Hepatitis B Virus Protein X-induced Expression of the CXCChemokine IP-10 Is Mediated through Activation of NF-�Band Increases Migration of Leukocytes*

Received for publication, September 23, 2009, and in revised form, January 14, 2010 Published, JBC Papers in Press, February 17, 2010, DOI 10.1074/jbc.M109.067629

Yu Zhou‡, Shuo Wang§, Jing-Wei Ma‡, Zhang Lei¶, Hui-Fen Zhu‡, Ping Lei‡, Zhuo-Shun Yang¶, Biao Zhang¶,Xin-Xin Yao‡, Chuan Shi‡, Li-Fang Sun‡, Xiong-Wen Wu‡, Qin Ning�, Guan-Xin Shen‡1, and Bo Huang¶2

From the Departments of ‡Immunology, ¶Biochemistry and Molecular Biology, and �Infectious Disease, Tongji Medical College,Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China and the §Lady Davis Institute, McGillUniversity, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada

Interferon-� inducible protein 10 (IP-10) involves inflamma-tory cell recruitment and cellular immune damage during virusinfection. Although an increase of the peripheral IP-10 level isknown in HBV-infected patients, the molecular basis of HBVinfection inducing IP-10 expressionhas remained elusive. In thepresent study, we demonstrate that hepatitis B virus protein X(HBx) increases IP-10 expression in a dose-dependent manner.Transfection of the HBx-expressing vector into HepG2 cellsresults in nuclear translocation of NF-�B, which directly bindsthe promoter of IP-10 at positions from �122 to �113, thusfacilitating transcription. The addition of the NF-�B inhibitorblocks the effect of HBx on IP-10 induction. In parallel, increaseof NF-�B subunits p65 and p50 in HepG2 cells also augmentsIP-10 expression. Furthermore, we show that HBx induces acti-vation of NF-�B through the TRAF2/TAK1 signaling pathway,leading to up-regulation of IP-10 expression. As a consequence,up-regulation of IP-10maymediate themigration of peripheralblood leukocytes in a NF-�B-dependentmanner. In conclusion,we report anovelmolecularmechanismofHBV infection induc-ing IP-10 expression, which involves viral protein HBx affectingNF-�B pathway, leading to transactivation of the IP-10 pro-moter. Our study provides insight into the migration of leuko-cytes in response to HBV infection, thus causing immune path-ological injury of liver.

Hepatitis B virus (HBV)3 infection is a main health problemworldwide by causing acute and chronic liver disease. Although

there is no direct cytopathic effect on hepatocytes, HBV infec-tion induces the infiltration of immune cells, leading toformation of necroinflammatory foci, thus mediating diseaseprocesses (1, 2). To date, migration of immune cells to theinflammatory site is known to be triggered by chemokine sig-naling (3).Chemokines are a family of small chemotactic cytokines that

contain between two and four highly conserved NH2-terminalcysteine amino acid residues. They function to recruit and acti-vate immune cells to inflammatory sites through binding to asubset of G protein-coupled receptors (4). IFN-� inducible pro-tein 10 (IP-10, CXCL10) is a CXC chemokine that can besecreted by hepatocytes and sinusoidal endothelium in the liverof hepatitis patients (5, 6). By binding CXCR3 receptor, IP-10exerts the chemoattracting effect on NK cells, activated T cells,and dendritic cells (7). In IP-10�/� mice, immune responses toinfection by neurotropic mouse hepatitis virus are reduced,concomitant with decreasedCD4� andCD8� lymphocyte traf-ficking to the brain and reduced production of inflammatoryfactors (8). In HBV transgenic mice, the blockade of IP-10 sig-nificantly decreases migration of mononuclear cells and theseverity of liver lesions induced by HBV-specific cytotoxic Tlymphocytes (9). Interestingly, mRNA and protein levels ofIP-10 in PBMCs, sinusoidal endothelium, and plasma are allincreased in patients with chronic hepatitis B (10). Therefore,the induction of IP-10 is an important event for development ofhepatitis B. However, the molecular basis of how IP-10 is regu-lated for induction and function exertion still remains elusiveso far.The above analysis indicates that induction of IP-10 in hep-

atitis B patientsmay play an important role in driving leukocytemigration and causing the development of the immune-medi-ated injury. However, to date, the mechanism of how HBVinduces IP-10 expression still remains elusive. It is known thatthe HBV genome encodes DNA polymerase, surface antigen(HBs), core protein (HBc), and nonstructural regulatory pro-tein HBx (11). Among them, HBx is a pleiotropic proteininvolved in viral replication, signal transduction, andHBV-me-diated carcinogenesis (12, 13). HBx is located at either thecytosol or the nucleus. The former may activate the signaltransduction cascade, whereas the latter activates specific tran-scription factors (14). HBx does not bind DNA directly buttransactivates multiple transcription factors including AP1

* This work was supported by the National Key and Basic Research Develop-ment Program of China Grant 2007CB512900, State Project on Major Infec-tious Diseases Prevention Grant 2008ZX10002-009, Program for New Cen-tury Excellent Talents in University Grant NCET-08-0219, and SpecialResearch Foundation for Universities affiliated with China Ministry of Edu-cation Grant Z2009005.

1 To whom correspondence may be addressed: 13 Hangkong Rd., Wuhan430030, China. Fax: 86-27-83693500; E-mail: guanxin_shen@yahoo.com.cn.

2 To whom correspondence may be addressed: 13 Hangkong Rd., Wuhan430030, China. Fax: 86-27-83693500; E-mail: tjhuangbo@hotmail.com.

3 The abbreviations used are: HBV, hepatitis B virus; IFN-�, interferon-�; HBs,surface hepatitis B; HBc, core hepatitis B; nt, nucleotide; PBL, peripheralblood lymphocyte; EMSA, electrophoretic mobility shift assay; ChIP, chro-matin immunoprecipitation; MAPK, mitogen-activated protein kinase;siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehy-drogenase; ELISA, enzyme-linked immunosorbent assay; CMV, cytomega-lovirus; TNFR1, tumor necrosis factor receptor 1; TRAF, TNFR-associatedfactor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 16, pp. 12159 –12168, April 16, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

APRIL 16, 2010 • VOLUME 285 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 12159

by guest on February 10, 2020http://w

ww

.jbc.org/D

ownloaded from

(15), NF-�B (16, 17), HIF-1 (18), and ATF/cAMP-response ele-ment-binding protein (19, 20). Among them,NF-�B has gainedmuch attention due to its pivotal role in regulating an excep-tionally large number of genes, particularly those involved inimmune and inflammatory responses (21, 22).Multiple viruses may regulate the expression of IP-10, such

as HRV (23), Sendai virus (24), measles virus (25), rabies virus(26), severe acute respiratory syndrome coronavirus nsp-1 (27),and human immunodeficiency virus gp120 (28). This hints thatHBVprobably has the same effect. In this study, we hypothesizethat HBx regulates expression of IP-10 through the NF-�Bpathway. Our data show that the HBx protein increases IP-10expression by promoting binding of NF-�B on the �B1 bindingsite of the 5�-untranslated region of IP-10 in a dose-dependentmanner. The activation of NF-�B by HBx involves signal mol-eculesTRAF2 andTAK1, thus leading to up-regulation of IP-10expression. In turn, up-regulation of IP-10 mediates migrationof peripheral blood lymphocytes (PBLs). These findings pro-vide new insight into the regulatory mechanism of leukocyterecruitment and infiltration to the HBV infection site.

