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Originally published online as doi:10.1189/jlb.0106014 on June 22, 2006

Published online before print June 22, 2006
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(Journal of Leukocyte Biology. 2006;80:424-432.)
© 2006 by Society for Leukocyte Biology

Up-regulation of ERK and p38 MAPK signaling pathways by hepatitis C virus E2 envelope protein in human T lymphoma cell line

Lan-Juan Zhao*, Xiao-Lian Zhang{dagger}, Ping Zhao*, Jie Cao*, Ming-Mei Cao*, Shi-Ying Zhu*, Hou-Qi Liu{ddagger} and Zhong-Tian Qi*,1

* State Key Laboratory of Medical Immunology and Departments of Microbiology and
{ddagger} Histology and Embryology, Second Military Medical University, Shanghai, China; and
{dagger} Department of Immunology, Wuhan University School of Medicine, China

1Correspondence: Department of Microbiology, Second Military Medical University, 800 Xiang-Yin Road, Shanghai 200433, China. E-mail: qizt53{at}hotmail.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatitis C virus (HCV) infection correlates with human immune disorders characterized by abnormal activation and proliferation of lymphocytes. Interaction of HCV major envelope protein E2 with susceptible cells occurs at an early stage of the viral infection. HCV tropism for susceptible cells may elicit cellular signaling events implicated in the viral pathogenicity, and E2 protein is known to be responsible for the tropism. We documented previously that HCV E2 protein was capable of activating extracellular signal-regulated kinase (ERK) in human hepatoma Huh-7 cells. Here, ERK and p38 mitogen-activated protein kinase (MAPK) signaling pathways were investigated in human T lymphoma cell line Molt-4 in response to HCV E2 protein. Binding of HCV E2 protein to Molt-4 cells was detectable, and such interaction was a determinant for recognition and delivery of the E2 signal to intracellular pathways. Activation of ERK and p38 MAPK was specifically induced following the HCV E2-cell interaction. CD81 and low-density lipoprotein receptor (LDLR), proposed cellular receptors for HCV, were expressed naturally on Molt-4 cells. CD81 and LDLR were shown to mediate HCV E2-induced activation of ERK and p38 MAPK. In CD81-deficient U937 cells, levels of ERK and p38 MAPK activation and cell proliferation induced by HCV E2 protein were lower than those in Molt-4 cells. Furthermore, cell proliferation and secretion of interferon-{gamma} and interleukin-10 by Molt-4 cells were promoted by HCV E2 protein. Therefore, ERK and p38 MAPK signaling pathways were up-regulated by HCV E2 protein without synergetic stimulation, which was accompanied by alterations of cell behavior.

Key Words: Molt-4 cells • low-density lipoprotein receptor • CD81 molecules • transmembrane signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There are complex signal transduction networks in mammalian cells. Corresponding to the importance of mitogen-activated protein kinase (MAPK) pathways in controlling cellular functions, perturbation of MAPK signaling cascades has severe, pathological consequences. Intracellular signaling events are triggered rapidly during viral infection of mammalian cells. Modulation of MAPK pathways by a series of viruses such as reovirus, rabies virus, Epstein-Barr virus, and hepatitis B virus has recently been shown to be an important implication for the viral pathogenesis at a molecular level [1 2 3 4 ].

Hepatitis C virus (HCV) is regarded as a causing agent of human liver diseases throughout the world. However, evidence has indicated that HCV infection also correlates closely with human immune manifestations such as cryoglobulinemia, non-Hodgkin lymphoma, and diabetes [5 6 7 ]. Deep insights into HCV pathogenesis are limited as a result of the lack of putative culture systems for HCV propagation in vitro. Currently, attention has been particularly paid to HCV-related, complicated, clinical phenomena on the basis of cellular signaling events. Studies suggested that the interference of HCV proteins with MAPK signaling cascades might account for the manifestations including chronic infection, liver cirrhosis, carcinomas, and interferon (IFN) resistance. For example, under the stimulation of epidermal growth factor, sustained activation of the extracellular signal-regulated kinase (ERK) signaling pathway was detectable in stable cell lines expressing HCV core protein [8 ]. In transgenic mice, HCV core protein induced activation of ERK and p38 MAPK, two members of MAPK signaling pathways, in cooperation with ethanol [9 ]. HCV nonstructural protein 5A was described to inhibit transcription factor-activating protein-1 function by perturbing the ERK pathway [10 ]. The above work has focused primarily on disturbance of cellular signal transduction caused by HCV core and nonstructural proteins. As interaction of the viral envelope proteins with relevant receptors on host cells is a key process for HCV infection, regulation of MAPK signaling pathways by HCV envelope proteins via cellular receptors has implications for understanding the pathogenesis of HCV infection.

