Activation of Focal Adhesion Kinase by Hepatitis B
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病菌学杂志 2006年第9期
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102
Department of Microbiology, New York University School of Medicine, New York, New York 10016
ABSTRACT
The hepatitis B virus (HBV) X protein (HBx) is a multifunctional regulator of cellular signal transduction and transcription pathways and has a critical role in HBV replication. Much of the cytoplasmic signal transduction activity associated with HBx expression and its stimulation of viral replication is attributable to HBx-induced activation of calcium signaling pathways involving Pyk2 and Src tyrosine kinases. To further characterize upstream signal transduction pathways that are required for HBx activity, including activation of Src and mitogen-activated protein kinase (MAPK) cascades, we determined whether focal adhesion kinase (FAK), a known regulator of Src family kinases and the other member of the Pyk2/FAK kinase family, is activated by HBx. We report that HBx activates FAK and that FAK activation is important for multiple HBx functions. Dominant inhibiting forms of FAK blocked HBx activation of Src kinases and downstream signal transduction, HBx stimulation of NF-B and AP-1-dependent transcription, and HBV DNA replication. We also demonstrate that HBx-induced activation of FAK is dependent on cellular calcium signaling, which is modulated by HBx. Moreover, prolonged expression of HBx increases both FAK activity and its level of expression. FAK activation may play a role in cellular transformation and cancer progression. HBx stimulation of FAK activity and abundance may also be relevant as a potential cofactor in HBV-associated hepatocellular carcinoma.
INTRODUCTION
It is estimated that there are 350 million people chronically infected with hepatitis B virus (HBV), which is significantly associated with development of hepatocellular carcinoma (HCC), one of the most common forms of cancer worldwide (reviewed in references 3 and 21). HBV encodes a small genome, consisting of a partially double-stranded, circular DNA that is encapsidated within an enveloped particle (57). The HBV genome contains four open reading frames, encoding the viral envelope proteins (also known as surface antigens), the viral core protein which comprises the viral capsid, a polymerase/reverse transcriptase, and the nonstructural regulatory protein known as HBx. While the precise function of HBx is unresolved, studies have established functions in cellular physiology, viral replication, transcription, and viral pathogenesis (reviewed in reference 10). The activity of HBx in HBV replication may depend on its direct interaction with cellular proteins such as UVDDB (ultraviolet damaged DNA binding protein), HBx-induced transcriptional activation, HBx modulation of intracellular calcium signaling, and stimulation of cellular signal transduction pathways (8, 9, 27, 30, 36, 39).
Numerous studies have identified HBx-responsive transcription factors including NF-B, NF-AT, and AP-1 among others, as well as HBx-responsive transcription elements such as the human immunodeficiency virus long terminal repeat and cyclic AMP response elements (4, 18, 30, 39, 42, 59). Recent reports suggest that at least some HBx-mediated transcriptional activation results from its ability to stimulate cellular calcium signaling pathways, resulting in activation of proline-rich tyrosine kinase 2 (Pyk2) and Src tyrosine kinases and, in turn, downstream signal transduction pathways such as the mitogen-activated protein kinase (MAPK) pathway (4, 6, 8, 9, 28, 34). HBx might also directly interact with components of the basal transcription machinery, such as ribosome binding protein 5 and TATA-binding protein, as well as the transcriptional activator CREB/ATF (16, 42, 58, 59). This provides another mechanism through which HBx stimulates transcription and possibly HBV replication. HBx can deregulate cell cycle progression checkpoints by inducing activation of the cyclin-dependent kinases CDK2 and CDK1 and association of these kinases with cyclins E and A or cyclin B, respectively, although the exact influence of HBx on cellular proliferation can vary depending on the transformed state of the cell (5, 26, 34; reviewed in reference 41). Under certain conditions, HBx can modulate cellular apoptotic pathways, although both pro- and antiapoptotic effects of HBx have been reported (reviewed in reference 10). The effect of HBx-associated activities varies in different cellular contexts, and the exact molecular mechanism for its activity is currently still very poorly defined, both when HBx is expressed alone and in the context of HBV replication. Which of the myriad HBx functions are required for HBV replication during natural infection and which activities, if any, influence HBV-associated development of HCC remains to be determined.
Only the mammalian HBVs are associated with HCC, and only the mammalian HBVs have been shown to encode an HBx protein (reviewed in references 1 and 2). An avian HBV has been reported to encode a divergent HBx-like protein, but whether it is expressed during natural infection like mammalian HBV HBx is not certain. The function of a putative avian HBx protein is also not required for replication of the avian virus, unlike that of the mammalian viruses (14, 21, 44). While there is some discrepancy among different HBx-transgenic mouse models, the majority of available evidence suggests a correlation between the presence of HBx and the development of HCC (25, 29, 35, 40). The association between HBx and HCC has therefore stimulated interest in defining the primary function and mechanism of HBx-induced activation of signal transduction pathways and transcription and how these activities could impact cancer etiology. Moreover, the demonstration that HBx is essential for in vivo replication of the woodchuck hepatitis virus and potentially so for replication of human HBV has stimulated interest in elucidating the primary function of this protein during viral replication (8, 15, 45, 60, 62). To identify activities associated with HBx expression that might contribute to its role in viral replication and possibly oncogenesis, we have studied HBx-induced activation of cellular signaling pathways, such as those that translate modulation of cytosolic calcium levels to activation of MAPK pathways, to determine what role these may have in the various activities that have been ascribed to HBx function. We previously demonstrated that HBV replication requires HBx-induced activation of cellular calcium signaling pathways, including stimulation of proline-rich tyrosine kinase 2 (Pyk2) and Src kinases (8, 9), and that HBx expression regulates multiple steps in viral DNA replication through the phosphorylation of HBV core protein (46). Members of the Pyk2/focal adhesion kinase (FAK) family are known to play a key role in cellular signaling pathways which are important for transcriptional regulation, cell cycle progression, modulation of apoptosis, control of cell migration, and metastasis of transformed cells (23, 47, 48, 49). HBx-induced activation of this family of kinases could therefore be important for many of its reported activities. For example, it was previously reported that HBx can modulate migration of human liver cells and regulate cellular adherens junctions in an Src kinase-dependent manner (32, 33). Stimulation of FAK also modulates similar activities, and HBx regulation of FAK could conceivably contribute to a potential involvement of FAK activation in the development of HBV-associated HCC.
The proline-rich tyrosine kinase, Pyk2, and the closely related kinase FAK, comprise a family of cytoplasmic, nonreceptor tyrosine kinases that can be regulated by extracellular stimuli and can activate Src family kinases (reviewed in references 23, 49, and 52). Pyk2 and FAK are activated by numerous mechanisms, one of which is an increase in cytosolic calcium levels. Following activation, FAK and Pyk2 recruit and activate members of the Src kinase family; this is ultimately linked to stimulation of targets such as the ERKs (extracellular-regulated kinases) and c-JUN N-terminal kinases/MAPKs (51, 54). Pyk2 and FAK activity is also induced by stimulation of G protein-coupled receptors, integrin engagement, and various cytokines, such as tumor necrosis factor alpha (reviewed in reference 52). Pyk2 expression can compensate for a subset, though clearly not all of the functions of FAK in cells derived from FAK knockout mice (56). Pyk2 and FAK also share homology, and both proteins contain similarly organized domains such as a central kinase domain flanked by extensive N-terminal and C-terminal regions (reviewed in reference 2). The N-terminal domains contain the major autophosphorylation sites (tyrosine 397 in FAK, tyrosine 402 in Pyk2). Upon activation, phosphorylation of this site creates an SH2 domain binding site for Src kinases. Binding of Src kinases to this site leads to the activation of Src, which further activates Pyk2 or FAK. The subsequent phosphorylation and enhanced activation of Pyk2/FAK is a complex process in which phosphorylation of multiple FAK or Pyk2 amino acid residues recruits other signaling proteins such as Shc, Grb2, and p130-Cas, ultimately activating MAPK cascades. FAK is largely but not entirely located in focal adhesions, which are sites of contact between the cell and the extracellular matrix, although studies demonstrate that Pyk2 can also localize to focal adhesions (38). Activation of FAK, and an increase in its level of expression, have been identified in a large number of transformed cells, including those of breast cancer, prostate cancer, melanoma, and liver cancer, and is thought to play a role in the transformation process (reviewed in references 24 and 43). Activation of FAK by HBx would therefore be of interest in virus-mediated carcinogenesis.