EXPERIMENTAL PROCEDURES

Patients—Twelve patients (8male, 4 female; age range 32–65years, average 48 � 9) with chronic hepatitis B who underwentsurgery or cancer treatmentwere selected from theUnionHos-pital of Tongji Medical College for IP-10 detection in liver tis-sues by real time PCR and immunohistochemistry. Thesepatients were positive for HBsAg, HBeAg, anti-HBc, and HBV-specific DNA in serum. Other causes of chronic liver diseasehad been excluded by clinical and laboratory assessments.Seven patientswith non-HBV infection that underwent surgeryfor cancer treatment were also selected. Five normal non-HBV-infected control liver tissues were obtained from a macroscop-ically normal area during liver hemangioma resection.Cell Lines and Cell Culture—Human hepatocellular cell lines

HepG2 and HepG2.2.15 (expressing the whole HBV proteinspersistently) were cultured in Dulbecco’s modified Eagle’smedium supplemented with 10% fetal bovine serum at 37 °C ina 5% CO2 incubator. The HBx-expressing HepG2 cell line,transfected with the pCMV-HBx plasmid and that stablyexpressed the HBx protein, was cultured with 500 �g/ml ofG418 and 10% fetal bovine serum at 37 °C in a 5% CO2incubator.RNA Extraction and Real Time PCR—Total RNA of fresh

tissue samples was extracted using TRIzol (Invitrogen) accord-ing to the manufacturer’s protocol. cDNA was synthesized asdescribed previously (29). Equal amounts of cDNA were sub-mitted to PCR in the presence of the SYBR-PCRMaster mix kit(Applied Biosystems, Foster City, CA) and real time PCR detec-tion machine Mx3000PTM (Stratagene, La Jolla, CA). IP-10specific primers were as follows: forward primer, 5�-GCCTCT-CCCATCACTTCCCTAC-3�; reverse primer, 5�-GAAGCAG-GGTCAGAACATCCAC-3�. The housekeeping gene GAPDHwas used as an internal control.Immunohistochemistry—Immunohistochemical staining was

described previously (30). In brief, after deparaffinization andhydration, the slides were treated with endogenous peroxidasein 0.3% H2O2 for 30 min. Then, the sections were blocked for

2 h at room temperature with 1.5% blocking serum. Sectionswere incubated with anti-IP-10 antibody (Abcam, Cambridge,MA) overnight at 4 °C. Labeled horseradish peroxidase wasapplied for 30 min at room temperature, followed by applica-tion of diaminobenzidine solution until color developed. Slideswere counterstained with hematoxylin. The results wereobserved under a light microscope.Preparation of Promoter Constructs—A 1050-bp IP-10 pro-

moter construct, corresponding to the sequence from �953 to�97 (relative to the transcriptional start site) of the 5�-flankingregion of the human IP-10 gene was generated from humangenomic DNA using forward 5�-TTTGCTAGCCAGTTATC-ACTGTTACTAGC-3�, and reverse 5�-AAACTCGAGGGCA-GCAAATCAGAATGGCA-3� primers. Using the (�953/�97)IP-10 construct as a template, several deletion constructs of theIP-10 promoter, including �534/�97, �239/�97, �191/�97,and �97/�97 were similarly generated by the correspondingforward primers: 5�-TTTGCTAGCACTTGCCAGTTCCAG-ATCTT-3�, 5�-TTTGCTAGCGAAACAGTTCATGTTTTG-GAA-3�, 5�-TTTGCTAGCAAAAGAGGAGCAGAGGGA-AAT-3�, and 5�-TTTGCTAGCGGTTTTGCTAAGTCAACT-GTAA-3�. Point mutations in two NF-�B sites, NF-�B1 andNF-�B2, were generated in the (�534/�97) IP-10 constructusing standard site-directed mutagenesis procedures. ForNF-�B1, a mutant reverse primer 5�-GTTCCTGGtGAAGT-CaCATGTTGCAGAC-3� was annealed in combination withthe previously described forward primer and subjected to PCRamplification.Meanwhile, a mutant forward primer 5�-GCAA-CATGtGACTTCaCCAGGAACAGCC-3� was annealed incombination with the previously described reverse primer andPCR amplification. The amplified product was gel purified andligated into pGL3-Basic vector. A point mutant construct forNF-�B2 was generated by the same strategy with mutatedprimers 5�-GGAGCAGAGtGAAATTaCGTAACTTGGA-3�and 5�-CAAGTTACGtAATTTCaCTCTGCTCCTC-3�. Allconstructs were sequenced for success.Vectors containing individual viral structural genes of HBV

and NF-�B subunits were kindly provided by Dr. Wen-JieHuang (State Key Laboratory of Virology, Wuhan Institute ofVirology, Chinese Academy of Sciences, Wuhan, People’sRepublic of China). The pNF-�B-Luc (Stratagene, La Jolla, CA)containing four copies of the binding sequence of NF-�B andfirefly luciferase gene was used in NF-�B activity detection.RNA Interference—A series of double-stranded small inter-

fering RNA targeting HBs, HBc, and HBx protein of HBV hadbeen cloned into a siRNA-expressing vector pGenesil-1. Thevalidity and efficiency of these siRNA plasmids were identifiedby determining the mRNA and protein levels of the corre-sponding target gene after transfecting them or mock vectorinto HepG2.2.15 cells. For the assay, cells were plated at a den-sity of 1 � 105 cell/well in a 24-well plate. After 24 h, cells weretransfected 0.8 �g of siRNA plasmids per well or mock vectorusing Lipofectamine 2000 (Invitrogen) according to the manu-facturer’s instructions. HBx-expressing HepG2 cells wereplated at a density of 5 � 105 cells/well in a 6-well plate. TRAF2siRNA or TAK1 siRNA (Santa Cruz, Santa Cruz, CA) per wellwas used to knock down TRAF2 or TAK1 expression in HBx-

HBx-induced Expression

12160 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 16 • APRIL 16, 2010

by guest on February 10, 2020http://w

ww

.jbc.org/D

ownloaded from

expressing HepG2 cells according to the manufacturer’sinstructions.Transfections and Reporter Gene Assays—HepG2 cells were

plated at a density of 1 � 105 cell/well in a 24-well plate. After24 h, cells were transfectedwith 0.8�g of the full-length gene ofHBV (pBlue-HBV), differentHBVprotein expressing plasmids,and p50 or p65 expressing plasmids, respectively. For the lucif-erase assay, cells were co-transfected with 0.6 �g of expressionvector plasmids, 0.18 �g of promoter reporter plasmids, and0.02 �g of the pRL-TK plasmids. 5 h later, cells were washedand allowed to recover in fresh medium supplemented with 1%fetal bovine serum. 48 h later, luciferase activity was detectedwith the Dual Luciferase� Reporter Assay System (Promega,Madison, WI) according to the manufacturer’s instructions.The relative luciferase activity was determined by aModulusTMLaboratory Luminometer (Turner Biosystems, Sunnyvale CA),and transfection efficiencywas normalized byRenilla luciferaseactivity.Electrophoretic Mobility Shift Assay (EMSA)—Nuclear ex-

