HBx Protein of Hepatitis B Virus Activates Jak1-STAT Signaling*

The X-gene product (HBx) of the hepatitis B virus plays essential roles in viral replication and the generation of hepatocellular carcinoma. Although the mechanism for HBx action is unclear, HBx may exert its pleiotropic functions through the stimulation of signal transduction pathways including the Ras/mitogen-activated protein kinase cascade and/or inactivation of the p53 function. Here, we investigated whether HBx has the ability to activate the Jak-STAT signaling pathway. As a first step, we established stable cell lines constitutively expressing HBx. In these HBx-expressing stable cells, the tyrosine phosphorylation of various STATs, including STAT3 and -5, was constitutively enhanced by HBx, and the concomitant increase in STAT-dependent DNA binding and transcriptional activation was observed. Furthermore, HBx specifically elevated tyrosine phosphorylation and in vitro kinase activity of Jak1, but not Jak2 or Tyk2, through protein to protein interaction with Jak1. These results clearly establish HBx as the inducer of the Jak-STAT signaling pathway, and at the same time, HBx-mediated Jak-STAT activation may provide a novel mechanism for the pleiotropic functions of HBx, including transformation and promiscuous transcriptional activation.

The X-gene product (HBx) of the hepatitis B virus plays essential roles in viral replication and the generation of hepatocellular carcinoma. Although the mechanism for HBx action is unclear, HBx may exert its pleiotropic functions through the stimulation of signal transduction pathways including the Ras/mitogen-activated protein kinase cascade and/or inactivation of the p53 function. Here, we investigated whether HBx has the ability to activate the Jak-STAT signaling pathway. As a first step, we established stable cell lines constitutively expressing HBx. In these HBx-expressing stable cells, the tyrosine phosphorylation of various STATs, including STAT3 and -5, was constitutively enhanced by HBx, and the concomitant increase in STAT-dependent DNA binding and transcriptional activation was observed. Furthermore, HBx specifically elevated tyrosine phosphorylation and in vitro kinase activity of Jak1, but not Jak2 or Tyk2, through protein to protein interaction with Jak1. These results clearly establish HBx as the inducer of the Jak-STAT signaling pathway, and at the same time, HBx-mediated Jak-STAT activation may provide a novel mechanism for the pleiotropic functions of HBx, including transformation and promiscuous transcriptional activation.
The hepatitis B virus is known as a causative agent of hepatitis, cirrhosis, and hepatocellular carcinoma (1). Among the four proteins originated from the hepatitis B virus genome, the X-gene product (HBx) 1 has drawn much attention for its pleiotropic functions as a viral transactivator. HBx plays essential roles in viral replication as shown in an in vivo animal study (2) and in a transfection-based replication assay (3). Moreover, HBx induces hepatocellular carcinoma in transgenic mice (4) and deregulates cell cycle checkpoint controls (5). A possible mechanism of HBx-mediated cell cycle activation is the disruption of p53 function as a tumor suppressor through the direct protein to protein interaction (3,6). Alternatively, HBx may promote cell proliferation by activating the signal transduction cascades including the Ras/mitogen-activated protein kinase (7), c-Jun N-terminal kinase (8), nuclear factor-B (9), and Src-dependent (10) pathways. Stimulation of these signaling pathways leads to the activation of Activating Protein-1 and nuclear factor-B-dependent transcriptional activation (7)(8)(9)(10).
Supposedly, the combined actions of these mechanisms may be responsible for the HBx-mediated tumorigenesis. On the other hand, HBx induces apoptosis in a p53-dependent manner and sensitizes cells to apoptosis by tumor necrosis factor (11)(12)(13), even though the role of HBx-mediated apoptosis in Hepatitis B virus pathogenesis is still not clear.
Many cytokine receptors lack intrinsic tyrosine kinase domains. Instead, they are associated with the Janus kinase (Jak) family of protein-tyrosine kinases to couple ligand binding to tyrosine phosphorylation of intracellular signaling molecules (14,15). Up to now, four members of the Jak family, including Jak1, Jak2, Jak3, and Tyk2, have been characterized. STAT proteins are a family of transcription factors of 90 -110 kDa that are activated upon tyrosine phosphorylation by Jak kinase (14,15). Growth factor or cytokine treatment leads to the oligomerization of receptor subunits, and receptorassociated Jaks phosphorylate STATs recruited to the receptors by binding to the phosphotyrosine residues through their SH2 domains. In turn, activated STATs are dimerized or multimerized through their SH2 domains, transported into the nucleus, and then bind to the specific STAT-binding DNA sequences leading to the activation of various genes.
