JVI Accepts, published online ahead of print on 26 November 2014 J. Virol. doi:10.1128/JVI.03106-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Hepatitis B core protein sensitizes hepatocytes to tumor necrosis factor-induced apoptosis
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by suppression of the phosphorylation of mitogen-activated protein kinase kinase 7
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Baosen Jia,a Minggao Guo,b Gaiyun Li,a Demin Yu,c Xinxin Zhang,c Ke Lan,a# Qiang Denga#
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Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese
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Academy of Sciences, Shanghai, China a
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Department of General Surgery, Sixth People’s Hospital, Shanghai Jiaotong University School
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of Medicine, Shanghai, China b
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Department of Infectious Diseases, Ruijin Hospital, Shanghai Jiaotong University School of
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Medicine, Shanghai, China d
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#Address correspondence to Dr. Qiang Deng, E-mail: [email protected]; or Dr. Ke Lan,
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E-Mail, [email protected]
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Running Title:
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HBc is pro-apoptotic by blocking MKK7
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1
15
Abstract
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Hepatitis B, which caused by Hepatitis B virus (HBV) infection, remains a major health threat
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worldwide. Hepatic injury and regeneration from chronic inflammation is the main driving
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factor of liver fibrosis and cirrhosis in chronic hepatitis B. Pro-inflammatory tumor necrosis
19
factor (TNF)-α has been implicated as a major inducer of liver cell death during viral hepatitis.
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Here, we report that in hepatoma cell lines and in primary mouse and human hepatocytes,
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expression of hepatitis B core (HBc) protein made cells susceptible to TNF-α-induced
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apoptosis. We found that receptor of activated protein kinase C (RACK) 1 interacted with HBc
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by tandem affinity purification and mass spectrometry. RACK1 was recently reported as a
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scaffold protein that facilitates the phosphorylation of mitogen-activated protein kinase kinase
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7 (MKK7) by its upstream activators. Our study showed that HBc abrogated the interaction
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between MKK7 and RACK1 by competitively binding to RACK1, thereby downregulating
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TNF-α-induced phosphorylation of MKK7 and the activation of c-Jun N-terminal kinase. In
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line with this finding, specific knockdown of MKK7 increased the sensitivity of hepatocytes to
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TNF-α-induced apoptosis, while overexpression of RACK1 counteracted the pro-apoptotic
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activity of HBc. Capsid particle formation was not obligatory for HBc pro-apoptotic activity, as
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analyzed using an assembly-defective HBc mutant. In conclusion, the expression of HBc
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sensitized hepatocytes to TNF-α-induced apoptosis by disrupting the interaction between
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MKK7 and RACK1. Our study is thus a first indication of the pathogenic effects of HBc in
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liver injury during hepatitis B.
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Importance:
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Our study revealed a previously unappreciated role of HBc in TNF-α-mediated apoptosis. The
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pro-apoptotic activity of HBc is important for understanding hepatitis B pathogenesis. In
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particular, severe hepatitis-associated HBV variants may upregulate apoptosis of hepatocytes 2
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through enhanced HBc expression. Our study also found that MKK7 is centrally involved in
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TNF-α-induced hepatocyte apoptosis and revealed a multifaceted role for JNK signaling in this
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process.
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Key words:
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Hepatitis B core protein; apoptosis; TNF-α; MKK7; RACK1
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3
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Introduction
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Hepatitis B virus (HBV) infection remains a major public health threat affecting an estimated
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400 million individuals worldwide, with high risk of developing severe liver diseases,
48
including cirrhosis and hepatocellular carcinoma. The host protective immune response is also
49
responsible for liver pathogenesis during HBV infection.
50
Pro-inflammatory tumor necrosis factor (TNF)-α is critical for controlling HBV in clinical
51
settings and in model systems, possibly by destabilizing cytoplasmic viral nucleocapids and
52
reducing nuclear viral DNA (1, 2). Recently, TNF-α was reported to reduce host cell
53
susceptibility to HBV entry via activation-induced cytidine deaminase (3). On the other hand,
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TNF-α has also been implicated as a mechanism of hepatic injury through induction of cellular
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apoptosis during viral hepatitis (4-9). Apoptosis is organized, genetically controlled
56
programmed cell death. Clearance of apoptotic debris stimulates transforming growth factor-β
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expression and induces collagen I secretion by hepatic stellate cells. These processes have been
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suggested as the core machinery for liver fibrogenesis and cirrhosis (10, 11).
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Overexpressing HBV core (HBc), precore, X, small and middle envelope proteins (S2S), as
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well as the viral replication, is not associated with liver disease in transgenic mouse models
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(12). Hence HBV itself is not considered to be directly cytopathic for infected hepatocytes.
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This notion has to be re-examined, however, in a pathophysiological setting. Of particular
63
interests, naturally occurring mutations within the basic core promoter of HBV are implicated
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in severe liver diseases including fulminant hepatitis, which results in up to 15-fold increased
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HBc synthesis (13-17). The fulminant hepatitis-associated mutant induces apoptosis in primary
66
hepatocytes and is not reversed by inhibition with nucleoside analogs (15). This finding
67
suggests that HBc might be directly responsible for virus-induced hepatocyte death, but the
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underlying mechanism remains undefined.
4
69
HBc is a 21 kDa viral structural protein with an N-terminal domain that is responsible for viral
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capsid assembly and a C-terminal arginine-rich polypeptide that interacts with HBV
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pregenomic RNA. HBc is found to be distributed in both the nucleus and the cytoplasm in
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HBV-producing hepatocytes and transgenic mice. Clinically, a nucleus-dominant distribution
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of HBc is associated with minor hepatitis activity while a cytoplasmic distribution of HBc is
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associated with chronic active liver disease (18). In the present study, we report that expression
75
of HBc increased susceptibility of hepatocytes to TNF-α-induced apoptosis by disrupting the
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interaction between receptor for activated c kinase 1 (RACK1) and mitogen-activated protein
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kinase kinase 7 (MKK7) (19), leading to downregulation of TNF-α-induced phosphorylation of
78
MKK7 and c-Jun-N-terminal kinase (JNK) (Fig. 1). Our results therefore revealed a previously
79
unappreciated role for HBc in liver pathogenesis during viral hepatitis.
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5
Materials and Methods
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Plasmid DNA and viral vectors
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Plasmids encoding HBV Polymerase, X protein, S2S, and HBc, as well as HA-tagged RACK1
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and Flag-tagged MKK7, were constructed by inserting the appropriate genes into the multiple
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cloning site (MCS) of pcDNA3.1(-) (Invitrogen). Genes encoding His-tagged RACK1 or
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His-tagged HBc were subcloned into the MCS of pET-30a(+) (Novagen) for prokaryotic
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protein expression. For GST pull-down assay, plasmids encoding GST-tagged HBc
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truncations and GST-tagged RACK1 truncations were constructed by inserting the appropriate
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genes into the MCS of pGEX-4T-1 (Promega).
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Recombinant adenovirus (rAd) expressing HBc (rAd-HBc/GFP, with a GFP gene separated by
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an IRES sequence) was constructed by Obio Technology (Shanghai), using E1-deleted human
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rAd vector of serotype 5. The lentiviral vector expressing HA-tagged RACK1 was constructed
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by inserting the sequence into the MCS of pCDH-CMV-MCS-EF1-Puro (SBI) and packaging
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the expression constructs into pseudoviral particles.
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siRNAs
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siRNA oligos against human and mouse MKK7 were synthesized by GenePharma: siMKK7-1:
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5’-CAGGACAGUUUCCCUACAATT-3’, siMKK7-2: 5’-GCAAGAUGACAGUGGCGAUTT
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-3’. siRNAs were transfected into cells by Lipofectamine 2000 (Invitrogen). Subsequent
99
treatments were performed 48 hours after transfection.
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Cells
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Huh-7, HepG2, HepG2.2.15 and 293T cells were cultured in DMEM containing 10% fetal
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bovine serum (FBS) and 1% Penicillin-Streptomycin. Primary mouse hepatocytes (PMH) were
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isolated from male C57BL/6 mice as described below. Primary human hepatocytes (PHH) were
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from Mucyte Biotech (Nanjing). PMH and PHH were cultured in Williams’ Media E
6
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containing 10% FBS, 2 mM L-glutamine, 1× nonessential amino acids, and 1%
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Penicillin-Streptomycin. HepG2 or Huh-7 cells constitutively expressing HBc were constructed
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by transfecting cells with pcDNA3.1-HBc and selecting with 50 μg/mL Geneticin.
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Stimulation with TNF-α
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To induce massive apoptosis, Huh-7 or HepG2 cells were plated into 12-well plates at 0.25-0.5
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million cells per well. The next day, cells were pretreated with 0.5 μg/mL actinomycin D
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(Sigma) for 30 minutes and cultured for 14 hours with 100 ng/mL hTNF-α (PeproTech) in the
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presence of 12 μM (for Huh-7 cells) or 20 μM (for HepG2 cells) of Bay11-7082 (Sigma). For
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co-treatment with pyrrolidine dithiocarbamic acid (PDTC, Beyotime), 50 μM of PDTC and 10
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ng/mL hTNF-α were used (for HepG2 cells).