tracts were prepared as previously described (31) from HepG2cell-transfected relative expression vector plasmids. Biotinend-labeled oligonucleotides were synthesized and annealed toobtain double-stranded DNA fragments. The oligonucleotidesequences of NF-�B1 were as follows: 5�-CATGGGACTTCC-CCAGGAACAGC-3� and 5�-GCTGTTCCTGGGGAAGTCC-CATG-3�. EMSA was performed using the LightShift� Chemi-luminescent EMSAkit (Pierce) according to themanufacturer’sprotocol. Briefly, the binding reactionswere performed in 20-�lsamples containing 1 �l of biotin-labeled oligonucleotides, 4mg of nuclear extracts, 2mg of poly(dI-dC), 2�l of 10� bindingbuffer, 0.1 mM EDTA, and 10% glycerol. After incubation atroom temperature for 20 min, samples were separated on 6%polyacrylamide gels with 0.5� TBE buffer and transferred to anylonmembrane (AmershamBiosciences) at 380mA (�100V)for 15 min. A chemiluminescent detection of membranes wasscanned by Image Station 4000R (Kodak, Rochester, NY). Spec-ificity of the protein-DNA interaction was confirmed by com-petition with 20- or 100-fold of unlabeled probes of the samesequence, or the NF-�B1 mutated probes: 5�-CATGt-GACTTCaCCAGGAACAGC-3�.Chromatin Immunoprecipitation Assay (ChIP)—ChIP assays

were carried out using a commercial ChIP assay kit (UpstateBiotechnology, Billerica, MA). Briefly, HepG2 cells transfectedwith relative plasmids were cross-linked to histones of DNAusing 1% formaldehyde at 37 °C for 10 min. After washing withcold phosphate-buffered saline, cells were scraped into a coni-cal tube and sonicated to shear the chromatins. The sonicatedchromatins were incubated with anti-p65 antibody, anti-p50antibody, or an isotype control IgG for 2 h. The chromatin-antibody complex was precipitated with protein A-agarosebeads. The DNA isolated from the complex was subject to PCRamplification using the following primers flanking the NF-�B1site in IP-10 promoter: 5�-AACTTGGAGGCTACAATAAA-3� and 5�-GAGGAATGTCTCAGAAAACG-3�.Confocal Laser ScanningMicroscopy—HepG2 cells were cul-

tured on coverslips at a density of 5 � 104 cells/coverslip. Aftertransfecting the relative plasmids for 48 h, cells were fixed withice-cold methanol for 10 min, blocked with 1% bovine serum

albumin in phosphate-buffered saline for 1 h, and probed for 90min at room temperature with anti-p65 antibody (Santa Cruz).After probing, the signal was visualized with fluorescein iso-thiocyanate-conjugated second antibody (Sigma). Nuclei werestained using 4�,6-diamidino-2-phenylindole (Sigma). Micro-graphs were acquired with a BX51 system equipped with DP70(Olympus, Japan).ELISAs—The amounts of IP-10 present in culture superna-

tants were determined using a specific rat anti-human IP-10ELISA kit (eBioscience, San Diego, CA) according the manu-facturer’s instructions.WesternBlotting—Total proteins, nuclear proteins, and cyto-

plasmic proteins were prepared as previously described (32),separated by stacking gel and SDS-PAGE with a Tris glycinesystemat 100V for 1h, and transferred topolyvinylidenedifluo-ride membranes (Millipore, Billerica, MA). The membranes

FIGURE 1. IP-10 expression in human liver tissues. A, real time PCR detec-tion of IP-10 mRNA expression in HBV-infected, non-HBV-infection, and nor-mal control liver tissues. Results are mean � S.D. of three experiments per-formed in duplicate and relative to the housekeeping gene GAPDH. Barsrepresent mean. B, detection of IP-10 protein expression in liver tissue byimmunohistochemistry. a, immunoreactive IP-10 protein was mainly visual-ized on sinusoidal endothelium and hepatocytes in HBV-infected liver cancertissue; b, isotype IgG was used as a negative control in HBV-infection livercancer tissue; c, immunoreactive IP-10 protein was scarcely visualized in non-HBV-infection liver cancer tissue; d, in normal control liver tissues, the IP-10protein could not be detected by immunohistochemistry.

HBx-induced Expression

APRIL 16, 2010 • VOLUME 285 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 12161

by guest on February 10, 2020http://w

ww

.jbc.org/D

ownloaded from

were blocked in 5% nonfat dry milk in phosphate-bufferedsaline containing 0.1% Tween 20 for 2 h at room temperature.Then the membranes were incubated with specific anti-p65antibody (Santa Cruz), anti-phospho-TAK1 (Thr184) antibody(Cell Signaling Technology, Boston, MA), or p-IKK� (Thr23)antibody (Santa Cruz) overnight at 4 °C. The membraneswere washed three times and incubated with horseradishperoxidase-conjugated secondary antibody. Proteins werevisualized by ECL Western blotting substrate (ThemoPierce, Rockford, IL).Migration Assay—Migratory activity was quantified using

24-well Transwell inserts (5-�m pore size, Costar, Bethesda,MD). PBLs (1 � 105) in 200 �l of RPMI medium were added tothe upper chamber. 1-ml of culture supernatant from the cellstransfected with the relative plasmids was added to the lowerchamber. To investigate the role of IP-10 in cell migration, 10�g of neutralizing anti-IP-10 (Abcam, Cambridge, MA) iso-type-matched control antibody was added 30 min prior to themigration assay. Supernatants of HepG2 cells transfected withpCMV-HBx following treated with the NF-�B inhibitor SN-50(Upstate Biotechnology, Lake Placid, NY) were also investi-gated. The chambers were incubated for 4 h at 37 °C in 5%CO2,then the Transwell inserts were removed and migration cellswere hematoxylin and eosin stained and counted.Data Analysis—Data were expressed asmean� S.D. of three

determinations. Statistical analysis data were analyzed usingthe Statistical Package for Social Sciences (SPSS) software (ver-

sion 13.0). Statistical comparisons were made using Student’s ttest, with p � 0.05 being considered significant.

RESULTS

Up-regulation of IP-10 Expression in HBV-infected LiverTissues—The up-regulation of IP-10 expression has beenreported in the PBMCs, plasma, and liver of patients withchronic hepatitis B (10). To confirm the previous report, weanalyzed the expression of IP-10 in HBV-infected liver tissue,and found that the mRNA levels of IP-10 were much higher inHBV-positive liver cancer tissues, compared with HBV-nega-tive liver cancer tissues or normal liver tissues (Fig. 1A). Thesimilar protein result was also confirmed by immunohisto-chemical staining (Fig. 1B). These data suggested that IP-10 iselevated in HBV-infected liver tissue, and may play an impor-tant role in HBV infection-induced hepatitis.HBx Induces IP-10GeneExpression inHepG2Cells—Toeval-

uate the effect of HBV on IP-10 expression, HepG2 cells weretransfected with the full-length HBV gene (pBlue-HBV) for48 h and protein levels of IP-10 in the supernatants were meas-ured. As shown in Fig. 2A, IP-10 protein was significantlyincreased in the pBlue-HBV-transfected group relative to con-trol. We then evaluated the effect of different HBV proteinsby transfecting pCMV-HBs, pCMV-HBc, and pCMV-HBx,respectively. The result showed that transfection ofHBx, ratherthan HBs, or HBc-expressing vector increased IP-10 proteinlevels (Fig. 2A). Consistently, using siRNA technology, we