We previously reported that HBx activates Pyk2 in HepG2 cells in a calcium-dependent manner (8, 9). We now demonstrate that HBx also stimulates FAK activity in HepG2 cells and stably increases its level of expression. Similar to HBx induction of Pyk2 activity, stimulation of FAK by HBx is dependent on cytosolic calcium signaling pathways. Our results suggest a mechanism by which HBx might contribute to the development of HCC in chronically HBV-infected individuals by activating the Pyk2/FAK family of kinases. HBx activation of FAK could in turn enhance cell migration and invasiveness, stimulated during the process of transformation and associated with upregulation of FAK activity (2, 12, 13, 19, 23, 47, 55, 61).
MATERIALS AND METHODS
Cell culture. NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium containing gentamicin (5 μg/ml), supplemented with 10% bovine calf serum. HepG2 cells were maintained in minimal essential medium supplemented with gentamicin (5 μg/ml), sodium pyruvate, glutamine, and 10% fetal bovine serum. All cells were maintained at 37°C in 5% CO2.
Plasmids. The HBx expressing plasmids pAdCMVX and pCEP4XFlag and the NF-B and AP-1-promoter responsive luciferase (Luc) reporter plasmids have been described previously (21, 31). The FAK plasmids pFAKKD and pFAK397 were a gift of J.-L. Guan (Cornell University). High-concentration stocks of plasmids were purified using the Concert High-Purity Plasmid Maxiprep system (Life Technologies) according to the manufacturer's directions.
Luciferase assays. Transient transfections were performed with Lipofectamine Plus (Life Technologies) for NIH 3T3 cells and Fugene 6 (Roche) for HepG2 cells, both according to manufacturer directions. For induction of NF-B and AP-1 luciferase, cells were transfected overnight with 2 μg expression plasmids, allowed to recover for 12 h, and then serum starved for 16 h prior to analysis of luciferase activity. Luciferase reporter assays were performed using the Promega Luciferase assay system according to the manufacturer's directions.
FAK phosphorylation. For FAK phosphorylation analyses, NIH 3T3 cells were transfected with expression plasmids or vector control plasmid using Lipofectamine Plus, allowed to recover for 12 h, and then serum starved for 16 h. Medium containing serum was then added, and samples were collected at the indicated times. Either 50 μm BAPTA-AM (Molecular Probes) or 3 mM EGTA (Sigma) was added as indicated and cells collected at the indicated time points. Cells were lysed in modified RIPA buffer (1% Triton X-100, 0.1% sodium dodecyl sulfate, 1% deoxycholate, 50 mM HEPES [pH 7.4], 150 mM sodium chloride, 10% glycerol, 1.5 mM magnesium chloride, 1 mM EGTA, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, and protease inhibitors), protein concentrations were determined, and equal amounts of protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis using a monoclonal antibody that specifically recognizes the phosphorylated tyrosine at amino acid position 397 [Y397(P)] of FAK (Biosource International) (54). Confirmation that equal amounts of FAK had been loaded was performed with a polyclonal anti-FAK antibody (Santa Cruz Biotechnology, Inc.).
Kinase assays. Cells were transfected and processed as described above. Equal amounts of FAK were immunoprecipitated and kinase assays performed as previously described (54). A portion of the immunoprecipitate was used to confirm equal amounts of protein had been purified.
FAK activation measured by binding to Grb2. Activation of FAK also results in phosphorylation of FAK amino acid residue 925, generating a binding site for Grb2 (53, 54). NIH 3T3 cells were transfected with control or HBx expression plasmids as described above, cells were lysed, and FAK was immunoprecipitated. Association of Grb2 with FAK was monitored by immunoblot analysis with an anti-Grb2 antibody (Santa Cruz Biotechnology, Inc.). Total FAK and phosphorylation of FAK at residue 397 were determined as described above.
HBV replication assays. HBV replication assays were conducted as previously described (8, 45). Briefly, 4 days after transfection of wild-type or HBx mutant HBV replicons, cytosolic HBV core particles were precipitated with polyethylene glycol and digested with proteinase K, and core-associated HBV DNA was resolved by electrophoresis in a 1% agarose gel, as described previously (8, 9). DNA was transferred to a nylon membrane, and Southern blot analysis was performed with an HBV-specific probe. Endogenous polymerase assays were carried out as previously described (8, 9).
RESULTS
HBx-mediated activation of FAK. We previously demonstrated that HBx activates Pyk2 and that this activation is dependent on cytosolic calcium signaling pathways (8, 9). To determine whether HBx can also activate FAK, we examined FAK for autophosphorylation mediated by HBx expression. Studies utilized HepG2 cells, a human hepatoblastoma cell line in which HBV replicons can carry out DNA replication, and NIH 3T3 cells, which have been used widely for strong FAK responsiveness. Cells were transfected with HBx expression vectors or control vectors, placed in normal growth medium to allow for HBx expression, and then serum starved overnight. Cells were collected at different times after readdition of normal growth medium, and FAK activity was analyzed. Activity was determined by two methods: immunoblot analysis of activating phosphorylation using a phosphospecific antibody to FAK Y397, the major site of activating phosphorylation (51), and analysis of immunoprecipitated FAK kinase activity. Measurement of the major activating phosphorylation site of FAK is a measure of the increased autophosphorylation kinase activity of FAK (51). Autophosphorylation kinase assays were performed as previously described (54).
Expression of HBx led to FAK Y397 phosphorylation (activation) of about three- to fourfold in both HepG2 and NIH 3T3 cells in an HBx dose-responsive manner (Fig. 1). As a control, FAK Y397 phosphorylation was stimulated by the addition of the phorbol ester tetradecanoyl phorbol acetate (TPA) to the growth medium, which was similar in magnitude to HBx. HBx therefore strongly stimulates FAK activation measured by Y397 phosphorylation. While Y397 phosphorylation is an accepted surrogate for FAK activity, we also performed FAK in vitro kinase assays. Endogenous FAK was immunoprecipitated from equal amounts of lysates obtained from vector control or HBx-expressing HepG2 or NIH 3T3 cells, normalized to equal amounts of total FAK protein, and incubated with [-32P]ATP to measure FAK autophosphorylation (activation) in vitro (Fig. 2). HBx induced 3-fold activation of FAK in both HepG2 and NIH 3T3 cells (upper panels) when equal amounts of FAK protein were assayed (lower panel). Thus, these results are similar in magnitude to the Y397(P) immunoblot analysis with FAK phosphorylation-specific antibody. HBx therefore activates FAK, similar to its activation of Pyk2.
Studies were next carried out to determine whether HBx activation of FAK is sufficient to stimulate downstream signaling pathways. Upon activation of FAK and recruitment of Src kinases, FAK is typically further phosphorylated, including phosphorylation of amino acid residue 925, which creates a binding site for Grb2 (45, 46). Therefore, increased binding of FAK to Grb2 is another indicator of FAK activation (53, 54). We therefore characterized the HBx-induced upregulation of the interaction between FAK and the signal transduction scaffolding protein Grb2. Cells were transfected with HBx or a control expression plasmid and lysed, and FAK was immunoprecipitated (Fig. 3). Equal levels of FAK are apparent in the anti-FAK samples. Immunoblot analysis was used to determine the levels of Grb2 that coimmunoprecipitate with FAK and total Grb2 levels in the lysates. Studies demonstrated that HBx strongly increased the association of Grb2 with FAK (by four- to fivefold), consistent with FAK Y397 phosphorylation. These results provide another independent line of evidence for HBx activation of FAK and assembly of FAK complexes capable of stimulating downstream signal transduction.