Regulation of Jak-STAT is linked to various biological aspects like cell transformation, development, differentiation, apoptosis, etc. Previous reports have suggested that transformation by several oncogenes like src and abl accompanies the activation of the Jak-STAT signaling pathway (16 -18), and especially in the liver, the constitutive elevation of Jak-STAT signaling is associated with hepatocyte proliferation in response to growth factor/cytokine or partial hepatectomy (20 -22).
Insomuch as HBx is known to stimulate cell proliferation, and Jak-STAT is associated with the proliferation signal of liver cells, we tested the effect of HBx on Jak-STAT signaling pathway in liver cells. We demonstrate that HBx activates various STATs through the specific stimulation of Jak1 tyrosine kinase. Combined with the fact that HBx activates several growth factor-dependent signaling, including the Ras/mitogenactivated protein kinase, c-Jun N-terminal kinase, and nuclear factor-B pathways (7-10), the observed Jak-STAT activation establishes HBx as an "internal ligand" of the growth factor/ cytokine-dependent signal transduction.

EXPERIMENTAL PROCEDURES
Plasmids-The HBx expression plasmid, pcDNA-X, has been described (3). pMFG-HBx, a retroviral expression plasmid, was constructed by subcloning the NcoI/BglII fragment of pcDNA-X into the NcoI/BamHI site of MFG-puro. Eight deletion mutants of HBx (XD1-XD8) were constructed by site-directed mutagenesis using pcDNA-X as the template. The mutant clones were selected by DNA sequencing, and the size of the translated product was confirmed by in vitro translation in the presence of [ 35 S]methionine.
Generation of Stable Cell Lines Expressing HBx-Stable cell lines were obtained by Lipofectin-mediated transfection of a mouse hepatoma cell line, Hepa 1-6 with pMFG-HBx plasmid. Twenty-four hours after transfection, the medium was replaced with medium containing puromycin (1 g/ml for Hepa 1-6). The candidate clones were analyzed by Western blot analysis using anti-HBx antiserum. Anti-HBx antiserum was obtained by immunizing two different keyhole limpet hemocyanin-conjugated HBx peptides into rabbits. The peptide sequences used for immunizations are LSAMSTTDLEAYFKDC (corresponding to amino acids 100 -115 of HBx) and SPAPCNFFTSA (corresponding to amino acids 144 -154 of HBx). These peptides were previously reported as highly immunogenic (24).
Luciferase Assay-One g of SIE-Luc reporter was cotransfected with 1 g of pcDNA-X or a series of mutant expression plasmids. Cells were incubated with DNA-Lipofectin for 20 h in serum-free Dulbecco's modified Eagle's medium. After the addition of 0.5% serum, the cells were further incubated for an additional 20 h and harvested, and luciferase activity was measured.

RESULTS
Establishment of HBx-stable Cell Line-Here we investigated the effect of HBx on the Jak-STAT signaling pathway. As a first step, we established stable cell lines expressing HBx in a constitutive manner. The expression construct pMFG-HBx is based on the retroviral vector pMFG (23) and depicted in Fig.  1A. pMFG-HBx was transfected into Hepa 1-6 mouse hepatoma cell line, and stable clones were selected in the presence of puromycin. As a negative control, parental pMFG-puro vector was transfected to generate Hepa-puro cell line. The expression of HBx in these stable clones was tested by Western blot analysis using antiserum raised against the synthetic peptides derived from HBx (24). As shown in Fig. 1B, HBx was detected as a 17-kDa protein, and two clones, Hepa-X1 and Hepa-X2, were selected for further study. These two clones were maintained for several months without losing HBx expression. In contrast to the Hepa 1-6 case, the efforts to establish HBxstable clones in human liver cell lines HepG2 and the Chang liver cell failed because of apparent cell death (data not shown). This result probably reflects the previously reported apoptosis phenomena of HBx (11)(12)(13).
HBx Enhances Tyrosine Phosphorylation of STATs in Liver Cells-As a next step, we tested the possibility of HBx inducing the tyrosine phosphorylation of STATs, because the tyrosine phosphorylation of STATs is essential for their dimerization and activation (14,15). Generally, activated STATs show the reduced mobility in SDS-PAGE because of phosphorylation and translocate into the nucleus (14,15). Therefore, nuclear extracts of Hepa-puro and Hepa-X cells were subjected to Western analysis using antibodies against 5 different STATs including STAT1 through -5 ( Fig. 2A). Surprisingly, all 5 antibodies tested detected the appearance of bands with reduced mobility in Hepa-X cells reflecting the post-translational modification, probably resulting from the tyrosine phosphorylation of all 5 STATs tested by HBx. The band shift of the STAT5 case was most prominent.