FIGURE 2. HBx protein induces IP-10 expression. A, the full-length gene of HBV (pBlue-HBV) and plasmids expressing different HBV proteins were transfectedinto HepG2 cells, respectively. The protein levels of IP-10 were measured by the ELISA kit. Transfection efficiency was 35.8 � 6.2%. Results are mean � S.D. ofthree experiments performed in duplicate. Error bars represent S.D. siRNA-expressing vectors targeting different HBV proteins and mock plasmids weretransfected into HepG2.2.15 cells, respectively. The supernatant protein levels of IP-10 were analyzed by the ELISA kit. The transfection efficiency was 41.6 �7.4%. The results are representative of three experiments. C, transfection efficiency of GFP-expressing HBx siRNA plasmids into HepG2.2.15 cells were observedunder a fluorescence microscope. D, siRNA-expressing vectors targeting HBx proteins and mock plasmids were transfected into HepG2.2.15 cells, respectively.The HBx mRNA levels were analyzed by real time PCR after 48, 72, and 96 h. The mRNA levels of HBx in HepG2.2.15 cells transfected with mock plasmids wereset to 1.0 and results relative to the housekeeping gene GAPDH. E, HepG2 cells were co-transfected with the IP-10 promoter luciferase reporter vector anddifferent HBV protein-expressing vectors, respectively. Relative luciferase activity was determined by standard procedures. The luciferase activity of the mockpCMV-tag group was designated as 1.00. F, HepG2 cells were co-transfected with different doses of HBx-expressing vector and the reporter vector. 48 h later,relative luciferase activity was determined. G, HepG2 cells were transfected with different doses of pCMV-HBx-FLAG, in which the sequence for the FLAGepitope (DYKDDDDK) was tagged to the 3� end of the HBx sequence, and the sequence of HBx-FLAG was inserted into the pCMV-tag2 vector. The increasedprotein levels of HBx were determined by Western blot with anti-FLAG antibody (Sigma).

HBx-induced Expression

12162 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 16 • APRIL 16, 2010

by guest on February 10, 2020http://w

ww

.jbc.org/D

ownloaded from

found that knockdown of the HBx gene significantly decreasedthe protein levels of IP-10 in HepG2.2.15 cells (Fig. 2B). Theefficiencies of transfection and gene silencing were determinedin Fig. 2, C and D. Interestingly, we found that knockdown ofeither the HBs or HBc genes had no effect on IP-10 expressionin HepG2.2.15 cells (Fig. 2B). Together, these data suggestedthatHBx is a critical regulator for IP-10 expression duringHBVinfection.To determinewhetherHBx induces IP-10 gene transcription

through a promoter, a human IP-10 promoter reporter plasmidcontaining the sequence from �953 to �97 (relative to thetranscriptional start site) of the 5�-flanking regionwas co-trans-fected with individual HBV protein-expressing plasmids(pCMV-HBs, pCMV-HBc, or pCMV-HBx) or mock plasmid(pCMV-tag2) intoHepG2 cells. The results show that the IP-10promoter wasmarkedly activated by the HBx protein in a dose-dependent manner (Fig. 2, E and F). In line with this, a gradualincreased level of HBx protein inHepG2 cells was confirmed byWestern blot (Fig. 2G).

NF-�BActivity Is Required forHBx-induced IP-10 Expression—Analysis using the Genomatix MatInspector program andGene2Promoter software revealed the presence of many con-sensus cis-elements including GAS, ISRE, AP-1, and NF-�Bbinding sites on the IP-10 promoter (Fig. 3A). To investigate therole of these cis-elements in regulating IP-10 expression by theHBx protein, a series of 5� deletions of the IP-10 promoter wereconstructed and co-transfected with pCMV-HBx, respectively,into HepG2 cells. Luciferase activity was measured 48 h aftertransfection. As shown in Fig. 3B, the deletion from nt �953 to�190 did not affect HBx-induced luciferase activity, however,deletion to nt �96 significantly decreased HBx-induced lucif-erase activity, suggesting that the sequence between nt�190 to�96 is critical for activation of the IP-10 promoter by the HBxprotein. Coincidently, two NF-�B binding sites (NF-�B1 andNF-�B2) were found in this region, and could be the reason forIP-10 activation. Thus, we mutated these two NF-�B sites bymultiple rounds of PCR, and found that only mutation ofNF-�B1 but not the NF-�B2 binding site effectively reduced

FIGURE 3. The �B site in the IP-10 promoter involved in HBx-induced IP-10 expression. A, potential binding sites for cis-acting elements in the 5�-flankingregion of the human IP-10 gene are shown. Gene2Promoter software revealed several transcription factor binding sites, including NF-�B, ISRE, and GAS. Thetranscription start site and translation start site are also indicated in the figure. B, effect of HBx protein on the truncated IP-10 promoter constructs. HepG2 cellswere co-transfected with serial truncated IP-10 promoter constructs and pCMV-HBx, and relative luciferase activity was determined. The schematic constructsshown (left) and the bar graphs are the relative levels of luciferase activity in each of the transfected samples (right). C, effect of mutated �B sites on activity ofthe IP-10 promoter. HepG2 cells were co-transfected with pCMV-HBx and the construct with mutated to NF-�B1 or NF-�B2 sites. The relative luciferase activitywas determined. D, NF-�B activity was necessary for HBx-induced IP-10 expression. HepG2 cells were co-transfected with pCMV-HBx and the reporter vectorfollowing treatment with the NF-�B inhibitor SN50 at different concentrations as indicated. After 48 h, luciferase activity was measured. Results are mean � S.D.of three experiments performed duplicate. Error bars represent S.D.

HBx-induced Expression

APRIL 16, 2010 • VOLUME 285 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 12163

by guest on February 10, 2020http://w

ww

.jbc.org/D

ownloaded from

HBx-induced activation of the IP-10 promoter (Fig. 3C). Thesedata suggested that theNF-�B1 binding site is required for acti-vation of the IP-10 promoter regulated by the HBx protein.To further determine the roles of NF-�B in activation of the

IP-10 promoter by the HBx protein, HepG2 cells were treatedwith different concentrations of theNF-�B inhibitor SN50 afterco-transfection with pCMV-HBx and the �953 to �97 IP-10reporter plasmid. Results from the luciferase activity assayshowed that blockingNF-�B by SN50 resulted in inhibition of theIP-10promoteractivity (Fig.3D), suggesting thatNF-�Bactivationis required to activate the IP-10 promoter by the HBx protein.NF-�B Binds to the Promoter of HBx-induced IP-10 Directly—

Next, we askedwhether the activatedNF-�Bbound to the IP-10promoter directly. For this purpose, we performed EMSA witha probe of biotin-labeled NF-�B1 binding oligonucleotides inthe IP-10 promoter (�125 to �102), and found that the DNAbinding activity of NF-�B was significantly increased in cellstransfected with pCMV-HBx, compared with mock plasmid(Fig. 4A). To determine the specificity of the NF-�B bindingactivity, nuclear extracts were incubated with labeled NF-�B1binding oligonucleotides in the presence of either an unlabeledwild type NF-�B binding probe or a mutated probe. As shownin Fig. 4A, the wild type NF-�B-binding oligonucleotides, butnot the mutated oligonucleotides abrogated NF-�B complexes.Consistently, the addition of NF-�B inhibitor SN50 also inhib-ited the DNA binding activity of NF-�B (Fig. 4A). These dataindicate that NF-�B binds to the NF-�B1 binding site in theIP-10 promoter.