HBx-induced activation of FAK is calcium dependent. An important activity of HBx is modulation of cytosolic (intracellular) calcium stores. Activation of Pyk2 by HBx is dependent on cytosolic calcium signaling (8, 9), and FAK can be activated by an increase in cytosolic calcium levels (reviewed in reference 52). To determine whether HBx-induced activation of FAK is dependent on cytosolic calcium signaling, HBx transfected HepG2 cells were treated with BAPTA-AM or EGTA (Fig. 4A). BAPTA-AM is a cell-permeable, intracellular chelator of calcium that can block intracellular calcium signaling, whereas EGTA is not cell permeable and chelates extracellular calcium, inhibiting the influx of calcium from the extracellular medium. As a positive control, vector-alone-transfected cells were treated for 20 min with 20 mM TPA to activate FAK. In control cells (vector alone), TPA stimulated FAK Y397 phosphorylation by 4-fold, compared to 3-fold activation by HBx. Treatment of cells with BAPTA-AM inhibited activation of FAK by both TPA and HBx, demonstrating the requirement for mobilization of intracellular calcium stores. Treatment of cells with EGTA, however, partially blocked TPA activation of FAK, whereas it had no effect on HBx activation of the kinase. Thus, influx of extracellular calcium is important for TPA stimulation of FAK, as expected, whereas HBx activation of FAK utilizes intracellular stores. The calcium-dependent activation of FAK is not cell type specific, as shown in Fig. 4B. In both HepG2 and NIH 3T3 cells, treatment of cells with EGTA did not inhibit HBx-induced activation of FAK, whereas BAPTA-AM treatment reduced HBx activation of FAK in both cell lines by three- to fourfold, measured by reduction in FAK Y397 phosphorylation (Fig. 4B). These results demonstrate that HBx stimulates calcium-dependent signals in a variety of cell types that lead to activation. Moreover, they point to the alteration of intracellular calcium caused by the release from intracellular calcium stores, rather than the influx of calcium from the extracellular medium, as a primary component of HBx activity.
Continuous expression of HBx increases the abundance of FAK. Both increased expression of FAK and increased levels of FAK activity have been observed in numerous cancers where FAK may control or influence the invasive and metastatic potential of tumor cells (reviewed in reference 24). Studies were therefore carried out to determine whether HBx constitutively stimulates FAK activity while also increasing its abundance. At early time points (6 h) following TPA stimulation and serum addition in control samples, increases in FAK activation persisted (indicated by phosphorylation) but were restored to baseline levels at 24 h. In contrast, HBx stimulated FAK similarly (3-fold) at the same time point post-serum addition, based on Y397 phosphorylation (Fig. 4C). There was no measurable increase in the total level of FAK expression in HepG2 or NIH 3T3 cells with HBx expression at 6 h post-serum addition (Fig. 4C). However, by 12 h, and persisting at 30 h (the last time point measured) in HBx-transfected cells, the level of activated FAK was increased by another twofold and the overall abundance of FAK was elevated approximately fourfold above that of the time zero control. Constitutive activation of FAK and increased FAK abundance by HBx were verified in NIH 3T3 and HepG2 cells transfected by an HBx expression construct controlled by either the simian virus 40 promoter in NIH 3T3 cells or the cytomegalovirus promoter in HepG2 cells, evaluated at 48 h after induction (Fig. 4D). In both NIH 3T3 and HepG2 cells, there was a significant increase in the total level of FAK protein expression at late time points as well as sustained FAK activation, whereas the level of the tubulin control was unchanged.
Dominant interfering mutants of FAK inhibit HBx activation of AP-1 and NF-B. Our previous studies demonstrated that a dominant inhibiting form of Pyk2 could block HBx activity (8, 9). To determine whether dominant inhibitors of FAK also impair HBx activities, we cotransfected HBx into NIH 3T3 or HepG2 cells with either an AP-1- or NF-B-dependent luciferase reporter vector and dominant inhibiting mutants of FAK and determined the effect on HBx activation of transcription (Fig. 5). Many previous studies have shown that HBx activates AP-1 and NF-B by three- to fivefold (6), as shown here. FAK mutants consisted of a protein that contains a Y397F mutation (FAK397) and cannot be activated, or a mutation in the kinase domain that blocks activity (FAKKD). The FAK mutants fully blocked HBx induction of AP-1- and NF-B-dependent transcription in both NIH 3T3 and HepG2 cells. These results support the conclusion that HBx-induced activation of the Pyk2/FAK family is essential for its induction of several transcription factors that are typically activated by HBx. However, it is not known whether activation of FAK is sufficient for AP-1/NF-B activation or if other HBx activities are involved as well.
Inhibition of FAK activity blocks HBV DNA replication. Inhibition of Pyk2 and Src kinases was shown to block HBV HBx stimulation of DNA replication in HepG2 cells (8, 27). To determine whether FAK is an important component of HBx-mediated HBV DNA replication, we tested whether dominant inhibiting forms of FAK block HBV DNA replication or transcription. HBV replicons, which consist of the HBV genome as a 130% reiteration (8) that either express HBx (wtHBV) or have a deletion of HBx [HBV (HBx–)], were cotransfected into HepG2 cells with either vector alone or a plasmid expressing the dominant inhibiting FAK397. Studies first determined whether HBx expressed from the HBV replicon activated FAK and the downstream MAPK, ERK2 (Fig. 6A). Activation of FAK and ERK2 were determined by immunoblotting using specific antibodies for the activating sites of phosphorylation. Immunoblot analysis of FAKY397(P) levels demonstrated activation in wtHBV replicon-transfected cells but not in the HBV (HBx–) samples, which was suppressed by inclusion of the dominant-inhibiting form of FAK. Increased levels of total FAK were also evident in the wtHBV samples but not in the HBV(HBx–) samples. Inclusion of the dominant-inhibiting form of FAK in the control wtHBV sample prevented the quantitation of FAK total abundance. Similarly, analysis of ERK2 activity demonstrated a strong increase only in the wtHBV samples, which was also prevented by inclusion of the dominant-inhibiting FAK397. There was no change in total abundance of ERK2 by HBx expression. Thus, HBx activates FAK and ultimately ERK2 in the context of replicating HBV.
HBV DNA replication was assayed by isolation of viral core particles (capsids), extraction of HBV replicative DNA intermediates, and analysis by Southern blot DNA hybridization, as previously described (8). We and others have previously shown that replication of HBV DNA in HepG2 cells is strongly influenced by HBx that is expressed in the context of the replicon, and we have used this system to define important functions of HBx in HBV replication. Both dominant-inhibiting FAK mutant proteins very strongly blocked HBV DNA replication (Fig. 6B). Analysis of viral mRNA levels by Northern blot analysis during the same period of time showed a slight reduction with expression of the FAK-interfering mutants. Inhibition of HBV DNA replication by FAK dominant-negative mutants averaged >15-fold, whereas reduction of viral mRNA levels averaged 2- to 3-fold. Finally, cytoplasmic viral core particles were isolated from cells transfected with wtHBV replicons in the presence or absence of the two FAK dominant inhibitors, and the DNA replication activity of the endogenous, encapsidated polymerase was determined using an in vitro endogenous polymerase assay and -32P-deoxynucleoside triphosphates. Analysis of the products of in vitro DNA elongation and strand completion showed a threefold reduction in polymerase activity obtained from FAK-inhibited cells compared to the noninhibited control (Fig. 6C). Examination of the level of isolated core particles (capsids) which were used for analysis showed them to be the same. These results therefore verify the importance of HBx activation of Pyk2/FAK kinases for HBV DNA replication.
DISCUSSION
It is well established that chronic HBV infections are associated with a several hundred-fold-increased incidence in development of primary liver cancer (reviewed in reference 21). Immune-mediated destruction of infected hepatocytes, the inflammatory response itself, and the subsequent continued turnover and replacement of hepatocytes clearly underlies the development of HBV-associated HCC. However, it is also likely that the virus itself encodes proteins that may be cofactors in the development or progression of liver cancer (17). HBx, the nonstructural regulatory protein of HBV, has been strongly implicated in the etiology or progression of HBV-associated liver disease and cancer in humans and in some HBx-transgenic mouse model systems. HBx expression is either directly associated with the development of liver cancer in some HBx-transgenic mouse strains or an increased progression to liver cancer in other toxin-exposed HBx-transgenic mouse strains (reviewed in reference 10). Many functions have been ascribed to HBx, but the precise molecular mechanism(s) responsible for its activities, and how these activities affect viral replication and possibly liver cell transformation, remain poorly defined. In the work presented here, we have focused on the effect of HBx activation of cellular calcium-dependent signal transduction pathways.