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The stimulation with TNF-α in PMH and PHH was described previously (27, 28). In brief,
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cells were pretreated with 0.5 μg/mL actinomycin D for 30 minutes, and cultured with 2 ng/mL
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of mTNF-α (PeproTech) (for PMH) or 100 ng/ml hTNF-α (for PHH) in the presence of 0.5
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μg/mL actinomycin D for 14 hours.
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Isolation of PMH
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PMH was isolated from C57BL/6 male mice by two step collagenase perfusion technique (29).
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The study was approved by the Animal Ethics Committee of Institut Pasteur of Shanghai (no.
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A2012008-2). Living hepatic parenchymal cells were separated by 35% percoll. After seeding
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for 4-6 hours, cells were refreshed with medium and cultured overnight. The next day, cells
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were transduced with rAd-HBc/GFP or a control rAd vector (rAd-GFP) at a MOI of 20. After
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culture for two days, cells were treated with mTNF-α as described above.
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Apoptosis analysis
127
Apoptosis was assessed 14 hours after stimulation with TNF-α, using PE Annexin V Apoptosis
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Detection Kit I (BD, 559763) and PE Active Caspase-3 Apoptosis Kit (BD, 550914)
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respectively according to the manufacturer’s protocol. 7
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Immunoblot
131
Cell lysates were prepared in radio immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl,
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pH 7.4, 150 mM NaCl, 0.5% Triton X-100) containing 1×Protease Inhibitor Cocktail (Roche)
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and 1×PhosSTOP Phosphatase Inhibitor Cocktail (Roche). Proteins were separated by
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SDS-PAGE and transferred to PVDF membranes for immunoblotting. Antibodies were against
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phospho-JNK (Thr183/Tyr185), JNK and MKK7 from Cell Signaling Technology (9251, 9252
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and 4172). Anti-phospho-MKK7 (Thr275) was from Sigma (SAB4504624). Anti-RACK1 was
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from BD (610178). Anti-HBc was from Dako (B0586). Antibodies against cleaved caspase 3
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and poly ADP-ribose polymerase (PARP) were from Cell Signaling Technology (9661 and
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9542).
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Tandem affinity purification (TAP)
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HepG2 cells stably expressing HBc fused with C-terminal Strep and Flag tags were lysed in
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RIPA buffer containing 1× Protease Inhibitor Cocktail. TAP of Strep and Flag-tagged HBc was
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performed by two rounds of affinity purification as previously described (30). Elutes were
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analyzed by SDS-PAGE and mass spectrometry (Shanghai Applied Protein Technology).
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Co-immunoprecipitation (Co-IP) and GST Pull-down
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Cells were lysed in RIPA buffer containing 1× Protease Inhibitor Cocktail for 1 hour on ice
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with brief vortexing every 10 minutes. Lysates were incubated with 1 μg antibody at 4 ºC
148
overnight with gentle rotation. After centrifugation, supernatants were incubated with protein
149
A/G beads (Life Technologies) at 4 ºC for 1 hour. The immunoprecipitates were separated by
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SDS-PAGE for immunoblotting.
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GST-fusion proteins or His-tagged proteins were expressed in Escherichia coli BL21, and
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purified using glutathione-sepharose 4B (GE Healthcare) or nickel-affinity resins (Qiagen)
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according to the manufacturers’ instructions. For the pull-down assays, GST-fusion proteins
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were immobilized on glutathione beads and incubated with purified His-tagged proteins in 8
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RIPA buffer containing 0.5% BSA, at 4 ºC overnight with gentle rotation. Bound proteins were
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analyzed by SDS–PAGE and immunoblotting.
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Immunofluorescence assay
158
Cells were fixed with 4% paraformaldehyde in Phosphate buffered saline (PBS) at room
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temperature for 15 minutes followed by permeabilization with 0.1% Triton X-100. After
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blocking with 5% goat serum, cells were incubated with 1:250 anti-RACK1 (BD, 610178) and
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1:200 anti-HBc (Dako, B0586) in PBS containing 1% goat serum and 0.05% Triton X-100 at 4
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ºC overnight. Alexa Fluor 555 Goat Anti-Mouse antibody and Alexa Fluor 488 Goat
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Anti-Rabbit antibody (Life Technologies) were used as secondary antibodies for anti-HBc or
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anti-RACK1 staining respectively.
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Statistics
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Data are expressed as means ± standard error of the mean (SEM). Student’s unpaired t-tests
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were performed with GraphPad Prism software.
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9
Results
169 170
HBc increases TNF-α-induced apoptosis in hepatoma cells and primary hepatocytes
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TNF-α is implicated in hepatic injury from a variety of liver diseases. In the presence of the
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NF-κB inhibitor Bay11-7082 (Fig. S1), stimulation with TNF-α significantly increased the
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percentage of annexin V-binding HepG2.2.15 cells compared with HBV-free HepG2 cells.
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Treatment with lamivudine did not significantly alter annexin V-binding to HepG2.2.15 cells,
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suggesting that the viral replication had little effect on TNF-induced apoptosis (Fig. 2 A-B).
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Upon TNF-α/Bay11-7082 treatment, annexin V-binding increased in Huh-7 cells that were
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constitutively expressing HBc, but not in cells with expression of the other viral proteins (Fig.
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2C). Expression of all these viral proteins could be readily detected by specific antibodies
179
respectively (data not shown), although they were likely to be at different levels. Mutant assays
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on the HBV genome also revealed that HBc might be directly responsible for increased
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apoptosis in HBV-producing cells (Fig. 2D). In either HepG2 (Fig. 2E) or Huh-7 cells (data
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not shown), ectopic expression of HBc increased the production of active caspase 3, a typical
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marker of apoptosis.
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rAd vectors were constructed for transduction assays in primary mouse or human hepatocytes.
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In the presence of actinomycin D (27, 28), an inhibitor of de novo gene transcription, TNF-α
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treatment upregulated active caspase 3 and increased apoptosis in the hepatocytes transduced
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with rAd-HBc/GFP, compared with that in rAd-GFP transduced cells (Fig. 2F-G). Collectively,
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these data indicated that HBc promoted TNF-α-induced apoptosis in cultured hepatoma cells
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and primary hepatocytes.
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HBc downregulates TNF-α-induced MKK7 and JNK phosphorylation
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TNF-α is a conditional death ligand. Its apoptotic capability is largely modulated by the NF-κB
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and JNK signaling cascades (Fig. 1). HBc did not affect NF-κB signaling pathway activity as
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measured by reporter assays (Fig. 3A). However, TNF-α-induced phosphorylation of MKK7
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and downstream JNK activation were substantially suppressed in HepG2 (Fig. 3B) and Huh-7
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(Fig. 3C) cells that constitutively expressed HBc, compared to HBc-negative cells. TNF-α also
196
induced phosphorylation of MKK4 in Huh-7 cells, but this was not affected by HBc (Fig. 3C).
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The inhibitory effect of HBc on TNF-α-induced MKK7/JNK activation occurred also in the
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context of an intact HBV genome. TNF-α-stimulated MKK7/JNK activation was reduced in
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HepG2 cells expressing the 1.2mer overlength HBV genome, but not in cells with an HBc-null
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mutant genome (Fig. 3D). These data clearly indicated that expression of HBc blocked
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TNF-α-stimulated activation of MKK7/JNK.
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Pro-apoptotic activity of HBc is mediated by blockage of MKK7 phosphorylation
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TNF-α alone stimulated rapid, transient activation of JNK (within 1 hour, Fig. 3), and does not
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cause apoptosis (data not shown). In the presence of the NF-κB inhibitor, however, TNF-α
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stimulation induced prolonged phosphorylation of MKK7/JNK (Fig. 4A). This prolonged JNK
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activation is thought to be essential in regulating TNF-induced apoptosis, although its precise
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role in this process remains controversial (20, 22, 24-26). While the activation of MKK7/JNK
208
decreased from hours 8 to12, cleaved caspase 3 and PARP were detected as early as 4 hours
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after stimulation, indicating the onset of programmed cell death (Fig. 4A). Of particular
210
interests, the prolonged MKK7/JNK activation was remarkably suppressed (mainly limited to
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1-4 hours after stimulation) in HBc-expressing HepG2 cells that underwent significantly
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enhanced apoptosis (Fig. 4A, B).
213
We then tested whether inactivation of MKK7/JNK was directly responsible for
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TNF-α-induced apoptosis. This was achieved by knockdown of MKK7 expression through
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RNA interference. HepG2 cells transfected with MKK7-specific siRNAs had increased
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apoptosis upon stimulation with TNF-α (Fig. 4C). Similar results were obtained by knockdown
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of MKK7 expression in mouse primary hepatocytes (Fig. 4D). Collectively, these data
11
218
suggested that the pro-apoptotic activity of HBc was achieved by blocking MKK7 activation.
