This would be responsible for
the direct effect of JAK activity by the HCV core protein, because the
tyrosine phosphorylation of JAK has been shown to be a primary event of the
JAK-STAT signaling cascade
(1722).
Therefore, we examined the binding of HCV core protein to the JAK and STAT
proteins in the CL2 core-I cells by means of the immunoprecipitation/Western
blot analysis (Fig.
5A). The HCV core protein was not found in the negative
control sample using nonspecific
-globulin (lane 1). On the
other hand, the HCV core protein was detected in the immunoprecipitates using
antibodies against JAK1 (lanes 24), JAK2 (lanes
57), and Tyk2 (data not shown), but not detected in those using
antibodies against STAT1 (lanes 810) and STAT3 (lanes
1113). This finding indicates the possible binding of HCV core
protein to murine JAK proteins. Similar results were observed in cells
stimulated with IL-6 (lanes 3, 6, 9, and 12) or IFN-
(lanes 4, 7, 10, and 13), suggesting that the binding of HCV
core protein to JAK proteins may occur in a phosphorylation-independent
manner. To confirm the interaction between HCV core protein and human JAK
proteins, we further carried out the assay using HepG2 extracts transiently
transfected with HCV core-expressing plasmid
(Fig. 5B). The HCV
core protein was found in the immunoprecipitates using antibodies against JAK1
(lane 4), JAK2 (lane 5), and Tyk2 (data not shown), but not
detected in those using nonspecific
-globulin (lane 3) and
antibodies to STAT1 and STAT3 (data not shown).
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Detection of the Interaction Site of HCV Core Protein to JAK ProteinsIt has been shown that various authentic JAK-binding proteins have the proline-rich PXXPXP sequence, designated box 1, as an interaction site with the JAK protein (3741). HCV core protein also bears the box 1-like PGYPWP sequence at amino acid positions 7984. Therefore, this region would be important for the binding of HCV core protein to JAKs. To confirm this possibility, we synthesized the mutant HCV core protein having three proline to alanine amino acid substitutions within the box 1-like region, and the deleted HCV core protein lacking the 6 amino acid residues of box 1-like region, as well as the wild-type HCV core protein, by means of the in vitro translation method (Fig. 6A). Similar levels of wild-type, mutant and deleted HCV core proteins were synthesized in vitro from plasmids pCore (1191)-V5, pCoreMut-V5 and pCoreDel-V5, respectively (Fig. 6B). Then, these HCV core proteins were mixed with the cellular lysate and immunoprecipitated with the antibody against JAK1 or JAK2. As shown in Fig. 6C, only wild-type HCV core protein was detected, whereas mutant and deleted HCV core proteins were not detectable in the immunoprecipitates using the JAK1 (lanes 13) or JAK2 (lanes 46) antibody. This result was obtained in both experiments using the murine CL2 lysate (Fig. 6C, upper panel) and the human HepG2 lysate (Fig. 6C, lower panel). According to these findings, the PGYPWP sequence of the HCV core protein may play a crucial role in its binding to both murine and human JAK proteins.
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Direct Effect on JAK-STAT Signaling Pathway Caused by the HCV Core-JAK
Interaction under IL-6 and IFN- StimuliTo examine
whether the interaction between HCV core protein and JAKs would directly
affect the activation of the JAK-STAT signaling cascade under IL-6 and
IFN-
stimuli, we conducted the reporter gene assay in both murine CL2
and human HepG2 cells by means of cotransfection of various effector plasmids
with the reporter plasmid. As effector plasmids, pCoreMut-V5 and pCoreDel-V5
encoding the HCV core protein without the functional JAK-binding site, as well
as pCore (1191)-V5 and the empty vector pcDNA3.1/V5-HisA, were used for
this experiment. In the CL2 cells under the stimulation with 100 ng/ml of IL-6
(Fig 7A), the
APRE-dependent transcription activity was 1.8-fold lower by transfection with
pCore (1191)-V5 than by that with pcDNA3.1/V5-HisA, as was the case for
the experiment using CL2 mock and core-I cells (see
Fig. 2A). In the case
of transfection with pCoreMut-V5 or pCoreDel-V5, however, the APRE-dependent
transcription activity was restored to the level of transfection with
pcDNA3.1/V5-HisA. HepG2 cells also displayed the same tendency for the
APRE-dependent transcription activity under IL-6 stimulation as the CL2 cells
(Fig. 7B). These
results indicate that HCV core-mediated inhibition of JAK-STAT signaling
pathway under IL-6 stimulation may be directly caused by the binding of HCV
core to the JAK proteins.