We previously reported that HBx expression activates Pyk2, a nonreceptor tyrosine kinase involved in the regulation of numerous cellular signal transduction pathways (8). Here, we demonstrate that HBx expression also activates the related kinase, FAK, a nonreceptor tyrosine kinase that has roles in signal transduction, cell cycle progression, apoptosis, cell migration, and cell invasion (reviewed in references 23, 47, 49, and 52). In contrast to our studies with Pyk2 in which HBx expression elevated Pyk2 kinase activity without influencing Pyk2 expression levels, in the work presented here, we have shown that HBx increases the expression level and activation of FAK. We also demonstrate that HBx requires cellular calcium signaling to activate FAK, similar to its activation of Pyk2 (8). Many of the activities that are associated with stimulation of the Pyk2/FAK family, such as regulation of cellular signal transduction cascades, cell proliferation, apoptosis, and cell migration (2, 12, 13, 19, 23, 50, 55, 61) have also been reported previously in HBx-expressing cells, long before it was known that HBx stimulates these tyrosine kinases (reviewed in reference 10). Whether all of these HBx activities correlate with its regulation of the Pyk2/FAK family has not been determined but could impact on our understanding of the consequence of HBx expression in different cell types and contexts.
The Pyk2/FAK family of nonreceptor tyrosine kinases are involved in many cellular processes ranging from control of cell migration to regulation of cellular proliferation and survival pathways (2, 12, 13, 19, 23, 47, 50, 55, 61). Both FAK and Pyk2 are activated by similar mechanisms, including stimulation by elevation in intracellular calcium levels. In the work presented here, when combined with our previous studies, we have now shown that HBx can modulate cellular calcium signals and activate both members of the Pyk2/FAK kinase family (8; this study). HBx activates both Pyk2 and FAK in a calcium-dependent manner. We also demonstrate that dominant-inhibiting mutants of FAK and Pyk2 can block many of the reported activities of HBx, including its stimulation of HBV replication (9; this study). Because of the extensive cross talk between FAK and Pyk2 as well as their ability to regulate similar pathways, the inhibition of HBx activities by dominant-negative forms of FAK likely also involve inhibition of Pyk2 activity (37). These observations are similar to the ability of various dominant-negative forms of Src kinases to inhibit HBx activities that are dependent on this family of kinases (27, 28). Pyk2 and FAK regulate similar pathways, and can, to some extent, compensate for the absence of the other protein as well as influence the activity of the other protein (56). Our results demonstrate that, similar to the ability of HBx to activate multiple members of the Src kinase family, HBx can also activate both members of the Pyk2/FAK family through regulation of cellular calcium signals. Activation of Pyk2 and FAK by HBx is likely to impact multiple cellular signal transduction cascades and could therefore contribute to cellular transformation and the development of HCC during HBV infections.
Activation of FAK by HBx could have profound affects on the cell. One function associated with activated FAK is regulation of cell migration. FAK modulates the assembly and disassembly of focal adhesions, sites of contact between cells, and the extracellular matrix (reviewed in reference 47). Importantly, these FAK activities can be regulated by calcium signals within the cell. It was recently reported that HBx can induce the migration of some liver cell lines (32, 33). HBx-enhanced migration was found to be dependent on activation of Src kinases and involved the regulation of adherens junctions, points of cell-cell contact that are modulated by members of the cadherin family of proteins. HBx expression was also shown to modulate matrix metalloproteinase expression which might be important for invasion of a three-dimensional matrix (31). Interestingly, FAK not only regulates focal adhesion formation but it can also regulate cadherin-mediated adhesion and matrix metalloproteinase levels (reviewed in reference 47). FAK-mediated regulation of adherens junctions proceeds through regulation of Src kinases similar to that observed for HBx-mediated regulation of adherens junctions. While our studies, and those reported by others, have not directly assessed the association between HBx activation of FAK and HBx stimulation of migration, our results combined with previous observations suggest a possible connection between HBx regulation of the Pyk2/FAK family and its stimulation of cell migration. Studies designed to directly assess this connection are ongoing, as these HBx functions could influence the rate of progression of hepatocyte transformation, invasiveness, or metastatic activity.
FAK activation has also been associated with stimulation of transcription pathways and modulation of cellular proliferation and survival (2, 12, 13, 19, 23, 47, 50, 55, 61). Many of the effects associated with FAK activation involve interactions with and stimulation of Src kinases. We previously demonstrated that HBx can activate Src kinases and that this activation is necessary for many of the reported properties of HBx, including its role in induction of cell proliferation, activation of transcription factors such as AP-1 and NF-B, and stimulation of HBV DNA replication (7, 8, 27, 28). The requirement for Src kinase activity in this HBx function, combined with our previous observation that dominant-inhibiting forms of Pyk2 can inhibit HBx activation of Src, suggests that the Pyk2/FAK kinase family are the effectors of HBx modulation of cellular calcium. We demonstrated that inhibition of Src kinases blocks HBV replication, HBx activation of various transcription factors, and HBx stimulation of cell proliferation (7, 28). Inhibiting forms of Pyk2 (8, 9) and FAK also block HBV replication (Fig. 6) and activation of AP-1 and NF-B (Fig. 5), suggesting a direct link between the ability of HBx to activate FAK/Pyk2 and activation of Src kinases (8; this report).
Elevation of FAK levels has been observed in various tumors including those in thyroid, prostate, cervix, rectum, and ovarian cancers (reviewed in references 24 and 43). More recently, it was reported that FAK is elevated in HCC and that elevated FAK could be used as a prognostic marker for HCC progression (20). While none of these studies have demonstrated a direct link between cellular transformation and elevation of FAK expression or activity, the fact that many types of cancers are associated with elevated levels of FAK suggests a strong correlation between FAK activity and expression levels and control of normal cellular physiology. It is likely that alterations of normal FAK levels and activities will impact cellular transformation mechanisms. In the studies reported here, we have demonstrated both a constitutive activation of FAK by HBx as well as an increase in FAK expression levels following prolonged expression of HBx (Fig. 4 and 6). The elevation of FAK levels was observed when HBx was expressed in the absence of other viral proteins (Fig. 4C and D) and, although to a lesser extent, when HBx was expressed in the context of viral replication (Fig. 6A, wtHBV). These differences may reflect variations in the levels of HBx expressed in these two systems, in that HBx is likely expressed at lower levels during viral replication. However, both results demonstrate that prolonged expression of HBx constitutively activates FAK and eventually increases its level of expression. Whether this occurs through stabilization of FAK protein levels or by modulating FAK transcription is currently unknown. Importantly, the activation of FAK that is observed in HBx-expressing cells is not simply a consequence of overexpressed FAK, as this activation is observed during early time points in our studies when FAK levels are not elevated by HBx expression (Fig. 4C). While FAK activation can regulate transcription pathways, whether it stimulates its own production is unclear (22). However, it is interesting that the promoter region of the FAK gene contains recognition elements for transcription factors that are activated by HBx, for example, NF-B (22, 39). Whether this is the mechanism through which HBx upregulates FAK expression is under investigation, but considering the strong activation of NF-kB by HBx, as well as the correlation between NF-kB activation and liver cancer, the confirmation of this HBx-FAK regulatory pathway would provide additional support for an association between HBV infection, HBx expression, and a contribution to development of liver cancer (reviewed in reference 1).
Finally, our results confirm the importance of calcium signaling pathways in HBx activities and support the notion that a fundamental activity of HBx, modulation of intracellular calcium, is responsible for many of its associated activities. Like FAK/Pyk2 signaling, alterations in cellular calcium levels are involved in many signaling pathways, including those involved in regulation of transcription, cell proliferation, cell migration, and cell survival (reviewed in reference 11). Through modulation of cellular calcium signals and subsequent activation of Pyk2/FAK and Src kinases, HBx could also regulate many of these pathways. In fact, our observations may help explain some of the inconsistent results that have been reported regarding HBx activities in various established cell lines. These seemingly discrepant reports could simply reflect cell-specific differences in response to calcium signals, expression and activation of Pyk2 or FAK, and subsequent activation of various Src kinase family members. Calcium signaling and activation of Pyk2 and FAK are the starting points for many complex signaling pathways, and the ultimate effects of their activation are likely to be determined by the availability of downstream signaling molecules in various cells. Differential regulation of these downstream signaling proteins, different expression levels, and different sites of intracellular localization could ultimately impact the consequence of HBx expression.
ACKNOWLEDGMENTS
We thank members of the Schneider lab for critical reviews of the manuscript.
This work was supported by NIH grants R01-CA 565633 (R.J.S.) and 5T32-AI07647 (M.J.B.).