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RACK1 is a cellular protein that interacts with HBc
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To understand the mechanism of the pro-apoptotic activity of HBc, we performed tandem
221
affinity purification to identify potential cellular interacting protein(s). RACK1, a multifaceted
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scaffold protein, was identified as a candidate for HBc interaction by mass spectrometry. In
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293T cells, HA-tagged RACK1 co-immunoprecipitated with Flag-tagged HBc, and vise versa
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(Fig. 5A, B). In HepG2.2.15 cells that stably produce HBV, endogenous RACK1
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co-immunoprecipitated with HBc (Fig. 5C). Consistent with their physical interaction, both
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HBc and endogenous RACK1 were distributed throughout the cytoplasm of HepG2.2.15 cells
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(Fig. 5D).
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HBc has an N-terminal domain (aa1–149) and a C-terminal arginine-rich polypeptide
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(aa150–183) (31), while RACK1 contains 7 Trp-Asp (WD) repeats (32). Interaction between
230
HBc and RACK1 was further verified by GST pull-down assay. In particular, His-tagged
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RACK1 co-precipitated with the GST-fused HBc N-terminal domain, but not the HBc
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C-terminal polypeptide (Fig. 5E), while His-tagged HBc co-precipitated only with GST-fused
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RACK1 WD6-7, but not WD1-3 or WD1-5 (Fig. 5F, G). Neither GST-fused RACK1 WD6 nor
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WD7 pulled down His-tagged HBc (Fig. 5G). These data indicated that RACK1 bound the
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HBc N-terminal domain directly through the WD6-7 region, probably through the RACK1
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knob structure between WD6 and WD7 domains (32).
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HBc pro-apoptotic activity is independent of capsid particle formation
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HBc is a structural protein of HBV and assembles into icosahedral capsid particles. We thus
239
investigated if free monomeric or dimeric protein, or the capsid particle accounted for HBc
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pro-apoptotic activity.
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We used an HBc mutant with a tyrosine-to-alanine substitution at aa 132 (Y132A). This
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mutation prevents HBc from self-assembly into capsid particles but retains ability to form 12
243
homodimers (33, 34). As revealed by native gel electrophoresis assays, ectopically expressed
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wild-type HBc formed capsid particles that were absent in cells transfected with the HBc
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mutant (Fig. 6A). In contrast to previous reports (1, 2), TNF-α treatment, with or without
246
NF-κB inhibitor, did not significantly reduce capsid particles in the cytosol. This result
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probably reflected a difference between the genuine nucleocapsids and the empty capsids that
248
lack HBV DNA/RNA (35). Expression of HBc Y132A was detected by SDS-PAGE with
249
immunoblotting, albeit with a relatively lower level compared to that of wild-type HBc. In the
250
presence of TNF-α/Bay11-7082, expression of the assembly-defective HBc mutant displayed
251
similar cytotoxicity to wild-type HBc (Fig. 6B). In this regard, the HBc mutant engaged in a
252
direct interaction with RACK1 (Fig. 6C). These data clearly suggested that the pro-apoptotic
253
activity of HBc was independent of capsid formation.
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HBc and MKK7 bind RACK1 competitively
255
RACK1 is reported to interact with MKK7 through its WD6-7 region, which facilitates MKK7
256
phosphorylation by upstream MAP3Ks (19). This suggested that HBc and MKK7 might bind
257
RACK1 competitively.
258
RACK1/MKK7 interaction was confirmed in our study by a GST pull-down assay. His-tagged
259
RACK1 co-precipitated with the GST-fused MKK7, which however was largely blocked in a
260
dose-dependent manner by co-incubation with purified His-tagged HBc (Fig. 7A). In 293T
261
cells expressing Flag-tagged-MKK7 (MKK7-Flag), co-expression of HBc led to a significant
262
decrease in the amount of MKK7-Flag co-precipitated RACK1 (about 4-fold, Fig. 7B).
263
Moreover, in HepG2 cells with high endogeneous expression of MKK7, RACK1 and
264
transforming growth factor beta-activated kinase 1 (TAK1, an MAP3K that phosphorylates
265
MKK7 upon TNF-α stimulation) co-immunoprecipitated with MKK7. Those interactions were
266
barely detectable when HBc was expressed (Fig. 7C). The proteins RACK1, TAK1, MKK7
267
and JNK were all detected in PHH, but with lower levels than in HepG2 or Huh-7 cells (Fig.
13
268
S4). Our results therefore indicated that HBc disrupted interaction between MKK7 and
269
RACK1, which might abrogate TNF-α-mediated MKK7/JNK activation.
270
Pro-apoptotic activity of HBc is reversed by RACK1 overexpression
271
Competitively binding to RACK1 thus appeared to be the means by which HBc affected
272
TNF-α-induced apoptosis. This hypothesis was verified by overexpression of RACK1 to
273
counteract the effect of HBc. As shown in Fig. 8A, expression of HBc in HepG2 cells resulted
274
in reduced MKK7/JNK phosphorylation upon TNF-α stimulation, which however was reversed
275
by ectopic expression of HA-tagged RACK1 in these cells through lentiviral transduction.
276
Moreover, in the presence of NF-κB inhibitor Bay11-7082 (data not shown) or PDTC (Fig. 8B),
277
overexpression of RACK1 prevented the upregulation of TNF-α-induced apoptosis by HBc in
278
these cells. These data suggested that HBc suppressed TNF-α-mediated MKK7/JNK activation
279
and promoted apoptosis mainly through RACK1 blocking.
280
14
281
Discussion
282
TNF-α mediates both cell survival and cell death signaling through TNF receptor-1, by
283
activation of NF-κB and caspase-8 respectively (Fig. 1). Alteration in NF-κB signaling largely
284
accounts for the change in TNF-α function from anti-apoptotic to pro-apoptotic. However, the
285
underlying mechanism and the physiological relevance of the NF-κB signaling alteration are
286
poorly understood. Nevertheless, TNF-mediated apoptosis can be experimentally induced in
287
the setting of global transcriptional or translational arrest, or selective inactivation of NF-κB
288
(21, 22, 24). Our study took advantage of these experimental conditions, based on previous
289
studies by others, to investigate the potential role of HBc in TNF-α-induced apoptosis.
290
Bay11-7082 has been widely used as a tool compound that inhibits NF-κB activation. In our
291
study, NF-κB inhibitor PDTC was also used in order to avoid possible off-target effects of
292
Bay11-7082 (Fig. S2). However, both Bay11-7082 and PDTC are thought to be
293
multi-target-directed compounds, and the mechanisms whereby these compounds affect NF-κB
294
signal transduction are not well-understood.
295
The role of TNF-α must be carefully examined in different pathophysiological settings. For
296
example, TNF-α is implicated in HBV clearance via non-cytotoxic mechanisms in which
297
TNF-α-stimulated NF-κB activation is indispensable (1-3). On the other hand, we propose a
298
scenario for the cytotoxic function of TNF-α (8, 9) in which the NF-κB activation might be
299
coordinately inhibited. For example, multiple apoptotic stimuli including from TNF-α and
300
professional death ligands such as FasL and Apo2L/TRAIL, might function cooperatively
301
during severe hepatitis. The professional death ligands directly induce caspase 8 activation but
302
poorly activate NF-κB (21, 36). It is noted that the apical caspase activation is weak to induce
303
apoptotic death of “type II cells” such as hepatocytes, and requires amplification through
304
crosstalk to the TNF-triggered JNK activation pathway (21, 22, 24). In particular, active
15
305
caspase 8 suppresses TNF-triggered NF-κB signaling, thereby accelerating the programmed
306
cell death (21). We propose that this scenario might occur during severe or fulminant hepatitis,
307
in which HBV mutant-related HBc overexpression is likely to be important in enhancing
308
TNF-α-mediated apoptosis.
309
JNK is an important regulator of TNF-α signaling and a member of the MAP kinase subfamily.
310
Inhibition of de novo protein synthesis or NF-κB activation sensitizes hepatocytes to
311
TNF-α-induced hepatocyte death as a result of sustained JNK activation (22, 24). Initial JNK
312
activation is thought to be transient and associated with cell survival and proliferation through
313
AP-1. Sustained JNK activation and ROS accumulation are associated with apoptotic and
314
necrotic cell death (24). Interestingly, we found that the pro-apoptotic activity of HBc was
315
associated with significant suppression of JNK phosphorylation. Possible explanations could
316
be: i). the precise role of JNK in TNF-α-mediated hepatocyte apoptosis is controversial (24,
317
25). In a recent report, JNK-1 transduced an antiapoptotic signal in TNF-α-mediated
318
hepatocyte apoptosis through Mcl-1 stabilization (26). ii). HBc might not have completely
319
abolished the sustained activation of JNK. MKK7 and MKK4 are both upstream activators of
320
JNK. Phosphorylation of MKK7 but not MKK4 was suppressed during HBc co-expression.