|
As for the experiment using the CL2 cells under 200 units/ml of IFN-
stimulation (Fig. 8A),
the GAS-dependent transcriptional activity was 2.2-fold higher by transfection
with pCore (1191)-V5 than by that with pcDNA3.1/V5-HisA, as was the
case for the experiment using CL2 mock and core-I cells (see
Fig. 2B). Unlike the
case of IL-6 stimulation, transfection with pCoreMut-V5 or pCoreDel-V5
revealed the same level of the GAS-dependent transcription activity as that
with pCore (1191)-V5. This tendency of the transcription activity under
IFN-
stimulation was also seen in the HepG2 cells
(Fig. 8B). Thus, the
mutated or deleted HCV core protein, which was not capable of binding to the
JAKs, could still activate the JAK-STAT signaling pathway under IFN-
stimulation. Therefore, HCV core-mediated enhancement of JAK-STAT signaling
pathway under IFN-
stimulation may not be responsible for the HCV
core-JAK interaction. To determine the region of HCV core protein contributing
to the augmentation of the JAK-STAT signaling pathway under IFN-
stimulation, we further constructed two plasmids encoding the C-terminal
deletion mutant forms of HCV core protein, pCore (1173)-V5 and pCore
(1122)-V5. Transfection with these two plasmids lost the ability of the
enhancement of GAS-mediated transcription activity in both CL2 and HepG2 cells
(data not shown), suggesting that the C-terminal region (amino acids positions
174191) of HCV core protein may be one of the important regions for the
activation of JAK-STAT signaling pathway under IFN-
stimulation.
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Influence of HCV Core Protein on Cytokine Receptor
ExpressionTo examine the expression level of each cytokine
receptor on the cell surface, we further carried out the flow cytometric
analysis. The IL-6 receptor subunits, gp-80 and gp-130, and IFN-
receptors, IFNGR1 and IFNGR2, were examined in cytokine-untreated CL2 mock and
core-I cells. The gp-80, gp-130, and IFNGR1 were expressed with no substantial
differences between the CL2 mock and core-I cells
(Fig. 9, AC).
However, the surface protein level of IFNGR2 was
3.3-fold higher in the
CL2 core-I cells than in the CL2 mock cells
(Fig. 9D). The
enhanced expression of IFNGR2 by HCV core protein was also seen in cells
stimulated with 200 units/ml of IFN-
for 1 h (data not shown),
suggesting that HCV core-mediated up-regulation of IFNGR2 expression can occur
irrespective of the presence or absence of IFN-
stimulation. When the
IFNGR2 mRNA level was investigated by Northern blot analysis, it was also
2.2-fold higher in the CL2 core-I cells than in the CL2 mock cells
(Fig. 9E). These
results showed that HCV core protein up-regulated both mRNA and surface
protein levels of IFNGR2. On the other hand, it did not affect the expression
levels of the IL-6 receptor subunits.
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DISCUSSION |
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We next examined the level of the tyrosine-phosphorylated JAK and STAT
proteins in both mock and HCV core-expressing cells under IL-6 and IFN-
stimuli. The phosphorylation levels of JAK1, JAK2, and STAT3 proteins were
suppressed by expression of HCV core protein under IL-6 stimulation. On the
other hand, the phosphorylation levels of JAK1, JAK2, and STAT1 were increased
by expression of HCV core protein under IFN-
stimulation. The HCV core
protein appeared to directly affect the kinase activity of JAK protein,
because the whole protein levels of JAK and STAT proteins were not
substantially influenced by its expression. Taken together, this demonstrated
that the HCV core protein regulates the JAK-STAT signaling pathway differently
under IL-6 and IFN-
stimuli.