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Department of Microbiology, New York University School of Medicine, New York, New York 10016
ABSTRACT
The hepatitis B virus (HBV) X protein (HBx) is a multifunctional regulator of cellular signal transduction and transcription pathways and has a critical role in HBV replication. Much of the cytoplasmic signal transduction activity associated with HBx expression and its stimulation of viral replication is attributable to HBx-induced activation of calcium signaling pathways involving Pyk2 and Src tyrosine kinases. To further characterize upstream signal transduction pathways that are required for HBx activity, including activation of Src and mitogen-activated protein kinase (MAPK) cascades, we determined whether focal adhesion kinase (FAK), a known regulator of Src family kinases and the other member of the Pyk2/FAK kinase family, is activated by HBx. We report that HBx activates FAK and that FAK activation is important for multiple HBx functions. Dominant inhibiting forms of FAK blocked HBx activation of Src kinases and downstream signal transduction, HBx stimulation of NF-B and AP-1-dependent transcription, and HBV DNA replication. We also demonstrate that HBx-induced activation of FAK is dependent on cellular calcium signaling, which is modulated by HBx. Moreover, prolonged expression of HBx increases both FAK activity and its level of expression. FAK activation may play a role in cellular transformation and cancer progression. HBx stimulation of FAK activity and abundance may also be relevant as a potential cofactor in HBV-associated hepatocellular carcinoma.
INTRODUCTION
It is estimated that there are 350 million people chronically infected with hepatitis B virus (HBV), which is significantly associated with development of hepatocellular carcinoma (HCC), one of the most common forms of cancer worldwide (reviewed in references 3 and 21). HBV encodes a small genome, consisting of a partially double-stranded, circular DNA that is encapsidated within an enveloped particle (57). The HBV genome contains four open reading frames, encoding the viral envelope proteins (also known as surface antigens), the viral core protein which comprises the viral capsid, a polymerase/reverse transcriptase, and the nonstructural regulatory protein known as HBx. While the precise function of HBx is unresolved, studies have established functions in cellular physiology, viral replication, transcription, and viral pathogenesis (reviewed in reference 10). The activity of HBx in HBV replication may depend on its direct interaction with cellular proteins such as UVDDB (ultraviolet damaged DNA binding protein), HBx-induced transcriptional activation, HBx modulation of intracellular calcium signaling, and stimulation of cellular signal transduction pathways (8, 9, 27, 30, 36, 39).
Numerous studies have identified HBx-responsive transcription factors including NF-B, NF-AT, and AP-1 among others, as well as HBx-responsive transcription elements such as the human immunodeficiency virus long terminal repeat and cyclic AMP response elements (4, 18, 30, 39, 42, 59). Recent reports suggest that at least some HBx-mediated transcriptional activation results from its ability to stimulate cellular calcium signaling pathways, resulting in activation of proline-rich tyrosine kinase 2 (Pyk2) and Src tyrosine kinases and, in turn, downstream signal transduction pathways such as the mitogen-activated protein kinase (MAPK) pathway (4, 6, 8, 9, 28, 34). HBx might also directly interact with components of the basal transcription machinery, such as ribosome binding protein 5 and TATA-binding protein, as well as the transcriptional activator CREB/ATF (16, 42, 58, 59). This provides another mechanism through which HBx stimulates transcription and possibly HBV replication. HBx can deregulate cell cycle progression checkpoints by inducing activation of the cyclin-dependent kinases CDK2 and CDK1 and association of these kinases with cyclins E and A or cyclin B, respectively, although the exact influence of HBx on cellular proliferation can vary depending on the transformed state of the cell (5, 26, 34; reviewed in reference 41). Under certain conditions, HBx can modulate cellular apoptotic pathways, although both pro- and antiapoptotic effects of HBx have been reported (reviewed in reference 10). The effect of HBx-associated activities varies in different cellular contexts, and the exact molecular mechanism for its activity is currently still very poorly defined, both when HBx is expressed alone and in the context of HBV replication. Which of the myriad HBx functions are required for HBV replication during natural infection and which activities, if any, influence HBV-associated development of HCC remains to be determined.
Only the mammalian HBVs are associated with HCC, and only the mammalian HBVs have been shown to encode an HBx protein (reviewed in references 1 and 2). An avian HBV has been reported to encode a divergent HBx-like protein, but whether it is expressed during natural infection like mammalian HBV HBx is not certain. The function of a putative avian HBx protein is also not required for replication of the avian virus, unlike that of the mammalian viruses (14, 21, 44). While there is some discrepancy among different HBx-transgenic mouse models, the majority of available evidence suggests a correlation between the presence of HBx and the development of HCC (25, 29, 35, 40). The association between HBx and HCC has therefore stimulated interest in defining the primary function and mechanism of HBx-induced activation of signal transduction pathways and transcription and how these activities could impact cancer etiology. Moreover, the demonstration that HBx is essential for in vivo replication of the woodchuck hepatitis virus and potentially so for replication of human HBV has stimulated interest in elucidating the primary function of this protein during viral replication (8, 15, 45, 60, 62). To identify activities associated with HBx expression that might contribute to its role in viral replication and possibly oncogenesis, we have studied HBx-induced activation of cellular signaling pathways, such as those that translate modulation of cytosolic calcium levels to activation of MAPK pathways, to determine what role these may have in the various activities that have been ascribed to HBx function. We previously demonstrated that HBV replication requires HBx-induced activation of cellular calcium signaling pathways, including stimulation of proline-rich tyrosine kinase 2 (Pyk2) and Src kinases (8, 9), and that HBx expression regulates multiple steps in viral DNA replication through the phosphorylation of HBV core protein (46). Members of the Pyk2/focal adhesion kinase (FAK) family are known to play a key role in cellular signaling pathways which are important for transcriptional regulation, cell cycle progression, modulation of apoptosis, control of cell migration, and metastasis of transformed cells (23, 47, 48, 49). HBx-induced activation of this family of kinases could therefore be important for many of its reported activities. For example, it was previously reported that HBx can modulate migration of human liver cells and regulate cellular adherens junctions in an Src kinase-dependent manner (32, 33). Stimulation of FAK also modulates similar activities, and HBx regulation of FAK could conceivably contribute to a potential involvement of FAK activation in the development of HBV-associated HCC.
The proline-rich tyrosine kinase, Pyk2, and the closely related kinase FAK, comprise a family of cytoplasmic, nonreceptor tyrosine kinases that can be regulated by extracellular stimuli and can activate Src family kinases (reviewed in references 23, 49, and 52). Pyk2 and FAK are activated by numerous mechanisms, one of which is an increase in cytosolic calcium levels. Following activation, FAK and Pyk2 recruit and activate members of the Src kinase family; this is ultimately linked to stimulation of targets such as the ERKs (extracellular-regulated kinases) and c-JUN N-terminal kinases/MAPKs (51, 54). Pyk2 and FAK activity is also induced by stimulation of G protein-coupled receptors, integrin engagement, and various cytokines, such as tumor necrosis factor alpha (reviewed in reference 52). Pyk2 expression can compensate for a subset, though clearly not all of the functions of FAK in cells derived from FAK knockout mice (56). Pyk2 and FAK also share homology, and both proteins contain similarly organized domains such as a central kinase domain flanked by extensive N-terminal and C-terminal regions (reviewed in reference 2). The N-terminal domains contain the major autophosphorylation sites (tyrosine 397 in FAK, tyrosine 402 in Pyk2). Upon activation, phosphorylation of this site creates an SH2 domain binding site for Src kinases. Binding of Src kinases to this site leads to the activation of Src, which further activates Pyk2 or FAK. The subsequent phosphorylation and enhanced activation of Pyk2/FAK is a complex process in which phosphorylation of multiple FAK or Pyk2 amino acid residues recruits other signaling proteins such as Shc, Grb2, and p130-Cas, ultimately activating MAPK cascades. FAK is largely but not entirely located in focal adhesions, which are sites of contact between the cell and the extracellular matrix, although studies demonstrate that Pyk2 can also localize to focal adhesions (38). Activation of FAK, and an increase in its level of expression, have been identified in a large number of transformed cells, including those of breast cancer, prostate cancer, melanoma, and liver cancer, and is thought to play a role in the transformation process (reviewed in references 24 and 43). Activation of FAK by HBx would therefore be of interest in virus-mediated carcinogenesis.