Using a comparable approach, we also performed a ChIPassay. Chromatin fragments were prepared from HepG2 cellstransfected with pCMV-HBx and immunoprecipitated withantibody against either the p50 or p65 subunit. A 177-bp DNAfragment was amplified by PCR from DNA isolated from cellstransfected with pCMV-HBx in the presence of anti-p50 oranti-p65 antibody, but theywere not detected in the presence ofmock plasmid or control antibody (Fig. 4B). Thus, these datasuggested that NF-�B binds to the NF-�B1 binding site in theIP-10 promoter.

FIGURE 4. NF-�B subunits bind to the IP-10 promoter directly. A, EMSAshowed direct binding of NF-�B to the NF-�B1 site of the IP-10 promoter.HepG2 cells were transfected with pCMV-HBx or mock plasmids for 48 h.Nuclear extracts were subjected to EMSA with biotin-labeled �B1 or �B2 oli-gonucleotides in the absence and presence of the indicated folds of unla-beled competitors or unlabeled mutated competitors. In addition, the NF-�Binhibitor SN50 was involved in the indicated case. B, the CHIP assay showeddirect binding of p65 and p50 to the IP-10 promoter. Amplification of a 177-bpDNA fragment containing the NF-�B1 site in the IP-10 promoter and the inputDNA are shown.

FIGURE 5. HBx protein induces the translocation of NF-�B subunits.A, HepG2 cells were transfected with HBx plasmids or co-transfected with HBxplasmids and TRAF2 or TAK1 siRNA for 48 h. Cells were then fixed, permeabi-lized, and stained with anti-p65 antibody followed by incubation with fluo-rescein isothiocyanate-conjugated goat anti-rabbit secondary antibody.Nuclei were counterstained with 4�,6-diamidino-2-phenylindole, and immu-nofluorescence was monitored by confocal microscopy. B, the levels ofectopic HBx protein in cells transfected with pCMV-HBx-FLAG are shown byWestern blot using the anti-FLAG antibody. The levels TRAF2 and TAK1 in cellstransfected with the respective siRNA and controls are also shown by Westernblots using the anti-TRAF2 antibody or anti-TAK1 antibody. C, levels of cyto-solic and nuclear p65 protein were determined at the indicated times byWestern blot using anti-p65 antibody after transfection with pCMV-HBx.DAPI, 4�,6-diamidino-2-phenylindole.

HBx-induced Expression

12164 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 16 • APRIL 16, 2010

by guest on February 10, 2020http://w

ww

.jbc.org/D

ownloaded from

HBx Induces Translocation of NF-�B Subunits p65 into theNucleus—NF-�B is composed of homo- and heterodimers, typ-ically p65 and p50 heterodimers. After the destruction of I�B,NF-�B is capable of translocating into the nucleus and transac-tivates target genes (33). We examined the effect of the HBxprotein on translocation of p65 from the cytosol to the nucleusafter transfecting pCMV-HBx into HepG2 cells. Nuclear trans-location of p65 was defined by confocal microscopy. As shownin Fig. 5A, transfection with pCMV-HBx, but not the pCMV-tag, caused a remarkable translocation of the p65 subunit ofNF-�B from the cytosol to the nucleus. In addition, we alsotested the HBV-transfected HepG2.2.15 cell line. A similarresult was observed; and knockdown of HBx expression bytransfecting the siRNA impaired p65 translocation (data notshown). Moreover, at 0, 24, and 48 h post-transfection ofpCMV-HBx, protein levels of p65 in cytosol and nucleus frac-tions were analyzed by Western blot using anti-p65 antibody.As shown in Fig. 5C, the levels of p65 protein decreased in thecytosol and increased in the nucleus in parallel with transfec-tion times. However, this phenomenon was not observed incells transfected with the pCMV-tag (data not shown).Together, these data suggest that HBx protein facilitates trans-location of p65 from the cytosol to the nucleus.NF-�B Subunits p65 and p50 Increase the Transcriptional

Activity of IP-10—NF-�B is composed of homo- and het-erodimers, typically p65 and p50 heterodimers. To investigatethe effect of p50 and p65 subunits of NF-�B on transcriptionalactivation of IP-10, HepG2 cells were transfected with the plas-mid expressing p65 or p50, and the protein levels of IP-10 weredetermined by ELISA. As shown in Fig. 6A, transfection of p50or p65 proteins increased the IP-10 protein levels, and IP-10reached the highest level when both p50 and p65 proteins werepresent. Furthermore, we co-transfected HepG2 cells withplasmids expressing p65 or p50 and IP-10 (�534/�97) reporterplasmids. Results from luciferase activity assays showed thattranscriptional activity of IP-10 was higher in the presence ofthe p50 or p65 proteins compared to the control (Fig. 6B). Thehighest level was reachedwhen both p50 and p65 proteins werepresent (Fig. 6B). However, mutating the NF-�B1 binding sitein the IP-10 promoter abolished the effect of the p50 and/or p65proteins on activation of the IP-10 promoter (Fig. 6B). There-fore, these data suggested that the p65/p50 heterodimer in-duces IP-10 expression.HBx-induced IP-10 Expression Is Mediated by the TRAF2/

TAK1/NF-�B Signaling Pathway—Mechanisms on the activa-tion of NF-�B byHBx have been reported before (16, 34). How-ever, the upstream molecular event still remains largelyunknown. Numerous studies have demonstrated that NF-�Bcan be activated through the Toll-like receptor signaling path-way. Toll-like receptors are a type of pattern recognition recep-tors and recognize molecules that are broadly shared by patho-gens but distinguishable from host molecules, collectivelyreferred to as pathogen-associated molecular patterns (35).After binding to ligand, Toll-like receptor signaling is trans-ducted and the scaffold intermediate kinase TAK1 is activated,which subsequently activates IKK, leading to activation ofNF-�B. Because HBx is a viral protein, it can also be considereda pathogen-associated molecular pattern. In this regard, we

hypothesized thatHBx induced activation ofNF-�B via activat-ing TAK1. After transfection of the HBx plasmid into theHepG2 cell line, phosphorylation of TAK1 and IKK� wasobserved in the HBx plasmid group, but not in mock or phos-phate-buffered saline groups (Fig. 7,A and B). (5Z)-7-Oxozeae-nol is a specific inhibitor of TAK1 by competing with ATP tobind TAK1, thus inhibiting TAK1 activity (36). Therefore, wefurther used (5Z)-7-Oxozeaenol to block the activity of HBx-inducedTAK1 inHepG2 cells. As shown in Fig. 7C, bothNF-�Bactivity and IP-10 promoter activity were reduced by TAK1inhibition. In parallel, knockdown of TAK1 by the siRNA alsocaused the decrease of NF-�B and IP-10 promoter activities(Fig. 7,D–F). Together, these data suggest thatHBx indeedmayactivate NF-�B via activating TAK1/IKK.To further investigate the upstream signal molecule(s) that

result in activation ofTAK1,we concentrated onTRAF2due to:1) TRAF family members such as TRAF6 and TRAF2 that par-ticipate in the activation of TAK1; and 2) that HBx may acti-vate TNFR1 (37) and TRAF2 is the adaptor for TNFR1 signal-ing. For this purpose, we knocked down the TRAF2 gene bytransfecting TRAF2 siRNA into cells. The efficiency of TRAF2