To further assess the influence of HCV core protein on JAK proteins, its
binding to JAK proteins was examined in HCV core-expressing cells. The result
strongly suggests that HCV core protein may interact with JAK1 and JAK2, but
not with STAT1 and STAT3 in cells. The binding of HCV core protein to JAK
proteins was observed in both the cytokine-untreated cells and those treated
with IL-6 and IFN-, indicating that it may occur irrespective of the
phosphorylation status of JAK proteins.
The authentic JAK-binding proteins have been shown to possess the proline-rich sequence, PXXPXP, designated box 1, which serves as a docking site for the JAK protein (3741). For example, PNVPDP for human and mouse gp130, the IL-6 receptor subunit (37, 41), PGIPSP for human erythropoietin receptor (38), and PSVPDP for granulocyte colony-stimulating factor receptor (39) have been identified. As for the HCV core protein, it also includes one box 1-like sequence, PGYPWP, at amino acid positions 7984. The in vitro protein-protein interaction analysis revealed that both the mutant HCV core protein having the AGYAWA sequence instead of the PGYPWP one and deleted HCV core protein lacking the PGYPWP sequence lost the binding activity to JAK proteins. This result indicates that this box-1 like PGYPWP sequence within the HCV core protein may play a major role in its direct interaction with JAK proteins. HCV is known to be susceptible to mutations and can be divided into six major genotypes (42). The rate of amino acid divergence of HCV has been shown to be 22.834.0% among the different genotypes (4346). Nevertheless, this PGYPWP box 1 motif of HCV core protein is well conserved among HCV strains (47). This suggests that the binding of the HCV core protein to JAK proteins may be a general event throughout all genotypes of HCV strains.
In the present study, the mouse cell line was mainly used, because cells constitutively expressing HCV core protein could not be established in the human liver-derived cell lines HepG2, Huh-7, and Hep3B. Therefore, the binding of HCV core protein to JAK proteins was also investigated using HepG2 cells transiently transfected with HCV core-expressing plasmid. The interaction of HCV core protein with JAK1 and JAK2 proteins was also found in human HepG2 cells. In addition, it was shown that the mutation and the deletion of the box 1-like PGYPWP sequence resulted in the loss of the binding activity to JAK proteins in the experiment using the human HepG2 lysate. Thus, the HCV core protein was shown to have binding activity for human JAK proteins, as well as murine JAK ones.
To assess the direct contribution of the HCV core-JAK interaction to the
HCV core-mediated modulation of JAK-STAT signaling pathway under IL-6 and
IFN- stimuli, we further carried out the reporter gene assay by means
of the cotransfection method. The results showed that the mutated or deleted
HCV core protein, which could not bind to the JAK proteins, lost the
inhibitory effect on the JAK-STAT pathway under IL-6 stimulation in both
murine CL2 and human HepG2 cells. Our finding strongly suggests that HCV
core-mediated inhibition of JAK-STAT pathway under IL-6 stimulation may be
directly caused by the interaction between HCV core and JAK. Such suppressive
effect of HCV core protein on the JAK-STAT pathway may be exhibited by its
competitive inhibition of interaction between the gp-130 and JAK proteins,
because the binding manner of HCV core protein to JAK proteins may be similar
to that of gp-130. Otherwise, the binding of HCV core protein to JAKs may lead
to the conformational change of JAK protein, resulting in decrease in the JAK
activity.
In contrast, HCV core-mediated augmentation of JAK-STAT pathway under
IFN- stimulation was not so much affected by introducing the mutation
or deletion of the box 1-like PGYPWP sequence within HCV core. This result
indicates that the interaction between HCV core and JAK may have no or minimal
effect on this pathway under IFN-
stimulation, unlike the case of IL-6
stimulation. To further investigate how HCV core protein would contribute to
the activation of JAK-STAT pathway under IFN-
stimulation, we examined
the differences in the expression levels of cytokine receptors between the
mock and HCV core-expressing cells. HCV core protein was found to be
considerably up-regulated the mRNA and cell surface expression levels of
IFNGR2. It is known that IFNGR1 can bind the ligand with high affinity and
that IFNGR2 is a crucial factor for signal transduction
(4850).