We previously reported that HBx activates Pyk2 in HepG2 cells in a calcium-dependent manner (8, 9). We now demonstrate that HBx also stimulates FAK activity in HepG2 cells and stably increases its level of expression. Similar to HBx induction of Pyk2 activity, stimulation of FAK by HBx is dependent on cytosolic calcium signaling pathways. Our results suggest a mechanism by which HBx might contribute to the development of HCC in chronically HBV-infected individuals by activating the Pyk2/FAK family of kinases. HBx activation of FAK could in turn enhance cell migration and invasiveness, stimulated during the process of transformation and associated with upregulation of FAK activity (2, 12, 13, 19, 23, 47, 55, 61).
MATERIALS AND METHODS
Cell culture. NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium containing gentamicin (5 μg/ml), supplemented with 10% bovine calf serum. HepG2 cells were maintained in minimal essential medium supplemented with gentamicin (5 μg/ml), sodium pyruvate, glutamine, and 10% fetal bovine serum. All cells were maintained at 37°C in 5% CO2.
Plasmids. The HBx expressing plasmids pAdCMVX and pCEP4XFlag and the NF-B and AP-1-promoter responsive luciferase (Luc) reporter plasmids have been described previously (21, 31). The FAK plasmids pFAKKD and pFAK397 were a gift of J.-L. Guan (Cornell University). High-concentration stocks of plasmids were purified using the Concert High-Purity Plasmid Maxiprep system (Life Technologies) according to the manufacturer's directions.
Luciferase assays. Transient transfections were performed with Lipofectamine Plus (Life Technologies) for NIH 3T3 cells and Fugene 6 (Roche) for HepG2 cells, both according to manufacturer directions. For induction of NF-B and AP-1 luciferase, cells were transfected overnight with 2 μg expression plasmids, allowed to recover for 12 h, and then serum starved for 16 h prior to analysis of luciferase activity. Luciferase reporter assays were performed using the Promega Luciferase assay system according to the manufacturer's directions.
FAK phosphorylation. For FAK phosphorylation analyses, NIH 3T3 cells were transfected with expression plasmids or vector control plasmid using Lipofectamine Plus, allowed to recover for 12 h, and then serum starved for 16 h. Medium containing serum was then added, and samples were collected at the indicated times. Either 50 μm BAPTA-AM (Molecular Probes) or 3 mM EGTA (Sigma) was added as indicated and cells collected at the indicated time points. Cells were lysed in modified RIPA buffer (1% Triton X-100, 0.1% sodium dodecyl sulfate, 1% deoxycholate, 50 mM HEPES [pH 7.4], 150 mM sodium chloride, 10% glycerol, 1.5 mM magnesium chloride, 1 mM EGTA, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, and protease inhibitors), protein concentrations were determined, and equal amounts of protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis using a monoclonal antibody that specifically recognizes the phosphorylated tyrosine at amino acid position 397 [Y397(P)] of FAK (Biosource International) (54). Confirmation that equal amounts of FAK had been loaded was performed with a polyclonal anti-FAK antibody (Santa Cruz Biotechnology, Inc.).
Kinase assays. Cells were transfected and processed as described above. Equal amounts of FAK were immunoprecipitated and kinase assays performed as previously described (54). A portion of the immunoprecipitate was used to confirm equal amounts of protein had been purified.
FAK activation measured by binding to Grb2. Activation of FAK also results in phosphorylation of FAK amino acid residue 925, generating a binding site for Grb2 (53, 54). NIH 3T3 cells were transfected with control or HBx expression plasmids as described above, cells were lysed, and FAK was immunoprecipitated. Association of Grb2 with FAK was monitored by immunoblot analysis with an anti-Grb2 antibody (Santa Cruz Biotechnology, Inc.). Total FAK and phosphorylation of FAK at residue 397 were determined as described above.
HBV replication assays. HBV replication assays were conducted as previously described (8, 45). Briefly, 4 days after transfection of wild-type or HBx mutant HBV replicons, cytosolic HBV core particles were precipitated with polyethylene glycol and digested with proteinase K, and core-associated HBV DNA was resolved by electrophoresis in a 1% agarose gel, as described previously (8, 9). DNA was transferred to a nylon membrane, and Southern blot analysis was performed with an HBV-specific probe. Endogenous polymerase assays were carried out as previously described (8, 9).
RESULTS
HBx-mediated activation of FAK. We previously demonstrated that HBx activates Pyk2 and that this activation is dependent on cytosolic calcium signaling pathways (8, 9). To determine whether HBx can also activate FAK, we examined FAK for autophosphorylation mediated by HBx expression. Studies utilized HepG2 cells, a human hepatoblastoma cell line in which HBV replicons can carry out DNA replication, and NIH 3T3 cells, which have been used widely for strong FAK responsiveness. Cells were transfected with HBx expression vectors or control vectors, placed in normal growth medium to allow for HBx expression, and then serum starved overnight. Cells were collected at different times after readdition of normal growth medium, and FAK activity was analyzed. Activity was determined by two methods: immunoblot analysis of activating phosphorylation using a phosphospecific antibody to FAK Y397, the major site of activating phosphorylation (51), and analysis of immunoprecipitated FAK kinase activity. Measurement of the major activating phosphorylation site of FAK is a measure of the increased autophosphorylation kinase activity of FAK (51). Autophosphorylation kinase assays were performed as previously described (54).
Expression of HBx led to FAK Y397 phosphorylation (activation) of about three- to fourfold in both HepG2 and NIH 3T3 cells in an HBx dose-responsive manner (Fig. 1). As a control, FAK Y397 phosphorylation was stimulated by the addition of the phorbol ester tetradecanoyl phorbol acetate (TPA) to the growth medium, which was similar in magnitude to HBx. HBx therefore strongly stimulates FAK activation measured by Y397 phosphorylation. While Y397 phosphorylation is an accepted surrogate for FAK activity, we also performed FAK in vitro kinase assays. Endogenous FAK was immunoprecipitated from equal amounts of lysates obtained from vector control or HBx-expressing HepG2 or NIH 3T3 cells, normalized to equal amounts of total FAK protein, and incubated with [-32P]ATP to measure FAK autophosphorylation (activation) in vitro (Fig. 2). HBx induced 3-fold activation of FAK in both HepG2 and NIH 3T3 cells (upper panels) when equal amounts of FAK protein were assayed (lower panel). Thus, these results are similar in magnitude to the Y397(P) immunoblot analysis with FAK phosphorylation-specific antibody. HBx therefore activates FAK, similar to its activation of Pyk2.
Studies were next carried out to determine whether HBx activation of FAK is sufficient to stimulate downstream signaling pathways. Upon activation of FAK and recruitment of Src kinases, FAK is typically further phosphorylated, including phosphorylation of amino acid residue 925, which creates a binding site for Grb2 (45, 46). Therefore, increased binding of FAK to Grb2 is another indicator of FAK activation (53, 54). We therefore characterized the HBx-induced upregulation of the interaction between FAK and the signal transduction scaffolding protein Grb2. Cells were transfected with HBx or a control expression plasmid and lysed, and FAK was immunoprecipitated (Fig. 3). Equal levels of FAK are apparent in the anti-FAK samples. Immunoblot analysis was used to determine the levels of Grb2 that coimmunoprecipitate with FAK and total Grb2 levels in the lysates. Studies demonstrated that HBx strongly increased the association of Grb2 with FAK (by four- to fivefold), consistent with FAK Y397 phosphorylation. These results provide another independent line of evidence for HBx activation of FAK and assembly of FAK complexes capable of stimulating downstream signal transduction.