321
However, MKK4 is reported to be only slightly activated by proinflammatory cytokines (37).
322
iii). another possibility is that inactivation of MKK7 itself might be directly pro-apoptotic by
323
an unknown mechanism independent of JNK signal transduction.
324
Direct evidence about MKK7 functions in TNF-α-induced hepatocyte death might come from
325
analysis of a mouse model with a specific genetic abrogation. However, mice lacking MKK7
326
or MKK4 die before birth (38). TAK1 is a direct activator of MKK7 that is critical for the
327
survival of hematopoietic cells and hepatocytes (39). Deletion of TAK1 results in bone marrow
328
and liver failure in mice, due to the massive apoptotic death of hematopoietic cells and
329
hepatocytes (39). Cultures of primary hepatocytes deficient in TAK1 exhibit spontaneous cell 16
330
death that is further increased in response to TNF-α (40). Similar to abrogation of MKK7
331
activation by HBc as described in our study, deletion of TAK1 in hepatocytes abolishes JNK
332
and NF-κB activation and confers susceptibility to TNF-α-mediated cell death.
333
Scaffolding of multicomponent regulatory systems is recognized as a major mechanism for
334
controlling signal transduction pathways (41). Specificity of regulation is achieved by
335
organization of MAPK modules, in part, through scaffolding and anchoring proteins. RACK1
336
is a widely expressed, highly conserved scaffolding protein of 7 WD repeat domains. It
337
interacts with a range of proteins and is involved in many cellular processes (32). A previous
338
study demonstrated that RACK1 is an adaptor for protein kinase C-mediated JNK activation
339
through direct binding to JNK1 and JNK2 (42). More recently, Guo and colleagues reported
340
that RACK1 engages directly with MKK7, enhancing the binding of MKK7 to its upstream
341
MAP3Ks and promoting MKK7 phosphorylation (19). The authors therefore proposed that
342
overexpression of RACK1 augments JNK activity, promoting growth of hepatocellular
343
carcinoma. Our study showed that HBc abrogated TNF-α-induced MKK7 phosphorylation by
344
competitively binding to RACK1. In-depth studies are needed to investigate whether
345
expression of HBc affects development of HBV-related hepatocellular carcinoma.
346
The pro-apoptotic activity of HBc might harm HBV infection. In this regard, induction of
347
apoptosis is an effective way to sacrifice virus-infected host cells and restrict pathogen spread
348
(43). Huang et al. recently found that virus-infected cells use the mitochondrial antiviral
349
signaling protein (MAVS)-MKK7-JNK2 signaling pathway to induce apoptosis as an innate
350
immune protection mechanism (44). HBc therefore could be a pathogen-associated molecular
351
pattern that sensitizes hepatocytes to TNF-α induced apoptosis. Supporting this possibility, Lin
352
et al. (33, 45) recently showed that HBc is a major viral factor for HBV clearance in a
353
hydrodynamics-based mouse model. This effect depends on the self-assembly of HBc into
354
capsid particles, which however is not obligatory for the pro-apoptotic activity of HBc 17
355
described in our present study.
356
HBV is believed to establish a stealthy infection without alerting significant host immunity. In
357
a chimpanzee model with acute viral hepatitis, the proportion of apoptotic and mitotic
358
hepatocytes is lower than 0.3% even at the viral clearance phase, although the turnover rate is
359
virtually accelerated (46). Nevertheless, it is the slow rate of cell destruction and long-term
360
persistent viral infection that cumulates fibrogenesis and cirrhosis in the liver. Apoptosis is
361
thought to be the nexus of liver injury and fibrosis (11). The pro-apoptotic activity of HBc
362
described in our study thus could be critical for understanding the pathogenesis of chronic
363
hepatitis B infection.
364
It will be interesting to discover whether HBc-related mutants of HBV have particular clinical
365
manifestations. Naturally occurring mutations in the HBV basic core promoter provide
366
evidence on this issue. A prevalent HBV variant is a double mutation of C-to-T at nucleotide
367
1768 and a T-to-A change at nucleotide 1770, resulting in highly upregulated core promoter
368
activity. This change is associated with a clinical outbreak of fatal fulminant hepatitis. In
369
particular, this HBV strain induces substantial apoptosis in primary tupaia hepatocytes by a
370
mechanism potentially independent of enhanced replication (15), possibly the strongly
371
upregulated expression of pro-apoptotic HBc.
372
In conclusion, our study found that HBc protein sensitizes hepatocytes to TNF-α-induced
373
apoptosis by competing with MKK7 for binding to RACK1, abrogating MKK7
374
phosphorylation. The study is thus a first indication of that HBc is pathogenic in liver injury
375
during hepatitis B.
376
18
377
Acknowledgement
378
This work was supported by grants from the National Science and Technology Major Projects
379
(2012ZX10002007), Natural Science Foundation (81171566), and National Key Basic
380
Research Program of China (2012CB519000). We also gratefully acknowledge the support of
381
SA-SIBS Scholarship Program.
382
19
Figure Legends
383
384
FIG. 1. Schema of TNF-α signaling pathway that induces apoptosis
385
TNF-α is capable of inducing apoptosis when NF-κB signaling is blocked (20-24). Upon
386
binding to TNF-receptor I, a protein Complex I forms rapidly that activates NF-κB and JNK
387
signaling pathways. Complex I undergoes ligand-dissociated internalization with formation of
388
Complex II. Complex II is composed of FADD and procaspases 8, and triggers apoptosis if
389
NF-κB signaling fails to induce expression of anti-apoptotic genes. JNK is an important
390
regulator of TNF-α-mediated apoptosis (20, 22, 24-26), and is activated via the sequential
391
activation of protein kinases including two MAP kinase kinases (MKK4 and MKK7) and
392
multiple MAP kinase kinase kinases (MAP3Ks). MKK7 is an essential component of the JNK
393
signaling pathway activated by TNF-α. RACK1 is a scaffold protein that facilitates
394
phosphorylation of MKK7 by upstream MAP3Ks (19). It is widely reported that sustained JNK
395
activation mediates TNF-α-induced cell death, although in hepatocytes JNK has both
396
pro-apoptotic and anti-apoptotic aspects (22, 24). We propose that HBc (red) disrupts
397
interactions between MKK7 and RACK1, rendering cells susceptible to TNF-α-mediated
398
apoptosis.
399
FIG. 2. HBc confers susceptibility to TNF-α-induced apoptosis in hepatoma cells and
400
primary hepatocytes
401
(A) Left, annexin V staining of HepG2 or HepG2.2.15 cells 14 hours after co-treatment with
402
TNF-α and Bay11-7082. Cells treated with Bay11-7082 alone, or mock treated, were used as
403
controls. To block HBV replication, HepG2.2.15 cells were pretreated with 20 g/mL
404
Lamivudine (LAM) for 48 hours. Right, representative flow cytometry analysis of cells treated
405
with TNF-α/Bay11-7082. Cells were double-stained with 7-amino actinomycin D (7-AAD) and
406
annexin V. Annexin V positive populations were calculated. (B) Upper, Southern blot of HBV 20
407
replication in HepG2, HepG2.2.15 and LAM-treated HepG2.2.15 cells. Replication
408
intermediates of HBV DNA are RC, relaxed circular; DSL, double-stranded linear; SS, single
409
strand; Lower, HBc levels detected by immunoblotting. (C) Annexin V staining of Huh-7 cells
410
stably transfected with a control plasmid or the plasmid encoding HBc, polymerase (Pol), X
411
protein (HBx), or S2S proteins of HBV respectively. Cells were treated with
412
TNF-α/Bay11-7082 as described in A. (D) Annexin V staining of HepG2 cells stably
413
transfected with a 1.2mer genome of HBV (payw1.2), or mutant HBV genome deficient in
414
HBe and HBc (payw1.2-e/C-null). Mock-transfected cells were used as controls. (E) Left, flow
415
cytometry of active caspase 3 expression after TNF-α/Bay11-7082 treatment in HepG2 cells
416
stably transfected with a control plasmid, or the plasmid encoding HBc (pcDNA3.1-HBc).
417
Right, representative flow cytometry analysis of cells treated with TNF-α/Bay11-7082.