It has also been reported that elevated expression of IFNGR2 by external
stimuli increased sensitivity to IFN-
and JAK-STAT activation
(51). Thus, regulation of
IFNGR2 expression has been shown to be one of important factors in determining
IFN-
responsiveness. According to these, the enhancement of JAK-STAT
pathway by HCV core protein under IFN-
stimulation may be mainly due to
elevated expression of IFNGR2. Under IFN-
stimulation, HCV
core-mediated up-regulation of IFNGR2 may have a stronger effect on the
JAK-STAT signal transduction than the suppressive action caused by the
interaction of HCV core with JAK proteins.
The influence of HCV expression on the JAK-STAT signaling pathway has been
studied. Naganuma et al.
(28) showed that the HCV core
protein enhanced the IFN--inducible 2'-5'-oligoadenylate
synthetase gene expression in the human hepatocyte-derived cells transiently
transfected with the HCV core gene. This does not agree with our findings that
persistent expression of HCV core protein caused no effects on the
ISRE-mediated transcription activity under IFN-
/
stimulation in
mouse normal liver cells. It has also been suggested by Markus et al.
(29) that the expression of
full-length HCV ORF strongly suppressed the IFN-
- or leukemia
inhibitory factor-mediated STAT/DNA binding activity without affecting the
STAT phosphorylation status in a human osteosarcoma cell line permanently
transfected with HCV cDNA. Such mutually conflicting results may have arisen
due to different expression levels of HCV protein and/or different kinds of
cultured cells. In addition, HCV proteins, other than the core protein, may
also play a substantial role in the modification of the JAK-STAT signaling
pathway. Further studies should offer better understandings of this
problem.
In summary, this is the first report indicating that the JAK protein is one
of the target molecules of HCV core protein. Our findings also showed that HCV
core protein modulates the JAK-STAT signaling pathway differently under IL-6
and IFN- stimuli, which may be due to the total effects of its binding
to the JAK proteins and the enhanced expression of the IFN-
receptor.
It is known that IL-6-induced STAT3 activation stimulates hepatocytes to
produce a variety of acute-phase proteins, such as C reactive protein and
fibrinogen (52). Furthermore,
liver regeneration after partial hepatectomy has been shown to be delayed in
IL-6-deficient mice, suggesting that IL-6 may play an important role in
hepatocyte proliferation (53).
As for the role of IFN-
in hepatocytes, IFN-
has also been shown
to contribute to the inhibition of hepatitis B virus expression in hepatitis B
virus transgenic mice (54). In
a clinical setting, the clearance of HCV viremia has been reported to be
associated with the enhanced antigen-specific IFN-
production in
chronic hepatitis C patients who underwent the antiviral treatment
(55). In addition, severe
hepatitis has been shown to arise in IFN-
transgenic mice
(56). Thus, IFN-
may be
closely associated with both virus eradication from the liver and the
development of hepatitis. Taken together, HCV core-mediated modulation of the
JAK-STAT signaling pathway under IL-6 and IFN-
stimuli may have a
substantial role in the pathogenesis of HCV-related liver diseases.
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FOOTNOTES |
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** To whom correspondence should be addressed: Dept. of Molecular Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3440; Fax: 81-6-6879-3449; E-mail: hayashin{at}moltx.med.osaka-u.ac.jp.
1 The abbreviations used are: HCV, hepatitis C virus; ORF, open reading
frame; JAK, Janus kinase; STAT, signal transducer and activator transcription
factor; IL, interleukin; IFN, interferon; ISRE, interferon stimulatory
response element; IFNGR, IFN- receptor; EF, elongation factor; APRE,
acute phase response element; GAS,
-interferon activation site; IRF,
interferon regulatory factor.
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ACKNOWLEDGMENTS |
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REFERENCES |
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