The tyrosine phosphorylation of STATs by HBx was further confirmed by immunoprecipitation-phosphotyrosine blotting experiments. As representative examples, STAT3 and -5 were immunoprecipitated from Hepa-X cells, and the tyrosine phosphorylation status was investigated by Western blot analysis with anti-phosphotyrosine antibodies (Fig. 2B). As expected, the tyrosine phosphorylation of STAT3 and -5 was constitutively elevated in Hepa-X cells indicating that HBx possesses the ability of inducing the tyrosine phosphorylation of STAT3 and -5 in the liver cells. Even though we have not shown directly the tyrosine phosphorylation of STAT1, -2, and -4, it is most likely that these STATs are also phosphorylated upon expression of HBx because the retarded migration is observed for all five STATs (Fig. 2A). The activation of multiple STATs may explain part of the pleiotropic functions of HBx in liver cells.
HBx Activates Jak1 Tyrosine Kinase-In the liver, three members of the Jak kinase, Jak1, Jak2, and Tyk2, are present (14,15). To determine the upstream events leading to the activation of STATs, we tested whether any of the Jak kinase members are involved in the signaling by HBx. Immunoprecipitation of Jak1/Jak2 and subsequent Western blot analysis with anti-phosphotyrosine antibody revealed that the tyrosine phosphorylation of Jak1, but not that of Jak2, is specifically enhanced in Hepa-X cells (Fig. 3A, left panel). Western analysis of the same immunoprecipitate with anti-Jak1 antibody detected an approximately equal intensity of the signal in both control and Hepa-X cells (Fig. 3A, right panel) ruling out the possibility that the increased tyrosine phosphorylation of Jak1 by HBx is because of the change in the protein level of Jak1. Of note is that, in addition to the approximately 130-kDa bands representing Jak, additional phosphoprotein bands of Ͼ200 and 90 kDa are detected at a low stringency immunoprecipitation condition (Fig. 3A, left panel). As previously reported (25), these associated bands may represent the Jak1-associated proteins, potentially the receptors or STATs. Insomuch as tyrosine phosphorylation of Jaks was shown to result in the increased kinase activity, we investigated whether the expression of HBx enhances the in vitro kinase activity of Jak1 (Fig.  3B). Jak1, Jak2, and Tyk2 were immunoprecipitated, incubated with ␥-[ 32 P]ATP, and the autophosphorylation was assayed as an indicator of kinase activity. We observed that the autophosphorylation of Jak1 was specifically enhanced in Hepa-X cells compared with the control, whereas those of Jak2 and Tyk2 were not altered. Based on the above results, we conclude that HBx induces the tyrosine phosphorylation of Jak1 leading to the enhancement of Jak1 tyrosine kinase activity and subsequent phosphorylation of multiple STATs.
Association between HBx and Jak1 Kinase-Previously, viral transforming proteins like Src or Abl were shown to activate the STAT pathway through direct interaction with Jak kinase (18,19). To define the mechanism for the HBx-mediated Jak1 activation, we tested the possibility of interaction between HBx and Jak1. HBx was prepared by in vitro translation in the presence of [ 35 S]Met (Fig. 4, left panel). Jak1 and Jak2 proteins were expressed using the baculoviruses kindly provided by Dr. J. Ihle (26). The Sf9 cell lysates were prepared and subjected to immunoprecipitation with Jak1 or Jak2 antibody, and protein A-agarose-bound Jak1 or Jak2 proteins were incubated with in vitro translated HBx. Subsequently, protein A-bound fractions were washed with buffer, resolved by SDS-PAGE, and exposed to x-ray film. As shown in Fig. 4, right panel, the HBx band was specifically coprecipitated with Jak1 protein but not with Jak2. Based on these results, we propose that HBx stimulates the Jak1-STAT pathway through protein to protein interaction with Jak1.