418
Co-treatment with TNF-α and the NF-κB inhibitor PDTC was also tested, with similar results
419
(Fig. S2). (F-G) PMHs (F) or PHHs (G) transduced with rAd-HBc/GFP, or rAd-GFP as control,
420
for 48 hours at a MOI of 20:1. Cells were then treated with mouse or human-derived TNF-α
421
and actinomycin D (ActD), with active caspase 3 expression analyzed by flow cytometry. The
422
error bars indicate SEM. *, P
1
Hepatitis B core protein sensitizes hepatocytes to tumor necrosis factor-induced apoptosis
2
by suppression of the phosphorylation of mitogen-activated protein kinase kinase 7
3
Baosen Jia,a Minggao Guo,b Gaiyun Li,a Demin Yu,c Xinxin Zhang,c Ke Lan,a# Qiang Denga#
4
Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese
5
Academy of Sciences, Shanghai, China a
6
Department of General Surgery, Sixth People’s Hospital, Shanghai Jiaotong University School
7
of Medicine, Shanghai, China b
8
Department of Infectious Diseases, Ruijin Hospital, Shanghai Jiaotong University School of
9
Medicine, Shanghai, China d
10
#Address correspondence to Dr. Qiang Deng, E-mail: [email protected]; or Dr. Ke Lan,
11
E-Mail, [email protected]
12
Running Title:
13
HBc is pro-apoptotic by blocking MKK7
14
1
15
Abstract
16
Hepatitis B, which caused by Hepatitis B virus (HBV) infection, remains a major health threat
17
worldwide. Hepatic injury and regeneration from chronic inflammation is the main driving
18
factor of liver fibrosis and cirrhosis in chronic hepatitis B. Pro-inflammatory tumor necrosis
19
factor (TNF)-α has been implicated as a major inducer of liver cell death during viral hepatitis.
20
Here, we report that in hepatoma cell lines and in primary mouse and human hepatocytes,
21
expression of hepatitis B core (HBc) protein made cells susceptible to TNF-α-induced
22
apoptosis. We found that receptor of activated protein kinase C (RACK) 1 interacted with HBc
23
by tandem affinity purification and mass spectrometry. RACK1 was recently reported as a
24
scaffold protein that facilitates the phosphorylation of mitogen-activated protein kinase kinase
25
7 (MKK7) by its upstream activators. Our study showed that HBc abrogated the interaction
26
between MKK7 and RACK1 by competitively binding to RACK1, thereby downregulating
27
TNF-α-induced phosphorylation of MKK7 and the activation of c-Jun N-terminal kinase. In
28
line with this finding, specific knockdown of MKK7 increased the sensitivity of hepatocytes to
29
TNF-α-induced apoptosis, while overexpression of RACK1 counteracted the pro-apoptotic
30
activity of HBc. Capsid particle formation was not obligatory for HBc pro-apoptotic activity, as
31
analyzed using an assembly-defective HBc mutant. In conclusion, the expression of HBc
32
sensitized hepatocytes to TNF-α-induced apoptosis by disrupting the interaction between
33
MKK7 and RACK1. Our study is thus a first indication of the pathogenic effects of HBc in
34
liver injury during hepatitis B.
35
Importance:
36
Our study revealed a previously unappreciated role of HBc in TNF-α-mediated apoptosis. The
37
pro-apoptotic activity of HBc is important for understanding hepatitis B pathogenesis. In
38
particular, severe hepatitis-associated HBV variants may upregulate apoptosis of hepatocytes 2
39
through enhanced HBc expression. Our study also found that MKK7 is centrally involved in
40
TNF-α-induced hepatocyte apoptosis and revealed a multifaceted role for JNK signaling in this
41
process.
42
Key words:
43
Hepatitis B core protein; apoptosis; TNF-α; MKK7; RACK1
44
3
45
Introduction
46
Hepatitis B virus (HBV) infection remains a major public health threat affecting an estimated
47
400 million individuals worldwide, with high risk of developing severe liver diseases,
48
including cirrhosis and hepatocellular carcinoma. The host protective immune response is also
49
responsible for liver pathogenesis during HBV infection.
50
Pro-inflammatory tumor necrosis factor (TNF)-α is critical for controlling HBV in clinical
51
settings and in model systems, possibly by destabilizing cytoplasmic viral nucleocapids and
52
reducing nuclear viral DNA (1, 2). Recently, TNF-α was reported to reduce host cell
53
susceptibility to HBV entry via activation-induced cytidine deaminase (3). On the other hand,
54
TNF-α has also been implicated as a mechanism of hepatic injury through induction of cellular
55
apoptosis during viral hepatitis (4-9). Apoptosis is organized, genetically controlled
56
programmed cell death. Clearance of apoptotic debris stimulates transforming growth factor-β
57
expression and induces collagen I secretion by hepatic stellate cells. These processes have been
58
suggested as the core machinery for liver fibrogenesis and cirrhosis (10, 11).
59
Overexpressing HBV core (HBc), precore, X, small and middle envelope proteins (S2S), as
60
well as the viral replication, is not associated with liver disease in transgenic mouse models
61
(12). Hence HBV itself is not considered to be directly cytopathic for infected hepatocytes.
62
This notion has to be re-examined, however, in a pathophysiological setting. Of particular
63
interests, naturally occurring mutations within the basic core promoter of HBV are implicated
64
in severe liver diseases including fulminant hepatitis, which results in up to 15-fold increased
65
HBc synthesis (13-17). The fulminant hepatitis-associated mutant induces apoptosis in primary
66
hepatocytes and is not reversed by inhibition with nucleoside analogs (15). This finding
67
suggests that HBc might be directly responsible for virus-induced hepatocyte death, but the
68
underlying mechanism remains undefined.
4
69
HBc is a 21 kDa viral structural protein with an N-terminal domain that is responsible for viral
70
capsid assembly and a C-terminal arginine-rich polypeptide that interacts with HBV
71
pregenomic RNA. HBc is found to be distributed in both the nucleus and the cytoplasm in
72
HBV-producing hepatocytes and transgenic mice. Clinically, a nucleus-dominant distribution
73
of HBc is associated with minor hepatitis activity while a cytoplasmic distribution of HBc is
74
associated with chronic active liver disease (18). In the present study, we report that expression
75
of HBc increased susceptibility of hepatocytes to TNF-α-induced apoptosis by disrupting the
76
interaction between receptor for activated c kinase 1 (RACK1) and mitogen-activated protein
77
kinase kinase 7 (MKK7) (19), leading to downregulation of TNF-α-induced phosphorylation of
78
MKK7 and c-Jun-N-terminal kinase (JNK) (Fig. 1). Our results therefore revealed a previously
79
unappreciated role for HBc in liver pathogenesis during viral hepatitis.
80
5
Materials and Methods
81 82
Plasmid DNA and viral vectors
83
Plasmids encoding HBV Polymerase, X protein, S2S, and HBc, as well as HA-tagged RACK1
84
and Flag-tagged MKK7, were constructed by inserting the appropriate genes into the multiple
85
cloning site (MCS) of pcDNA3.1(-) (Invitrogen). Genes encoding His-tagged RACK1 or
86
His-tagged HBc were subcloned into the MCS of pET-30a(+) (Novagen) for prokaryotic
87
protein expression. For GST pull-down assay, plasmids encoding GST-tagged HBc
88
truncations and GST-tagged RACK1 truncations were constructed by inserting the appropriate
89
genes into the MCS of pGEX-4T-1 (Promega).
90
Recombinant adenovirus (rAd) expressing HBc (rAd-HBc/GFP, with a GFP gene separated by
91
an IRES sequence) was constructed by Obio Technology (Shanghai), using E1-deleted human
92
rAd vector of serotype 5. The lentiviral vector expressing HA-tagged RACK1 was constructed
93
by inserting the sequence into the MCS of pCDH-CMV-MCS-EF1-Puro (SBI) and packaging
94
the expression constructs into pseudoviral particles.
95
siRNAs
96
siRNA oligos against human and mouse MKK7 were synthesized by GenePharma: siMKK7-1:
97
5’-CAGGACAGUUUCCCUACAATT-3’, siMKK7-2: 5’-GCAAGAUGACAGUGGCGAUTT
98
-3’. siRNAs were transfected into cells by Lipofectamine 2000 (Invitrogen). Subsequent
99
treatments were performed 48 hours after transfection.
100
Cells
101
Huh-7, HepG2, HepG2.2.15 and 293T cells were cultured in DMEM containing 10% fetal
102
bovine serum (FBS) and 1% Penicillin-Streptomycin. Primary mouse hepatocytes (PMH) were
103
isolated from male C57BL/6 mice as described below. Primary human hepatocytes (PHH) were
104
from Mucyte Biotech (Nanjing). PMH and PHH were cultured in Williams’ Media E
6
105
containing 10% FBS, 2 mM L-glutamine, 1× nonessential amino acids, and 1%
106
Penicillin-Streptomycin. HepG2 or Huh-7 cells constitutively expressing HBc were constructed
107
by transfecting cells with pcDNA3.1-HBc and selecting with 50 μg/mL Geneticin.
108
Stimulation with TNF-α
109
To induce massive apoptosis, Huh-7 or HepG2 cells were plated into 12-well plates at 0.25-0.5
110
million cells per well. The next day, cells were pretreated with 0.5 μg/mL actinomycin D
111
(Sigma) for 30 minutes and cultured for 14 hours with 100 ng/mL hTNF-α (PeproTech) in the
112
presence of 12 μM (for Huh-7 cells) or 20 μM (for HepG2 cells) of Bay11-7082 (Sigma). For
113
co-treatment with pyrrolidine dithiocarbamic acid (PDTC, Beyotime), 50 μM of PDTC and 10
114
ng/mL hTNF-α were used (for HepG2 cells).