FIGURE 6. NF-�B subunits increase the IP-10 expression. A, HepG2 cellswere transfected with p65 and/or p50 expression constructs. IP-10 proteinlevels in the supernatants were measured by the ELISA kit. Results are mean �S.D. of three experiments performed in duplicate. B, HepG2 cells were co-transfected with (�534/�97) IP-10 or NF-�B1 Mut reporter constructs andp65 or p50 expression constructs, respectively. After 48 h, luciferase activitywas measured. The luciferase activity of the cells co-transfected with pCMV-tag2 and the indicated reporter plasmids was set to 1.00, respectively. Resultsare mean � S.D. of three experiments performed in duplicate. Error bars rep-resent S.D. *, a statistically significant difference from mock plasmids (p �0.05). **, a statistically significant difference from mock plasmids (p � 0.01).

HBx-induced Expression

APRIL 16, 2010 • VOLUME 285 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 12165

by guest on February 10, 2020http://w

ww

.jbc.org/D

ownloaded from

siRNA was confirmed byWestern blot (Fig. 7, D and E). Undersuch conditions, we measured the NF-�B and IP-10 promoteractivity induced byHBx. The result showed thatTRAF2 knock-down impaired NF-�B activity induced by HBx (Fig. 7F). Con-sistently, the knockdown of TRAF2 or TAK1 resulted in inhibi-tion of nuclear translocation of the NF-�B subunit p65 (Fig.5A). The levels of ectopic HBx protein as well as the levelsTRAF2 and TAK1 in cells transfected with the respectivesiRNA and controls were shown by Western blots (Fig. 5B).Taken together, our data suggest that HBx-induced IP-10expressionmight bemediated by theTRAF2/TAK1/NF-�B sig-naling pathway.

HBx-induced IP-10 IncreasesMigration of PBLs—To define thefunctionality of the secreted IP-10,we performed a migration assay.With the 4-h time frame of theexperiment, minimal migration ofPBLs was observed in response tothe supernatant from HepG2 cellstransfected with mock plasmid;however, PBLs exhibited a decentincrease of migratory activity inresponse to supernatants from cellstransfected with plasmids express-ing HBx, p50, or p65 (Fig. 8). Theaddition of neutralizing anti-IP-10but not control antibody to thesupernatants significantly impairedthis migration (Fig. 8). Interestingly,we additionally found that treat-ment with the NF-�B inhibitorSN50 after transfection of pCMV-HBx could decrease the chemotac-tic activity (Fig. 8). Together, thesedata suggested that HBx-inducedIP-10 may mediate the migration ofPBLs and NF-�B activity is involvedin this process.

DISCUSSION

During HBV infection, the in-crease of chemokine IP-10 is a criti-cal step for inflammatory cell re-cruitment and hepatopathology. Inthe present study, we demonstratethe increase of IP-10 levels in HBV-infected patients, and elucidate themolecular mechanism of IP-10expression through the viral regula-tory protein HBx-activated NF-�Bsignaling pathway in the humanhepatocellular cell line HepG2. Wealso define that HBx-induced IP-10can attract inflammatory cells invitro.The regulation of IP-10 expres-

sion has been reported by varioustranscription factors and signal elements. For instance, thephosphorylation of STAT1 could transcriptionally activateIP-10 though ISRE-binding factors (38–40), and signal mole-cules such as PKC, PKA, Akt, and p38 MAPK may be involvedthe regulation (41–44). Regardless of these, the mechanism ofthe increase of IP-10 expression in individuals carrying HBV(10) so far remains elusive. Here, we demonstrate that HBxrather than HBs or HBc encoded by HBV activates IP-10expression. Different from HBs or HBc, HBx profoundly influ-ences various signaling pathways. The HBx protein interactswith and stimulatesmany kinases such as PKC, Jak1, IKK, phos-phatidylinositol 3-kinase, SAPK (stress-activated protein

FIGURE 7. TRAF2/TAK1/NF-�B signaling pathway is involved in HBx-induced IP-10 expression. A, HepG2cells were transfected with HBx expression plasmids or mock plasmids. After 48 h, phosphorylation of TAK1 andIKK� was detected by Western blot. HepG2 cells stimulated with 100 ng/ml of lipopolysaccharide (LPS) for 30min were used as positive control. B, semi-quantitative analysis of p-TAK1 and p-IKK� protein levels was per-formed by Band-Scan software 5.0, and expression of �-actin was used as control. This quantitative analysisrepresented the respective Western blot of the above (A). C, HepG2 cells were co-transfected with NF-�B orIP-10 promoter luciferase reporter plasmids and HBx expression plasmids or mock plasmids with or without theTAK1-specific inhibitor (5Z)-7-Oxozeaenol (50 nM). After 48 h, luciferase activity was measured. HepG2 cellsstimulated with 100 ng/ml of LPS for 30 min were used as positive control. The luciferase activity of the cellsco-transfected with mock plasmids pCMV-tag2 and the indicated report plasmids was set to 1.00, respectively.D and E, the knockdown of TAK1 and TRAF2 genes. HepG2 cells were co-transfected with the HBx expressionplasmid and TAK1 or TRAF2 siRNA for 48 h. The protein levels of TAK1 and TRAF2 were analyzed by Western blot,respectively (D), and quantified with Band-Scan software 5.0 against the internal control �-actin (E). F, HBx-expressing HepG2 cells were co-transfected with NF-�B or IP-10 promoter luciferase reporter plasmids andTAK1 siRNA or TRAF2 siRNA. 48 h later, luciferase activity was measured. The luciferase activity of cells co-transfected with mock siRNA and the indicated report plasmids was set to 1.00. In C and F, results are mean �S.D. of three experiments performed in duplicate. Error bars represent S.D. *, p � 0.05, compared with mockplasmids. **, p � 0.01, compared with mock plasmids.