HBx Enhances STAT-dependent Transcription Activation and DNA Binding-The observed tyrosine phosphorylation of STATs led us to test the effect of HBx on STAT-dependent transcriptional activation and DNA binding. The transcriptional activation was assayed in heterologous, human hepatoma cell line HepG2 to test whether the observed STAT activation by HBx is extended to liver cell lines other than Hepa 1-6 (Fig. 5A). The reporter SIE-Luc and its mutant version, mSIE-Luc, were described previously (27) and contain 3 repeats of SIE or mutant SIE, respectively. When SIE-Luc was transiently cotransfected with the HBx expression plasmid pcDNA-X, approximately a 4-fold activation of luciferase activity was observed compared with the negative control. The epidermal growth factor treatment of Hepa-puro cells used as a positive control resulted in 7-fold activation. On the other hand, mutant SIE (mSIE)-driven reporter activity was not elevated by the coexpression of HBx, indicating that HBx spe- HBx-expressing Hepa-X (ϩ) cells, tyrosine phosphorylation of Jak1 and Jak2 was analyzed by immunoprecipitation with anti-Jak antibody followed by Western blot with anti-phosphotyrosine antibody. The tyrosine phosphorylation of Jak1 but not Jak2 is enhanced in Hepa-X cells compared with Hepa-puro cells (left panel). To control the level of Jak1, the same immunoprecipitates were subjected to Western analysis with anti-Jak1 antibody (right panel). B, HBx induces the kinase activity of Jak1. The effects of HBx on the kinase activity of Jak1, Jak2, and Tyk2 were analyzed by the in vitro kinase assay of the immunoprecipitated proteins. The immunoprecipitates were washed with a kinase buffer and incubated with ␥-[ 32 P]ATP. cifically activates STAT-dependent transcription activation. In addition, these results suggest that STAT activation is not limited to Hepa 1-6 cells and is not caused by the secondary effect introduced during the establishment of Hepa-X stable cells.
Insomuch as STAT is known as a DNA-binding transcription factor, we tested the effect of HBx on the DNA-binding activity of STATs by electrophoretic mobility shift assay (Fig. 5B). Nuclear extracts were prepared from the control Hepa-puro and Hepa-X cells and incubated with a labeled APRE probe, which has been shown to bind STAT3 (28). As shown in Fig. 5B, the APRE-binding activity was enhanced by approximately 4-fold in Hepa-X cells. The observed DNA-binding complex was specifically competed by the 100-fold excess of unlabeled APRE but not by an unrelated Fos intragenic regulatory element. Moreover, the addition of anti-STAT3 antibody led to the appearance of a supershifted band indicating that the constitutive enhancement of DNA-binding activity in Hepa-X cells is attributable to STAT proteins.
The Central Region of HBx Is Responsible for STAT Activation-Next, to control the effect of wild type HBx and also to map the domain responsible for the STAT activation, we generated a series of HBx deletion mutants (Fig. 6A) and tested the effects of these mutants on SIE-Luc reporter activity (Fig. 6B). Except for the deletions in the N-(XD1) or C-terminal (XD8) region, all of the internal deletions of HBx resulted in the reduced activation of STAT-dependent transcription, suggesting that a relatively broad, central region of HBx is responsible for STAT activation. Perhaps these internal deletions of HBx alter the conformation of HBx necessary for the interaction with Jak1. The domain necessary for STAT-dependent transcriptional activation is overlapped with the previously identified transcriptional activation domains of HBx (29, 30) except for the difference in the XD2 case. These domain mapping results suggest that a certain cross-talk may exist in the activation mechanism of HBx.

DISCUSSION
In this report, we have shown that HBx interacts with Jak1tyrosine kinase leading to the activation of the Jak1-STAT signaling pathway. However, the exact mechanism through which HBx activates Jak1 needs further investigation. Insomuch as HBx can form a dimer (30), HBx dimerization might bring associated Jak1 molecules into close proximity allowing cross-phosphorylation and autoactivation, which in turn leads to the activation of multiple STATs. The full activation of STATs requires serine phosphorylation in addition to tyrosine phosphorylation (28). Considering that HBx activates serine/ threonine kinases like mitogen-activated protein kinase and Jun-N-terminal kinase (7,8), we do not exclude the possibility that HBx assists the activation of STATs through serine phosphorylation in addition to tyrosine phosphorylation (31).
Recently, Klein and Schneider (10) reported that HBx activates Src kinase, and this event is linked to HBx-mediated Ras pathway activation. Because Src is known to activate Jak1 and STAT3 (17,19), there is a possibility that HBx-mediated activation of Src and Jak/STAT is closely related. However, in the Hepa-X cell system, we could not detect any enhancement of Src tyrosine phosphorylation by HBx (data not shown). Although this might be a cell type-specific phenomenon, at least we propose that Jak-STAT activation in Hepa-X cells is not a result of Src activation.
Importantly, HBx-mediated Jak-STAT activation may explain the promiscuous actions of HBx as a viral transactivator. The observed Jak1-STAT activation, in combination with the FIG. 4. Association between HBx and Jak1 kinase in vitro. HBx was prepared by in vitro translation in the presence of [ 35 S]methionine. Jak1 or Jak2 baculovirus-infected Sf9 cells were cultured for 48 h, and cell lysates were prepared by sonication. Jak1 or Jak2 protein was precipitated by incubation with anti-Jak1-or anti-Jak2-bound protein A-agarose. Subsequently, the protein A-bound immunoprecipitates were coincubated with in vitro translated 35 S-labeled HBx for 4 h, washed with wash buffer, resolved by SDS-PAGE, and subjected to autoradiography.