115
The stimulation with TNF-α in PMH and PHH was described previously (27, 28). In brief,
116
cells were pretreated with 0.5 μg/mL actinomycin D for 30 minutes, and cultured with 2 ng/mL
117
of mTNF-α (PeproTech) (for PMH) or 100 ng/ml hTNF-α (for PHH) in the presence of 0.5
118
μg/mL actinomycin D for 14 hours.
119
Isolation of PMH
120
PMH was isolated from C57BL/6 male mice by two step collagenase perfusion technique (29).
121
The study was approved by the Animal Ethics Committee of Institut Pasteur of Shanghai (no.
122
A2012008-2). Living hepatic parenchymal cells were separated by 35% percoll. After seeding
123
for 4-6 hours, cells were refreshed with medium and cultured overnight. The next day, cells
124
were transduced with rAd-HBc/GFP or a control rAd vector (rAd-GFP) at a MOI of 20. After
125
culture for two days, cells were treated with mTNF-α as described above.
126
Apoptosis analysis
127
Apoptosis was assessed 14 hours after stimulation with TNF-α, using PE Annexin V Apoptosis
128
Detection Kit I (BD, 559763) and PE Active Caspase-3 Apoptosis Kit (BD, 550914)
129
respectively according to the manufacturer’s protocol. 7
130
Immunoblot
131
Cell lysates were prepared in radio immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl,
132
pH 7.4, 150 mM NaCl, 0.5% Triton X-100) containing 1×Protease Inhibitor Cocktail (Roche)
133
and 1×PhosSTOP Phosphatase Inhibitor Cocktail (Roche). Proteins were separated by
134
SDS-PAGE and transferred to PVDF membranes for immunoblotting. Antibodies were against
135
phospho-JNK (Thr183/Tyr185), JNK and MKK7 from Cell Signaling Technology (9251, 9252
136
and 4172). Anti-phospho-MKK7 (Thr275) was from Sigma (SAB4504624). Anti-RACK1 was
137
from BD (610178). Anti-HBc was from Dako (B0586). Antibodies against cleaved caspase 3
138
and poly ADP-ribose polymerase (PARP) were from Cell Signaling Technology (9661 and
139
9542).
140
Tandem affinity purification (TAP)
141
HepG2 cells stably expressing HBc fused with C-terminal Strep and Flag tags were lysed in
142
RIPA buffer containing 1× Protease Inhibitor Cocktail. TAP of Strep and Flag-tagged HBc was
143
performed by two rounds of affinity purification as previously described (30). Elutes were
144
analyzed by SDS-PAGE and mass spectrometry (Shanghai Applied Protein Technology).
145
Co-immunoprecipitation (Co-IP) and GST Pull-down
146
Cells were lysed in RIPA buffer containing 1× Protease Inhibitor Cocktail for 1 hour on ice
147
with brief vortexing every 10 minutes. Lysates were incubated with 1 μg antibody at 4 ºC
148
overnight with gentle rotation. After centrifugation, supernatants were incubated with protein
149
A/G beads (Life Technologies) at 4 ºC for 1 hour. The immunoprecipitates were separated by
150
SDS-PAGE for immunoblotting.
151
GST-fusion proteins or His-tagged proteins were expressed in Escherichia coli BL21, and
152
purified using glutathione-sepharose 4B (GE Healthcare) or nickel-affinity resins (Qiagen)
153
according to the manufacturers’ instructions. For the pull-down assays, GST-fusion proteins
154
were immobilized on glutathione beads and incubated with purified His-tagged proteins in 8
155
RIPA buffer containing 0.5% BSA, at 4 ºC overnight with gentle rotation. Bound proteins were
156
analyzed by SDS–PAGE and immunoblotting.
157
Immunofluorescence assay
158
Cells were fixed with 4% paraformaldehyde in Phosphate buffered saline (PBS) at room
159
temperature for 15 minutes followed by permeabilization with 0.1% Triton X-100. After
160
blocking with 5% goat serum, cells were incubated with 1:250 anti-RACK1 (BD, 610178) and
161
1:200 anti-HBc (Dako, B0586) in PBS containing 1% goat serum and 0.05% Triton X-100 at 4
162
ºC overnight. Alexa Fluor 555 Goat Anti-Mouse antibody and Alexa Fluor 488 Goat
163
Anti-Rabbit antibody (Life Technologies) were used as secondary antibodies for anti-HBc or
164
anti-RACK1 staining respectively.
165
Statistics
166
Data are expressed as means ± standard error of the mean (SEM). Student’s unpaired t-tests
167
were performed with GraphPad Prism software.
168
9
Results
169 170
HBc increases TNF-α-induced apoptosis in hepatoma cells and primary hepatocytes
171
TNF-α is implicated in hepatic injury from a variety of liver diseases. In the presence of the
172
NF-κB inhibitor Bay11-7082 (Fig. S1), stimulation with TNF-α significantly increased the
173
percentage of annexin V-binding HepG2.2.15 cells compared with HBV-free HepG2 cells.
174
Treatment with lamivudine did not significantly alter annexin V-binding to HepG2.2.15 cells,
175
suggesting that the viral replication had little effect on TNF-induced apoptosis (Fig. 2 A-B).
176
Upon TNF-α/Bay11-7082 treatment, annexin V-binding increased in Huh-7 cells that were
177
constitutively expressing HBc, but not in cells with expression of the other viral proteins (Fig.
178
2C). Expression of all these viral proteins could be readily detected by specific antibodies
179
respectively (data not shown), although they were likely to be at different levels. Mutant assays
180
on the HBV genome also revealed that HBc might be directly responsible for increased
181
apoptosis in HBV-producing cells (Fig. 2D). In either HepG2 (Fig. 2E) or Huh-7 cells (data
182
not shown), ectopic expression of HBc increased the production of active caspase 3, a typical
183
marker of apoptosis.
184
rAd vectors were constructed for transduction assays in primary mouse or human hepatocytes.
185
In the presence of actinomycin D (27, 28), an inhibitor of de novo gene transcription, TNF-α
186
treatment upregulated active caspase 3 and increased apoptosis in the hepatocytes transduced
187
with rAd-HBc/GFP, compared with that in rAd-GFP transduced cells (Fig. 2F-G). Collectively,
188
these data indicated that HBc promoted TNF-α-induced apoptosis in cultured hepatoma cells
189
and primary hepatocytes.
190
HBc downregulates TNF-α-induced MKK7 and JNK phosphorylation
191
TNF-α is a conditional death ligand. Its apoptotic capability is largely modulated by the NF-κB
192
and JNK signaling cascades (Fig. 1). HBc did not affect NF-κB signaling pathway activity as
10
193
measured by reporter assays (Fig. 3A). However, TNF-α-induced phosphorylation of MKK7
194
and downstream JNK activation were substantially suppressed in HepG2 (Fig. 3B) and Huh-7
195
(Fig. 3C) cells that constitutively expressed HBc, compared to HBc-negative cells. TNF-α also
196
induced phosphorylation of MKK4 in Huh-7 cells, but this was not affected by HBc (Fig. 3C).
197
The inhibitory effect of HBc on TNF-α-induced MKK7/JNK activation occurred also in the
198
context of an intact HBV genome. TNF-α-stimulated MKK7/JNK activation was reduced in
199
HepG2 cells expressing the 1.2mer overlength HBV genome, but not in cells with an HBc-null
200
mutant genome (Fig. 3D). These data clearly indicated that expression of HBc blocked
201
TNF-α-stimulated activation of MKK7/JNK.
202
Pro-apoptotic activity of HBc is mediated by blockage of MKK7 phosphorylation
203
TNF-α alone stimulated rapid, transient activation of JNK (within 1 hour, Fig. 3), and does not
204
cause apoptosis (data not shown). In the presence of the NF-κB inhibitor, however, TNF-α
205
stimulation induced prolonged phosphorylation of MKK7/JNK (Fig. 4A). This prolonged JNK
206
activation is thought to be essential in regulating TNF-induced apoptosis, although its precise
207
role in this process remains controversial (20, 22, 24-26). While the activation of MKK7/JNK
208
decreased from hours 8 to12, cleaved caspase 3 and PARP were detected as early as 4 hours
209
after stimulation, indicating the onset of programmed cell death (Fig. 4A). Of particular
210
interests, the prolonged MKK7/JNK activation was remarkably suppressed (mainly limited to
211
1-4 hours after stimulation) in HBc-expressing HepG2 cells that underwent significantly
212
enhanced apoptosis (Fig. 4A, B).