HBx-induced Expression

12166 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 16 • APRIL 16, 2010

by guest on February 10, 2020http://w

ww

.jbc.org/D

ownloaded from

kinase), and Akt/PKB (12, 37, 45–47). The 154 amino acids ofthe HBx protein contain regions important for transactivation,dimerization, p53 binding, and 14-3-3 protein binding (13). Inthis study, we find that HBx transactivates IP-10 by activatingand translocating NF-�B to the nucleus. Although interferons�/� or � are known as inducers for IP-10 via the STAT1 signal-ing pathway (38, 48), we confirm that the levels of IFN-�,IFN-�, IFN-� mRNAs, and proteins are not changed in HepG2cells after transfection of HBx vector.4 Thus, interferons areunlikely involved in HBx-induced IP-10 expression. Neverthe-less, whether other signaling pathway(s) contribute to HBx-induced IP-10 expression still needs to be clarified, if consider-ing that HBx targets multiple signaling pathways.NF-�B can be homo- or heterodimers, made up of different

members of the Rel family of proteins (p50/NF-�B1, p52/NF-�B2, c-Rel/Rel, p65/Rel-A, Rel-B, and Dorsal). Of these, thep50/p65 heterodimer (commonly referred to as NF-�B) is themost abundant and ubiquitous. In this study, we find the het-erodimer of p50 and p65 involved in HBx-induced IP-10expression, evaluated by EMSA and ChIP assays. These resultsare further confirmed by transfection of wild type p50 and p65into HepG2 cells and followed an increase of IP-10 expression.Previous studies have reported that HBx activated NF-�Bthrough multiple ways, including 1) HBx may induce the deg-radation of I�B� and the p105 via phosphorylation of theseproteins (16); 2) HBx directly interacts with I�B� and I�B�,

leading to translocation of NF-�B into the nucleus (34); 3) theHBxproteinmay activate hepaticNF-�BviaTNFR1 (37); and 4)collaboration of HBx with hepatitis B virus X-associated pro-tein is also capable of coactivating NF-�B signaling (49). Inaddition, several upstream signal transduction cascadesdefined to be involved in HBx-induced activation of NF-�B,including PKC, Src, Ras, and Akt have been identified (12, 14,39, 45). In this study, we define another upstream signalingpathway. HBxmay activate NF-�B activity though phosphoryl-ation of TAK1 and IKK. Blockade of TAK1 activity by its spe-cific inhibitor markedly inhibits NF-�B activity and IP-10 lucif-erase activity. As a pivotal factor for MAPK and NF-�Bactivation in response to the Toll-like receptor, interleukin-1R,and TNFR stimulation (40), TAK1 activation requires the par-ticipation ofTRAF familymembers such asTRAF2 andTRAF6.Here, we further confirm that TRAF2 is required for HBx-in-duced NF-�B activation.IP-10 shares receptor CXCR3 with CXCL9 (Mig) recruiting

inflammatory cells to the sites of infected tissues andmay exag-gerate the immune-mediated injury. In the mouse model ofhepatitis virus infection, treatment with either anti-IP-10 oranti-Mig antibody shows a decrease of T cell infiltration intothe brain (41, 42). Similarly, in HBV transgenic mice, blockadeof IP-10 significantly reduces the recruitment of host-derivedinflammatory cells into the liver and the severity of liver dam-age (9). Those findings demonstrate that CXCR3 signaling iscritical in leukocytic chemotaxis and the pathogenesis of liverdisease after HBV infection. Interestingly, in the present study,we find that such chemotaxis activity of HBx-induced IP-10requires NF-�B activity. Transfection of either p65 or p50expression vectors to cells significantly increased the che-moattractive effect, whereas the addition of NF-�B inhibi-tors to the culture medium significantly decreased the che-motaxis, suggesting that NF-�B is a critical mediator for theresulting HBx, IP-10-induced chemotaxis. Blockade of theNF-�B pathway has served as a potential strategy to treatinflammatory diseases and tumors, such as rheumatoidarthritis, myeloma, and T-cell leukemia/lymphoma (43, 44,50). Our findings further suggest that targeting the NF-�Bpathway is a potential therapeutic strategy against HBV-re-lated inflammatory damage, which not only blocks IP-10generation but also impairs its chemotatic function.In summary, the data reported here show for the first time

that theHBxprotein encoded byHBV induces IP-10 expressiondirectly in hepatocytes, and binding of NF-�B to the NF-�B1site in the IP-10 promoter is important for HBx-induced IP-10expression. The underlying molecular mechanism involvesTRAF2/TAK1-mediated signaling pathway. Our data also indi-cate that the NF-�B signaling pathway may play an importantrole in chemotaxis of the HBx-induced IP-10. These findingsmay shed light on the understanding of how leukocytesmigrateinto the liver and exaggerate host-derived immune responses,and probably provide a novel therapeutic target in severe acuteor fulminant hepatitis.

Acknowledgment—We thank Dr.Wen-jie Huang for the HBV proteinexpression vectors.

4 Y. Zhou, S. Wang, J.-W. Ma, Z. Lei, H.-F. Zhu, P. Lei, Z.-S. Yang, B. Zhang, X.-X.Yao, C. Shi, L.-F. Sun, X.-W. Wu, Q. Ning, G.-X. Shen, and B. Huang, unpub-lished data.

FIGURE 8. HBx-induced IP-10 expression increases the chemotaxis. Super-natants from HepG2 cells transfected with the indicated plasmid cells wereadded to the lower chamber of transwell plates, and PBLs were placed in theupper chamber. The lower chamber was in the presence of neutralizing anti-IP-10 antibody or control antibody. After a 4-h incubation, the number ofmigrated cells was counted in three randomly selected fields per well. Recom-binant IP-10 (10 ng/ml) and antibody alone (10 �g/ml) were used as positiveand negative controls, respectively. The number of migrated cells in responseto supernatants from HepG2 cells transfected with the pCMV-tag was set to100%. Results are mean � S.D. of three experiments performed in duplicate.*, a statistically significant difference from mock plasmids (p � 0.05). Error barsrepresent S.D. **, a statistically significant difference from mock plasmids (p �0.01).

HBx-induced Expression

APRIL 16, 2010 • VOLUME 285 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 12167

by guest on February 10, 2020http://w

ww

.jbc.org/D

ownloaded from

REFERENCES1. Chisari, F. V., and Ferrari, C. (1995) Annu. Rev. Immunol. 13, 29–602. Tiollais, P., Charnay, P., and Vyas, G. N. (1981) Science 213, 406–4113. Luster, A. D.,Wang, J., Zhao, J. H.,Wang, P. P., Xiang, G. J., Lee, Y.H., Yun,

Y., Doria, M., Klein, N., Lucito, R., and Schneider, R. J. (1998) N. Engl.J. Med. 338, 436–445

4. Rossi, D., and Zlotnik, A. (2000) Annu. Rev. Immunol. 18, 217–2425. Narumi, S., Tominaga, Y., Tamaru, M., Shimai, S., Okumura, H., Nishioji,

K., Itoh, Y., and Okanoue, T. (1997) J. Immunol. 158, 5536–55446. Nishioji, K., Okanoue, T., Itoh, Y., Narumi, S., Sakamoto, M., Nakamura,

H., Morita, A., and Kashima, K. (2001) Clin. Exp. Immunol. 123, 271–2797. Neville, L. F., Mathiak, G., and Bagasra, O. (1997) Cytokine Growth Factor

Rev. 8, 207–2198. Dufour, J. H., Dziejman,M., Liu,M.T., Leung, J. H., Lane, T. E., and Luster,

A. D. (2002) J. Immunol. 168, 3195–32049. Kakimi, K., Lane, T. E.,Wieland, S., Asensio, V. C., Campbell, I. L., Chisari,

F. V., and Guidotti, L. G. (2001) J. Exp. Med. 194, 1755–176610. Wang, J., Zhao, J. H., Wang, P. P., and Xiang, G. J. (2008) Hepatobiliary

Pancreat. Dis. Int. 7, 45–5011. Robinson, W. S., Clayton, D. A., and Greenman, R. L. (1974) J. Virol. 14,

384–39112. Kekule, A. S., Lauer, U., Weiss, L., Luber, B., and Hofschneider, P. H.