HBx-induced activation of FAK is calcium dependent. An important activity of HBx is modulation of cytosolic (intracellular) calcium stores. Activation of Pyk2 by HBx is dependent on cytosolic calcium signaling (8, 9), and FAK can be activated by an increase in cytosolic calcium levels (reviewed in reference 52). To determine whether HBx-induced activation of FAK is dependent on cytosolic calcium signaling, HBx transfected HepG2 cells were treated with BAPTA-AM or EGTA (Fig. 4A). BAPTA-AM is a cell-permeable, intracellular chelator of calcium that can block intracellular calcium signaling, whereas EGTA is not cell permeable and chelates extracellular calcium, inhibiting the influx of calcium from the extracellular medium. As a positive control, vector-alone-transfected cells were treated for 20 min with 20 mM TPA to activate FAK. In control cells (vector alone), TPA stimulated FAK Y397 phosphorylation by 4-fold, compared to 3-fold activation by HBx. Treatment of cells with BAPTA-AM inhibited activation of FAK by both TPA and HBx, demonstrating the requirement for mobilization of intracellular calcium stores. Treatment of cells with EGTA, however, partially blocked TPA activation of FAK, whereas it had no effect on HBx activation of the kinase. Thus, influx of extracellular calcium is important for TPA stimulation of FAK, as expected, whereas HBx activation of FAK utilizes intracellular stores. The calcium-dependent activation of FAK is not cell type specific, as shown in Fig. 4B. In both HepG2 and NIH 3T3 cells, treatment of cells with EGTA did not inhibit HBx-induced activation of FAK, whereas BAPTA-AM treatment reduced HBx activation of FAK in both cell lines by three- to fourfold, measured by reduction in FAK Y397 phosphorylation (Fig. 4B). These results demonstrate that HBx stimulates calcium-dependent signals in a variety of cell types that lead to activation. Moreover, they point to the alteration of intracellular calcium caused by the release from intracellular calcium stores, rather than the influx of calcium from the extracellular medium, as a primary component of HBx activity.
Continuous expression of HBx increases the abundance of FAK. Both increased expression of FAK and increased levels of FAK activity have been observed in numerous cancers where FAK may control or influence the invasive and metastatic potential of tumor cells (reviewed in reference 24). Studies were therefore carried out to determine whether HBx constitutively stimulates FAK activity while also increasing its abundance. At early time points (6 h) following TPA stimulation and serum addition in control samples, increases in FAK activation persisted (indicated by phosphorylation) but were restored to baseline levels at 24 h. In contrast, HBx stimulated FAK similarly (3-fold) at the same time point post-serum addition, based on Y397 phosphorylation (Fig. 4C). There was no measurable increase in the total level of FAK expression in HepG2 or NIH 3T3 cells with HBx expression at 6 h post-serum addition (Fig. 4C). However, by 12 h, and persisting at 30 h (the last time point measured) in HBx-transfected cells, the level of activated FAK was increased by another twofold and the overall abundance of FAK was elevated approximately fourfold above that of the time zero control. Constitutive activation of FAK and increased FAK abundance by HBx were verified in NIH 3T3 and HepG2 cells transfected by an HBx expression construct controlled by either the simian virus 40 promoter in NIH 3T3 cells or the cytomegalovirus promoter in HepG2 cells, evaluated at 48 h after induction (Fig. 4D). In both NIH 3T3 and HepG2 cells, there was a significant increase in the total level of FAK protein expression at late time points as well as sustained FAK activation, whereas the level of the tubulin control was unchanged.
Dominant interfering mutants of FAK inhibit HBx activation of AP-1 and NF-B. Our previous studies demonstrated that a dominant inhibiting form of Pyk2 could block HBx activity (8, 9). To determine whether dominant inhibitors of FAK also impair HBx activities, we cotransfected HBx into NIH 3T3 or HepG2 cells with either an AP-1- or NF-B-dependent luciferase reporter vector and dominant inhibiting mutants of FAK and determined the effect on HBx activation of transcription (Fig. 5). Many previous studies have shown that HBx activates AP-1 and NF-B by three- to fivefold (6), as shown here. FAK mutants consisted of a protein that contains a Y397F mutation (FAK397) and cannot be activated, or a mutation in the kinase domain that blocks activity (FAKKD). The FAK mutants fully blocked HBx induction of AP-1- and NF-B-dependent transcription in both NIH 3T3 and HepG2 cells. These results support the conclusion that HBx-induced activation of the Pyk2/FAK family is essential for its induction of several transcription factors that are typically activated by HBx. However, it is not known whether activation of FAK is sufficient for AP-1/NF-B activation or if other HBx activities are involved as well.
Inhibition of FAK activity blocks HBV DNA replication. Inhibition of Pyk2 and Src kinases was shown to block HBV HBx stimulation of DNA replication in HepG2 cells (8, 27). To determine whether FAK is an important component of HBx-mediated HBV DNA replication, we tested whether dominant inhibiting forms of FAK block HBV DNA replication or transcription. HBV replicons, which consist of the HBV genome as a 130% reiteration (8) that either express HBx (wtHBV) or have a deletion of HBx [HBV (HBx–)], were cotransfected into HepG2 cells with either vector alone or a plasmid expressing the dominant inhibiting FAK397. Studies first determined whether HBx expressed from the HBV replicon activated FAK and the downstream MAPK, ERK2 (Fig. 6A). Activation of FAK and ERK2 were determined by immunoblotting using specific antibodies for the activating sites of phosphorylation. Immunoblot analysis of FAKY397(P) levels demonstrated activation in wtHBV replicon-transfected cells but not in the HBV (HBx–) samples, which was suppressed by inclusion of the dominant-inhibiting form of FAK. Increased levels of total FAK were also evident in the wtHBV samples but not in the HBV(HBx–) samples. Inclusion of the dominant-inhibiting form of FAK in the control wtHBV sample prevented the quantitation of FAK total abundance. Similarly, analysis of ERK2 activity demonstrated a strong increase only in the wtHBV samples, which was also prevented by inclusion of the dominant-inhibiting FAK397. There was no change in total abundance of ERK2 by HBx expression. Thus, HBx activates FAK and ultimately ERK2 in the context of replicating HBV.
HBV DNA replication was assayed by isolation of viral core particles (capsids), extraction of HBV replicative DNA intermediates, and analysis by Southern blot DNA hybridization, as previously described (8). We and others have previously shown that replication of HBV DNA in HepG2 cells is strongly influenced by HBx that is expressed in the context of the replicon, and we have used this system to define important functions of HBx in HBV replication. Both dominant-inhibiting FAK mutant proteins very strongly blocked HBV DNA replication (Fig. 6B). Analysis of viral mRNA levels by Northern blot analysis during the same period of time showed a slight reduction with expression of the FAK-interfering mutants. Inhibition of HBV DNA replication by FAK dominant-negative mutants averaged >15-fold, whereas reduction of viral mRNA levels averaged 2- to 3-fold. Finally, cytoplasmic viral core particles were isolated from cells transfected with wtHBV replicons in the presence or absence of the two FAK dominant inhibitors, and the DNA replication activity of the endogenous, encapsidated polymerase was determined using an in vitro endogenous polymerase assay and -32P-deoxynucleoside triphosphates. Analysis of the products of in vitro DNA elongation and strand completion showed a threefold reduction in polymerase activity obtained from FAK-inhibited cells compared to the noninhibited control (Fig. 6C). Examination of the level of isolated core particles (capsids) which were used for analysis showed them to be the same. These results therefore verify the importance of HBx activation of Pyk2/FAK kinases for HBV DNA replication.
DISCUSSION
It is well established that chronic HBV infections are associated with a several hundred-fold-increased incidence in development of primary liver cancer (reviewed in reference 21). Immune-mediated destruction of infected hepatocytes, the inflammatory response itself, and the subsequent continued turnover and replacement of hepatocytes clearly underlies the development of HBV-associated HCC. However, it is also likely that the virus itself encodes proteins that may be cofactors in the development or progression of liver cancer (17). HBx, the nonstructural regulatory protein of HBV, has been strongly implicated in the etiology or progression of HBV-associated liver disease and cancer in humans and in some HBx-transgenic mouse model systems. HBx expression is either directly associated with the development of liver cancer in some HBx-transgenic mouse strains or an increased progression to liver cancer in other toxin-exposed HBx-transgenic mouse strains (reviewed in reference 10). Many functions have been ascribed to HBx, but the precise molecular mechanism(s) responsible for its activities, and how these activities affect viral replication and possibly liver cell transformation, remain poorly defined. In the work presented here, we have focused on the effect of HBx activation of cellular calcium-dependent signal transduction pathways.