213
We then tested whether inactivation of MKK7/JNK was directly responsible for
214
TNF-α-induced apoptosis. This was achieved by knockdown of MKK7 expression through
215
RNA interference. HepG2 cells transfected with MKK7-specific siRNAs had increased
216
apoptosis upon stimulation with TNF-α (Fig. 4C). Similar results were obtained by knockdown
217
of MKK7 expression in mouse primary hepatocytes (Fig. 4D). Collectively, these data
11
218
suggested that the pro-apoptotic activity of HBc was achieved by blocking MKK7 activation.
219
RACK1 is a cellular protein that interacts with HBc
220
To understand the mechanism of the pro-apoptotic activity of HBc, we performed tandem
221
affinity purification to identify potential cellular interacting protein(s). RACK1, a multifaceted
222
scaffold protein, was identified as a candidate for HBc interaction by mass spectrometry. In
223
293T cells, HA-tagged RACK1 co-immunoprecipitated with Flag-tagged HBc, and vise versa
224
(Fig. 5A, B). In HepG2.2.15 cells that stably produce HBV, endogenous RACK1
225
co-immunoprecipitated with HBc (Fig. 5C). Consistent with their physical interaction, both
226
HBc and endogenous RACK1 were distributed throughout the cytoplasm of HepG2.2.15 cells
227
(Fig. 5D).
228
HBc has an N-terminal domain (aa1–149) and a C-terminal arginine-rich polypeptide
229
(aa150–183) (31), while RACK1 contains 7 Trp-Asp (WD) repeats (32). Interaction between
230
HBc and RACK1 was further verified by GST pull-down assay. In particular, His-tagged
231
RACK1 co-precipitated with the GST-fused HBc N-terminal domain, but not the HBc
232
C-terminal polypeptide (Fig. 5E), while His-tagged HBc co-precipitated only with GST-fused
233
RACK1 WD6-7, but not WD1-3 or WD1-5 (Fig. 5F, G). Neither GST-fused RACK1 WD6 nor
234
WD7 pulled down His-tagged HBc (Fig. 5G). These data indicated that RACK1 bound the
235
HBc N-terminal domain directly through the WD6-7 region, probably through the RACK1
236
knob structure between WD6 and WD7 domains (32).
237
HBc pro-apoptotic activity is independent of capsid particle formation
238
HBc is a structural protein of HBV and assembles into icosahedral capsid particles. We thus
239
investigated if free monomeric or dimeric protein, or the capsid particle accounted for HBc
240
pro-apoptotic activity.
241
We used an HBc mutant with a tyrosine-to-alanine substitution at aa 132 (Y132A). This
242
mutation prevents HBc from self-assembly into capsid particles but retains ability to form 12
243
homodimers (33, 34). As revealed by native gel electrophoresis assays, ectopically expressed
244
wild-type HBc formed capsid particles that were absent in cells transfected with the HBc
245
mutant (Fig. 6A). In contrast to previous reports (1, 2), TNF-α treatment, with or without
246
NF-κB inhibitor, did not significantly reduce capsid particles in the cytosol. This result
247
probably reflected a difference between the genuine nucleocapsids and the empty capsids that
248
lack HBV DNA/RNA (35). Expression of HBc Y132A was detected by SDS-PAGE with
249
immunoblotting, albeit with a relatively lower level compared to that of wild-type HBc. In the
250
presence of TNF-α/Bay11-7082, expression of the assembly-defective HBc mutant displayed
251
similar cytotoxicity to wild-type HBc (Fig. 6B). In this regard, the HBc mutant engaged in a
252
direct interaction with RACK1 (Fig. 6C). These data clearly suggested that the pro-apoptotic
253
activity of HBc was independent of capsid formation.
254
HBc and MKK7 bind RACK1 competitively
255
RACK1 is reported to interact with MKK7 through its WD6-7 region, which facilitates MKK7
256
phosphorylation by upstream MAP3Ks (19). This suggested that HBc and MKK7 might bind
257
RACK1 competitively.
258
RACK1/MKK7 interaction was confirmed in our study by a GST pull-down assay. His-tagged
259
RACK1 co-precipitated with the GST-fused MKK7, which however was largely blocked in a
260
dose-dependent manner by co-incubation with purified His-tagged HBc (Fig. 7A). In 293T
261
cells expressing Flag-tagged-MKK7 (MKK7-Flag), co-expression of HBc led to a significant
262
decrease in the amount of MKK7-Flag co-precipitated RACK1 (about 4-fold, Fig. 7B).
263
Moreover, in HepG2 cells with high endogeneous expression of MKK7, RACK1 and
264
transforming growth factor beta-activated kinase 1 (TAK1, an MAP3K that phosphorylates
265
MKK7 upon TNF-α stimulation) co-immunoprecipitated with MKK7. Those interactions were
266
barely detectable when HBc was expressed (Fig. 7C). The proteins RACK1, TAK1, MKK7
267
and JNK were all detected in PHH, but with lower levels than in HepG2 or Huh-7 cells (Fig.
13
268
S4). Our results therefore indicated that HBc disrupted interaction between MKK7 and
269
RACK1, which might abrogate TNF-α-mediated MKK7/JNK activation.
270
Pro-apoptotic activity of HBc is reversed by RACK1 overexpression
271
Competitively binding to RACK1 thus appeared to be the means by which HBc affected
272
TNF-α-induced apoptosis. This hypothesis was verified by overexpression of RACK1 to
273
counteract the effect of HBc. As shown in Fig. 8A, expression of HBc in HepG2 cells resulted
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in reduced MKK7/JNK phosphorylation upon TNF-α stimulation, which however was reversed
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by ectopic expression of HA-tagged RACK1 in these cells through lentiviral transduction.
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Moreover, in the presence of NF-κB inhibitor Bay11-7082 (data not shown) or PDTC (Fig. 8B),
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overexpression of RACK1 prevented the upregulation of TNF-α-induced apoptosis by HBc in
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these cells. These data suggested that HBc suppressed TNF-α-mediated MKK7/JNK activation
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and promoted apoptosis mainly through RACK1 blocking.
280
14
281
Discussion
282
TNF-α mediates both cell survival and cell death signaling through TNF receptor-1, by
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activation of NF-κB and caspase-8 respectively (Fig. 1). Alteration in NF-κB signaling largely
284
accounts for the change in TNF-α function from anti-apoptotic to pro-apoptotic. However, the
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underlying mechanism and the physiological relevance of the NF-κB signaling alteration are
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poorly understood. Nevertheless, TNF-mediated apoptosis can be experimentally induced in
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the setting of global transcriptional or translational arrest, or selective inactivation of NF-κB
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(21, 22, 24). Our study took advantage of these experimental conditions, based on previous
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studies by others, to investigate the potential role of HBc in TNF-α-induced apoptosis.
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Bay11-7082 has been widely used as a tool compound that inhibits NF-κB activation. In our
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study, NF-κB inhibitor PDTC was also used in order to avoid possible off-target effects of
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Bay11-7082 (Fig. S2). However, both Bay11-7082 and PDTC are thought to be
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multi-target-directed compounds, and the mechanisms whereby these compounds affect NF-κB
294
signal transduction are not well-understood.
295
The role of TNF-α must be carefully examined in different pathophysiological settings. For
296
example, TNF-α is implicated in HBV clearance via non-cytotoxic mechanisms in which
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TNF-α-stimulated NF-κB activation is indispensable (1-3). On the other hand, we propose a
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scenario for the cytotoxic function of TNF-α (8, 9) in which the NF-κB activation might be
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coordinately inhibited. For example, multiple apoptotic stimuli including from TNF-α and
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professional death ligands such as FasL and Apo2L/TRAIL, might function cooperatively
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during severe hepatitis. The professional death ligands directly induce caspase 8 activation but
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poorly activate NF-κB (21, 36). It is noted that the apical caspase activation is weak to induce
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apoptotic death of “type II cells” such as hepatocytes, and requires amplification through
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crosstalk to the TNF-triggered JNK activation pathway (21, 22, 24). In particular, active
15
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caspase 8 suppresses TNF-triggered NF-κB signaling, thereby accelerating the programmed
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cell death (21). We propose that this scenario might occur during severe or fulminant hepatitis,
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in which HBV mutant-related HBc overexpression is likely to be important in enhancing
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TNF-α-mediated apoptosis.
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JNK is an important regulator of TNF-α signaling and a member of the MAP kinase subfamily.
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Inhibition of de novo protein synthesis or NF-κB activation sensitizes hepatocytes to
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TNF-α-induced hepatocyte death as a result of sustained JNK activation (22, 24). Initial JNK
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activation is thought to be transient and associated with cell survival and proliferation through
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AP-1. Sustained JNK activation and ROS accumulation are associated with apoptotic and
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necrotic cell death (24). Interestingly, we found that the pro-apoptotic activity of HBc was
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associated with significant suppression of JNK phosphorylation. Possible explanations could
316
be: i). the precise role of JNK in TNF-α-mediated hepatocyte apoptosis is controversial (24,
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25). In a recent report, JNK-1 transduced an antiapoptotic signal in TNF-α-mediated
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hepatocyte apoptosis through Mcl-1 stabilization (26). ii). HBc might not have completely
319
abolished the sustained activation of JNK. MKK7 and MKK4 are both upstream activators of
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JNK. Phosphorylation of MKK7 but not MKK4 was suppressed during HBc co-expression.