(1993) Nature 361, 742–74513. Diao, J., Garces, R., and Richardson, C. D. (2001) Cytokine Growth Factor

Rev. 12, 189–20514. Doria, M., Klein, N., Lucito, R., and Schneider, R. J. (1995) EMBO J. 14,

4747–475715. Benn, J., Su, F., Doria, M., and Schneider, R. J. (1996) J. Virol. 70,

4978–498516. Su, F., and Schneider, R. J. (1996) J. Virol. 70, 4558–456617. Lucito, R., and Schneider, R. J. (1992) J. Virol. 66, 983–99118. Moon, E. J., Jeong, C. H., Jeong, J. W., Kim, K. R., Yu, D. Y., Murakami, S.,

Kim, C. W., and Kim, K. W. (2004) FASEB J. 18, 382–38419. Maguire, H. F., Hoeffler, J. P., and Siddiqui, A. (1991) Science 252,

842–84420. Williams, J. S., andAndrisani, O.M. (1995)Proc. Natl. Acad. Sci. U.S.A. 92,

3819–382321. Ghosh, S., May, M. J., and Kopp, E. B. (1998) Annu. Rev. Immunol. 16,

225–26022. Karin, M., and Delhase, M. (2000) Semin. Immunol. 12, 85–9823. Spurrell, J. C.,Wiehler, S., Zaheer, R. S., Sanders, S. P., and Proud,D. (2005)

Am. J. Physiol. Lung Cell Mol. Physiol. 289, L85–L9524. LeGoffic, R.,Mouchel, T., Aubry, F., Patard, J. J., Ruffault, A., Jegou, B., and

Samson, M. (2002) Endocrinology 143, 1434–144025. Nazar, A. S., Cheng, G., Shin, H. S., Brothers, P. N., Dhib-Jalbut, S., Shin,

M. L., and Vanguri, P. (1997) J. Neuroimmunol. 77, 116–12726. Nakamichi, K., Saiki, M., Sawada, M., Takayama-Ito, M., Yamamuro, Y.,

Morimoto, K., and Kurane, I. (2005) J. Virol. 79, 11801–11812

27. Law, A. H., Lee, D. C., Cheung, B. K., Yim, H. C., and Lau, A. S. (2007)J. Virol. 81, 416–422

28. Asensio, V. C., Maier, J., Milner, R., Boztug, K., Kincaid, C., Moulard, M.,Phillipson, C., Lindsley, K., Krucker, T., Fox, H. S., and Campbell, I. L.(2001) J. Virol. 75, 7067–7077

29. Park, S. R., Lee, J. H., andKim, P.H. (2001)Eur. J. Immunol. 31, 1706–171530. Kim, S. J., Rabbani, Z. N., Dewhirst, M.W., Vujaskovic, Z., Vollmer, R. T.,

Schreiber, E. G., Oosterwijk, E., and Kelley, M. J. (2005) Lung Cancer 49,325–335

31. Takenaka,M., Preston, A. S., Kwon, H.M., andHandler, J. S. (1994) J. Biol.Chem. 269, 29379–29381

32. Kim, Y., and Fischer, S. M. (1998) J. Biol. Chem. 273, 27686–2769433. Hayden, M. S., and Ghosh, S. (2004) Genes Dev. 18, 2195–222434. Weil, R., Sirma, H., Giannini, C., Kremsdorf, D., Bessia, C., Dargemont, C.,

Brechot, C., and Israel, A. (1999)Mol. Cell. Biol. 19, 6345–635435. Aderem, A., and Ulevitch, R. J. (2000) Nature 406, 782–78736. Ninomiya-Tsuji, J., Kajino, T., Ono, K., Ohtomo, T., Matsumoto, M.,

Shiina, M., Mihara, M., Tsuchiya, M., andMatsumoto, K. (2003) J. Biol.Chem. 278, 18485–18490

37. Kim, W. H., Hong, F., Jaruga, B., Hu, Z., Fan, S., Liang, T. J., and Gao, B.(2001) FASEB J. 15, 2551–2553

38. Wenzel, J., Zahn, S., Bieber, T., and Tuting, T. (2009)Arch. Dermatol. Res.301, 83–86

39. Kim, H., Lee, Y. H., Won, J., and Yun, Y. (2001) Biochem. Biophys. Res.Commun. 286, 886–894

40. Sato, S., Sanjo, H., Takeda, K., Ninomiya-Tsuji, J., Yamamoto, M., Kawai,T., Matsumoto, K., Takeuchi, O., and Akira, S. (2005) Nat. Immunol. 6,1087–1095

41. Mahalingam, S., Farber, J. M., and Karupiah, G. (1999) J. Virol. 73,1479–1491

42. Liu, M. T., Chen, B. P., Oertel, P., Buchmeier, M. J., Armstrong, D., Ham-ilton, T. A., and Lane, T. E. (2000) J. Immunol. 165, 2327–2330

43. Feldmann, M., Maini, R. N., Bondeson, J., Taylor, P., Foxwell, B. M., andBrennan, F. M. (2001) Adv. Exp. Med. Biol. 490, 119–127

44. Harousseau, J. L., Shaughnessy, J., Jr., and Richardson, P. (2004)Hematol-ogy Am. Soc. Hematol. Educ. Program, 237–256

45. Chung, T. W., Lee, Y. C., and Kim, C. H. (2004) FASEB J. 18, 1123–112546. Diao, J., Khine, A. A., Sarangi, F., Hsu, E., Iorio, C., Tibbles, L. A.,

Woodgett, J. R., Penninger, J., and Richardson, C. D. (2001) J. Biol. Chem.276, 8328–8340

47. Lee, Y. H., and Yun, Y. (1998) J. Biol. Chem. 273, 25510–2551548. Rotondi,M., Lazzeri, E., Romagnani, P., and Serio,M. (2003) J. Endocrinol.

Invest. 26, 177–18049. Shamay, M., Barak, O., Doitsh, G., Ben-Dor, I., and Shaul, Y. (2002) J. Biol.

Chem. 277, 9982–998850. Horie, R., Watanabe, T., and Umezawa, K. (2006)Drug News Perspect. 19,

201–209

HBx-induced Expression

12168 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 16 • APRIL 16, 2010

by guest on February 10, 2020http://w

ww

.jbc.org/D

ownloaded from

Guan-Xin Shen and Bo HuangYang, Biao Zhang, Xin-Xin Yao, Chuan Shi, Li-Fang Sun, Xiong-Wen Wu, Qin Ning, Yu Zhou, Shuo Wang, Jing-Wei Ma, Zhang Lei, Hui-Fen Zhu, Ping Lei, Zhuo-Shun

B and Increases Migration of LeukocytesκMediated through Activation of NF-Hepatitis B Virus Protein X-induced Expression of the CXC Chemokine IP-10 Is

doi: 10.1074/jbc.M109.067629 originally published online February 17, 20102010, 285:12159-12168.J. Biol. Chem. 

  10.1074/jbc.M109.067629Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/285/16/12159.full.html#ref-list-1

This article cites 49 references, 23 of which can be accessed free at

by guest on February 10, 2020http://w

ww

.jbc.org/D

ownloaded from