FIG. 5. HBx up-regulates STAT-dependent transcriptional activation. A, activation of SIE (Sis-inducible element)-dependent reporter activity by HBx. HepG2 cells were transiently transfected with 1 g of SIE-Luc reporter in the presence or absence of 1 g of HBx expression plasmid, pcDNA-X. Epidermal growth factor-treated HepG2 cells were used as a positive control. As a negative control, a reporter containing mutant SIE (mSIE-Luc) was employed. Forty hours later, luciferase activity was assayed. The experiments were performed in duplicate and at least 3 times. B, DNA-binding activity of STAT was assayed by EMSA. Nuclear extracts were prepared from serum-starved control Hepa-puro and HBx-expressing Hepa-X cells. The same amount of nuclear extract was used for each EMSA with 10 fmol of ␥-32 P-labeled APRE (acute phase response element) as a probe. One pmol each of unlabeled wild type (W, APRE) or mutant (M, Fos intragenic regulatory element) oligonucleotide was used for competition assay. For antibody supershift assay, 1 g of STAT3 antibody (ST3) was included in the reaction mixture. The supershifted band (SS) is marked by an arrow. N.E., nuclear extract; Com, competitor; W, wild type competitor; M, mutant competitor; ST, STAT3 antibody. fact that HBx also activates the serine/threonine kinase pathway through the activation of Ras and Src (7,10), establishes that HBx behaves like a typical growth factor and acts at the membrane proximal level. As reported previously, activated Jak1 may recruit a number of molecules, including STATs, Shc, and Raf (33)(34)(35)(36). In addition, activated STAT3 can act as an adaptor protein, recruiting phosphatidylinositol 3-kinase to the receptor complex (37). Therefore, activation of Jak1 thereby mediates multiple impacts of HBx on cytoplasmic signaling pathways as well as on nuclear transcriptional events.
The finding in this report establishes hepatitis B virus as the first example of a transforming DNA virus, the gene product that activates the Jak/STAT signaling pathway. Up to now, Jak/STAT activation was limited for some oncogenic retroviruses like v-src, v-abl, and human T-cell lymphotrophic virus (16 -19). The activation of the Jak/STAT-signaling pathway may contribute to the transforming potential of HBx, even though the role of Jak/STAT signaling in mitogenesis is still under debate. Cumulating evidence indicates that the Jak/ STAT pathway is activated in the transformed cell (16 -19), and the genetic studies of the HOP/D-STAT pathway in Dro-sophila are suggestive of a role of Jak/STAT in cell proliferation and oncogenesis (32). Moreover, a cross-talk between Jak/ STAT and the oncogenic Ras pathway has been reported extensively (33)(34)(35)(36). In cytokine-growth factor signaling, activated Jaks tyrosine phosphorylates shc or raf, which in turn activates the Ras-Raf-mitogen-activated protein kinase pathway. Perhaps previously reported Ras/mitogen-activated protein kinase activation by HBx may be mediated through Jak/ STAT. Taking this information together, we propose that the HBx-mediated Jak-STAT activation contributes at least in part to the development of hepatocellular carcinoma.
In addition to the potential role in transformation, HBxmediated Jak/STAT activation may be linked to a variety of pathogenic phenomena caused by the infection of the hepatitis B virus. For example, constitutive activation of STAT3 leads to the dysregulation of the interleukin-6 gene, the main mediator of the acute phase response and liver cirrhosis (38,39).
In conclusion, we found that the constitutive expression of HBx results in the activation of the Jak1/STAT signaling pathway, which may potentially provide the mechanism for promiscuous transcription activation by HBx, cirrhosis, and hepatocellular carcinoma. Activation of Jak1/STAT by the viral transactivator may be a common strategy adopted by a number of viruses to exploit the host cell machinery.
FIG. 6. Central region of HBx is responsible for STAT activation. A, construction of HBx deletion mutants. Eight different internal deletion mutants of HBx were generated by site-directed mutagenesis and subcloned into the expression vector, pcDNAI/Amp. The domains deleted are indicated at the left. The identity of the HBx mutants was confirmed by DNA sequencing and in vitro translation using reticulocyte lysate as shown in the lower panel. B, the central region of HBx is responsible for STAT activation. The HBx mutants were cotransfected with SIE-Luc reporter into HepG2 cells. The experiments were also performed in duplicate and at least 3 times.