We previously reported that HBx expression activates Pyk2, a nonreceptor tyrosine kinase involved in the regulation of numerous cellular signal transduction pathways (8). Here, we demonstrate that HBx expression also activates the related kinase, FAK, a nonreceptor tyrosine kinase that has roles in signal transduction, cell cycle progression, apoptosis, cell migration, and cell invasion (reviewed in references 23, 47, 49, and 52). In contrast to our studies with Pyk2 in which HBx expression elevated Pyk2 kinase activity without influencing Pyk2 expression levels, in the work presented here, we have shown that HBx increases the expression level and activation of FAK. We also demonstrate that HBx requires cellular calcium signaling to activate FAK, similar to its activation of Pyk2 (8). Many of the activities that are associated with stimulation of the Pyk2/FAK family, such as regulation of cellular signal transduction cascades, cell proliferation, apoptosis, and cell migration (2, 12, 13, 19, 23, 50, 55, 61) have also been reported previously in HBx-expressing cells, long before it was known that HBx stimulates these tyrosine kinases (reviewed in reference 10). Whether all of these HBx activities correlate with its regulation of the Pyk2/FAK family has not been determined but could impact on our understanding of the consequence of HBx expression in different cell types and contexts.
The Pyk2/FAK family of nonreceptor tyrosine kinases are involved in many cellular processes ranging from control of cell migration to regulation of cellular proliferation and survival pathways (2, 12, 13, 19, 23, 47, 50, 55, 61). Both FAK and Pyk2 are activated by similar mechanisms, including stimulation by elevation in intracellular calcium levels. In the work presented here, when combined with our previous studies, we have now shown that HBx can modulate cellular calcium signals and activate both members of the Pyk2/FAK kinase family (8; this study). HBx activates both Pyk2 and FAK in a calcium-dependent manner. We also demonstrate that dominant-inhibiting mutants of FAK and Pyk2 can block many of the reported activities of HBx, including its stimulation of HBV replication (9; this study). Because of the extensive cross talk between FAK and Pyk2 as well as their ability to regulate similar pathways, the inhibition of HBx activities by dominant-negative forms of FAK likely also involve inhibition of Pyk2 activity (37). These observations are similar to the ability of various dominant-negative forms of Src kinases to inhibit HBx activities that are dependent on this family of kinases (27, 28). Pyk2 and FAK regulate similar pathways, and can, to some extent, compensate for the absence of the other protein as well as influence the activity of the other protein (56). Our results demonstrate that, similar to the ability of HBx to activate multiple members of the Src kinase family, HBx can also activate both members of the Pyk2/FAK family through regulation of cellular calcium signals. Activation of Pyk2 and FAK by HBx is likely to impact multiple cellular signal transduction cascades and could therefore contribute to cellular transformation and the development of HCC during HBV infections.
Activation of FAK by HBx could have profound affects on the cell. One function associated with activated FAK is regulation of cell migration. FAK modulates the assembly and disassembly of focal adhesions, sites of contact between cells, and the extracellular matrix (reviewed in reference 47). Importantly, these FAK activities can be regulated by calcium signals within the cell. It was recently reported that HBx can induce the migration of some liver cell lines (32, 33). HBx-enhanced migration was found to be dependent on activation of Src kinases and involved the regulation of adherens junctions, points of cell-cell contact that are modulated by members of the cadherin family of proteins. HBx expression was also shown to modulate matrix metalloproteinase expression which might be important for invasion of a three-dimensional matrix (31). Interestingly, FAK not only regulates focal adhesion formation but it can also regulate cadherin-mediated adhesion and matrix metalloproteinase levels (reviewed in reference 47). FAK-mediated regulation of adherens junctions proceeds through regulation of Src kinases similar to that observed for HBx-mediated regulation of adherens junctions. While our studies, and those reported by others, have not directly assessed the association between HBx activation of FAK and HBx stimulation of migration, our results combined with previous observations suggest a possible connection between HBx regulation of the Pyk2/FAK family and its stimulation of cell migration. Studies designed to directly assess this connection are ongoing, as these HBx functions could influence the rate of progression of hepatocyte transformation, invasiveness, or metastatic activity.
FAK activation has also been associated with stimulation of transcription pathways and modulation of cellular proliferation and survival (2, 12, 13, 19, 23, 47, 50, 55, 61). Many of the effects associated with FAK activation involve interactions with and stimulation of Src kinases. We previously demonstrated that HBx can activate Src kinases and that this activation is necessary for many of the reported properties of HBx, including its role in induction of cell proliferation, activation of transcription factors such as AP-1 and NF-B, and stimulation of HBV DNA replication (7, 8, 27, 28). The requirement for Src kinase activity in this HBx function, combined with our previous observation that dominant-inhibiting forms of Pyk2 can inhibit HBx activation of Src, suggests that the Pyk2/FAK kinase family are the effectors of HBx modulation of cellular calcium. We demonstrated that inhibition of Src kinases blocks HBV replication, HBx activation of various transcription factors, and HBx stimulation of cell proliferation (7, 28). Inhibiting forms of Pyk2 (8, 9) and FAK also block HBV replication (Fig. 6) and activation of AP-1 and NF-B (Fig. 5), suggesting a direct link between the ability of HBx to activate FAK/Pyk2 and activation of Src kinases (8; this report).
Elevation of FAK levels has been observed in various tumors including those in thyroid, prostate, cervix, rectum, and ovarian cancers (reviewed in references 24 and 43). More recently, it was reported that FAK is elevated in HCC and that elevated FAK could be used as a prognostic marker for HCC progression (20). While none of these studies have demonstrated a direct link between cellular transformation and elevation of FAK expression or activity, the fact that many types of cancers are associated with elevated levels of FAK suggests a strong correlation between FAK activity and expression levels and control of normal cellular physiology. It is likely that alterations of normal FAK levels and activities will impact cellular transformation mechanisms. In the studies reported here, we have demonstrated both a constitutive activation of FAK by HBx as well as an increase in FAK expression levels following prolonged expression of HBx (Fig. 4 and 6). The elevation of FAK levels was observed when HBx was expressed in the absence of other viral proteins (Fig. 4C and D) and, although to a lesser extent, when HBx was expressed in the context of viral replication (Fig. 6A, wtHBV). These differences may reflect variations in the levels of HBx expressed in these two systems, in that HBx is likely expressed at lower levels during viral replication. However, both results demonstrate that prolonged expression of HBx constitutively activates FAK and eventually increases its level of expression. Whether this occurs through stabilization of FAK protein levels or by modulating FAK transcription is currently unknown. Importantly, the activation of FAK that is observed in HBx-expressing cells is not simply a consequence of overexpressed FAK, as this activation is observed during early time points in our studies when FAK levels are not elevated by HBx expression (Fig. 4C). While FAK activation can regulate transcription pathways, whether it stimulates its own production is unclear (22). However, it is interesting that the promoter region of the FAK gene contains recognition elements for transcription factors that are activated by HBx, for example, NF-B (22, 39). Whether this is the mechanism through which HBx upregulates FAK expression is under investigation, but considering the strong activation of NF-kB by HBx, as well as the correlation between NF-kB activation and liver cancer, the confirmation of this HBx-FAK regulatory pathway would provide additional support for an association between HBV infection, HBx expression, and a contribution to development of liver cancer (reviewed in reference 1).
Finally, our results confirm the importance of calcium signaling pathways in HBx activities and support the notion that a fundamental activity of HBx, modulation of intracellular calcium, is responsible for many of its associated activities. Like FAK/Pyk2 signaling, alterations in cellular calcium levels are involved in many signaling pathways, including those involved in regulation of transcription, cell proliferation, cell migration, and cell survival (reviewed in reference 11). Through modulation of cellular calcium signals and subsequent activation of Pyk2/FAK and Src kinases, HBx could also regulate many of these pathways. In fact, our observations may help explain some of the inconsistent results that have been reported regarding HBx activities in various established cell lines. These seemingly discrepant reports could simply reflect cell-specific differences in response to calcium signals, expression and activation of Pyk2 or FAK, and subsequent activation of various Src kinase family members. Calcium signaling and activation of Pyk2 and FAK are the starting points for many complex signaling pathways, and the ultimate effects of their activation are likely to be determined by the availability of downstream signaling molecules in various cells. Differential regulation of these downstream signaling proteins, different expression levels, and different sites of intracellular localization could ultimately impact the consequence of HBx expression.
ACKNOWLEDGMENTS
We thank members of the Schneider lab for critical reviews of the manuscript.
This work was supported by NIH grants R01-CA 565633 (R.J.S.) and 5T32-AI07647 (M.J.B.).
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