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However, MKK4 is reported to be only slightly activated by proinflammatory cytokines (37).
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iii). another possibility is that inactivation of MKK7 itself might be directly pro-apoptotic by
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an unknown mechanism independent of JNK signal transduction.
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Direct evidence about MKK7 functions in TNF-α-induced hepatocyte death might come from
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analysis of a mouse model with a specific genetic abrogation. However, mice lacking MKK7
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or MKK4 die before birth (38). TAK1 is a direct activator of MKK7 that is critical for the
327
survival of hematopoietic cells and hepatocytes (39). Deletion of TAK1 results in bone marrow
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and liver failure in mice, due to the massive apoptotic death of hematopoietic cells and
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hepatocytes (39). Cultures of primary hepatocytes deficient in TAK1 exhibit spontaneous cell 16
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death that is further increased in response to TNF-α (40). Similar to abrogation of MKK7
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activation by HBc as described in our study, deletion of TAK1 in hepatocytes abolishes JNK
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and NF-κB activation and confers susceptibility to TNF-α-mediated cell death.
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Scaffolding of multicomponent regulatory systems is recognized as a major mechanism for
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controlling signal transduction pathways (41). Specificity of regulation is achieved by
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organization of MAPK modules, in part, through scaffolding and anchoring proteins. RACK1
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is a widely expressed, highly conserved scaffolding protein of 7 WD repeat domains. It
337
interacts with a range of proteins and is involved in many cellular processes (32). A previous
338
study demonstrated that RACK1 is an adaptor for protein kinase C-mediated JNK activation
339
through direct binding to JNK1 and JNK2 (42). More recently, Guo and colleagues reported
340
that RACK1 engages directly with MKK7, enhancing the binding of MKK7 to its upstream
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MAP3Ks and promoting MKK7 phosphorylation (19). The authors therefore proposed that
342
overexpression of RACK1 augments JNK activity, promoting growth of hepatocellular
343
carcinoma. Our study showed that HBc abrogated TNF-α-induced MKK7 phosphorylation by
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competitively binding to RACK1. In-depth studies are needed to investigate whether
345
expression of HBc affects development of HBV-related hepatocellular carcinoma.
346
The pro-apoptotic activity of HBc might harm HBV infection. In this regard, induction of
347
apoptosis is an effective way to sacrifice virus-infected host cells and restrict pathogen spread
348
(43). Huang et al. recently found that virus-infected cells use the mitochondrial antiviral
349
signaling protein (MAVS)-MKK7-JNK2 signaling pathway to induce apoptosis as an innate
350
immune protection mechanism (44). HBc therefore could be a pathogen-associated molecular
351
pattern that sensitizes hepatocytes to TNF-α induced apoptosis. Supporting this possibility, Lin
352
et al. (33, 45) recently showed that HBc is a major viral factor for HBV clearance in a
353
hydrodynamics-based mouse model. This effect depends on the self-assembly of HBc into
354
capsid particles, which however is not obligatory for the pro-apoptotic activity of HBc 17
355
described in our present study.
356
HBV is believed to establish a stealthy infection without alerting significant host immunity. In
357
a chimpanzee model with acute viral hepatitis, the proportion of apoptotic and mitotic
358
hepatocytes is lower than 0.3% even at the viral clearance phase, although the turnover rate is
359
virtually accelerated (46). Nevertheless, it is the slow rate of cell destruction and long-term
360
persistent viral infection that cumulates fibrogenesis and cirrhosis in the liver. Apoptosis is
361
thought to be the nexus of liver injury and fibrosis (11). The pro-apoptotic activity of HBc
362
described in our study thus could be critical for understanding the pathogenesis of chronic
363
hepatitis B infection.
364
It will be interesting to discover whether HBc-related mutants of HBV have particular clinical
365
manifestations. Naturally occurring mutations in the HBV basic core promoter provide
366
evidence on this issue. A prevalent HBV variant is a double mutation of C-to-T at nucleotide
367
1768 and a T-to-A change at nucleotide 1770, resulting in highly upregulated core promoter
368
activity. This change is associated with a clinical outbreak of fatal fulminant hepatitis. In
369
particular, this HBV strain induces substantial apoptosis in primary tupaia hepatocytes by a
370
mechanism potentially independent of enhanced replication (15), possibly the strongly
371
upregulated expression of pro-apoptotic HBc.
372
In conclusion, our study found that HBc protein sensitizes hepatocytes to TNF-α-induced
373
apoptosis by competing with MKK7 for binding to RACK1, abrogating MKK7
374
phosphorylation. The study is thus a first indication of that HBc is pathogenic in liver injury
375
during hepatitis B.
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18
377
Acknowledgement
378
This work was supported by grants from the National Science and Technology Major Projects
379
(2012ZX10002007), Natural Science Foundation (81171566), and National Key Basic
380
Research Program of China (2012CB519000). We also gratefully acknowledge the support of
381
SA-SIBS Scholarship Program.
382
19
Figure Legends
383
384
FIG. 1. Schema of TNF-α signaling pathway that induces apoptosis
385
TNF-α is capable of inducing apoptosis when NF-κB signaling is blocked (20-24). Upon
386
binding to TNF-receptor I, a protein Complex I forms rapidly that activates NF-κB and JNK
387
signaling pathways. Complex I undergoes ligand-dissociated internalization with formation of
388
Complex II. Complex II is composed of FADD and procaspases 8, and triggers apoptosis if
389
NF-κB signaling fails to induce expression of anti-apoptotic genes. JNK is an important
390
regulator of TNF-α-mediated apoptosis (20, 22, 24-26), and is activated via the sequential
391
activation of protein kinases including two MAP kinase kinases (MKK4 and MKK7) and
392
multiple MAP kinase kinase kinases (MAP3Ks). MKK7 is an essential component of the JNK
393
signaling pathway activated by TNF-α. RACK1 is a scaffold protein that facilitates
394
phosphorylation of MKK7 by upstream MAP3Ks (19). It is widely reported that sustained JNK
395
activation mediates TNF-α-induced cell death, although in hepatocytes JNK has both
396
pro-apoptotic and anti-apoptotic aspects (22, 24). We propose that HBc (red) disrupts
397
interactions between MKK7 and RACK1, rendering cells susceptible to TNF-α-mediated
398
apoptosis.
399
FIG. 2. HBc confers susceptibility to TNF-α-induced apoptosis in hepatoma cells and
400
primary hepatocytes
401
(A) Left, annexin V staining of HepG2 or HepG2.2.15 cells 14 hours after co-treatment with
402
TNF-α and Bay11-7082. Cells treated with Bay11-7082 alone, or mock treated, were used as
403
controls. To block HBV replication, HepG2.2.15 cells were pretreated with 20 g/mL
404
Lamivudine (LAM) for 48 hours. Right, representative flow cytometry analysis of cells treated
405
with TNF-α/Bay11-7082. Cells were double-stained with 7-amino actinomycin D (7-AAD) and
406
annexin V. Annexin V positive populations were calculated. (B) Upper, Southern blot of HBV 20
407
replication in HepG2, HepG2.2.15 and LAM-treated HepG2.2.15 cells. Replication
408
intermediates of HBV DNA are RC, relaxed circular; DSL, double-stranded linear; SS, single
409
strand; Lower, HBc levels detected by immunoblotting. (C) Annexin V staining of Huh-7 cells
410
stably transfected with a control plasmid or the plasmid encoding HBc, polymerase (Pol), X
411
protein (HBx), or S2S proteins of HBV respectively. Cells were treated with
412
TNF-α/Bay11-7082 as described in A. (D) Annexin V staining of HepG2 cells stably
413
transfected with a 1.2mer genome of HBV (payw1.2), or mutant HBV genome deficient in
414
HBe and HBc (payw1.2-e/C-null). Mock-transfected cells were used as controls. (E) Left, flow
415
cytometry of active caspase 3 expression after TNF-α/Bay11-7082 treatment in HepG2 cells
416
stably transfected with a control plasmid, or the plasmid encoding HBc (pcDNA3.1-HBc).
417
Right, representative flow cytometry analysis of cells treated with TNF-α/Bay11-7082.
418
Co-treatment with TNF-α and the NF-κB inhibitor PDTC was also tested, with similar results
419
(Fig. S2). (F-G) PMHs (F) or PHHs (G) transduced with rAd-HBc/GFP, or rAd-GFP as control,
420
for 48 hours at a MOI of 20:1. Cells were then treated with mouse or human-derived TNF-α
421
and actinomycin D (ActD), with active caspase 3 expression analyzed by flow cytometry. The
422
error bars indicate SEM. *, P