HCV, an enveloped RNA virus, has a precursor polyprotein, which generates various structural and nonstructural proteins with the processing of viral and cellular proteases. There are two HCV envelope glycoproteins, E1 and E2, on the virion surface. Viruses are obligate, intracellular parasites, and adsorption to susceptible cells is an essential step taken by viruses for entry and replication. In the case of HCV, its envelope glycoproteins are responsible for the tissue and cellular tropisms. In comparison with HCV E1, the E2 protein has been demonstrated to play a critical role in mediating HCV attachment to susceptible cells through interaction with the relevant cellular receptors [11 , 12 ]. During natural infection, HCV is thought to use multiple receptors or the receptor complex to enter host cells. Human CD81, belonging to the tetraspanin family, is identified as an HCV receptor for its abilities to bind specifically to E2 protein and bona fide viral particles [13 , 14 ]. Moreover, distinct features of CD81 have been outlined, such as its involvement of cell adhesion, morphology, activation, differentiation, and cellular signal transduction [15 ]. With the exception of CD81, low-density lipoprotein receptor (LDLR) is proposed to be a candidate receptor, as it mediates HCV invasion [16 , 17 ], whose distribution pattern is different from that of CD81. In addition, a number of cellular surface molecules are characterized as novel HCV receptors, including human scavenger receptor class B type I, heparan sulfate, and liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin [18 19 20 ]. Roles of these cellular receptors in HCV E2-triggered signaling events remain to be investigated further.

Host immune response correlates closely with the outcomes of HCV infection. Human lymphocytes, besides hepatocytes, are permissive cells for HCV infection [21 , 22 ]. It is reasonable to presume that HCV tropism for lymphocytes may elicit a cellular response implicated in the viral pathogenicity. We wondered whether HCV-related immunological alternations were partially associated with the modulation of cellular signal transduction by the E2 protein. In the present study, the MAPK signaling pathways initiated by HCV E2 protein and the contributions were explored in human T lymphoma Molt-4 cells. Furthermore, response to the E2 protein was analyzed in CD81-deficient human histiocytic lymphoma U937 cells to evaluate roles of related receptors in recognition of the E2 signal. We documented that the interaction of the HCV E2 protein with CD81 and LDLR on target cells resulted in the activation of ERK and p38 MAPK signaling pathways, which was accompanied by enhancement of the cell proliferation and production of IFN-{gamma} and interleukin-10 (IL-10) cytokines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fluorescein-activated cell sorter (FACS) analysis
Human T lymphoma cell line Molt-4 and histiocytic lymphoma cell line U937 were cultured in RPMI-1640 medium (Gibco, Grand Island, NY) plus 10% fetal bovine serum (FBS; HyClone Laboratories, Logan UT) under a 5% CO2 atmosphere at 37°C. HCV E2 protein expressed in Chinese hamster ovary (CHO) cells and mouse monoclonal antibody (mAb; 3E5-1) against the E2 protein were kindly provided by Dr. Michael Houghton (Chiron Corp., Emeryville, CA). Binding of HCV E2 protein to Molt-4 and U937 cells was determined by a FACS-based assay. After washes in phosphate-buffered saline (PBS) containing 1% FBS, Molt-4 and U937 cells under test were incubated with 6 µg/ml E2 protein at 37°C for 1 h. The cells were then washed twice with PBS and incubated with the HCV E2 mAb for an additional hour. The cells were washed again with PBS, and binding of the E2 protein to cells was detected with fluorescein isothiocyanate (FITC)-conjugated goat antimouse immunoglobulin G (IgG) by FACS analysis. In addition, levels of CD81 and LDLR expressed on the surface of Molt-4 and U937 cells were evaluated as described previously [23 ].

Immunocytochemistry
Molt-4 cells were serum-starved for 12 h in RPMI-1640 medium without FBS and then plated on poly-L-lysine-coated glass slides. The cells were stimulated with 1 µg/ml HCV E2 protein for 15 min at 37°C, washed twice with PBS, and fixed in 4% paraformaldehyde at 4°C for 30 min. The cells were washed with PBS and treated with 20% acetic acid to abrogate endogenous alkaline phosphatase activity. Following permeabilization in ice-cold methanol and blockage of 6% goat serum at room temperature, the cells were stained for phosphorylated MAPKs with rabbit polyclonal antibodies against phospho-ERK (1:250 dilution) or phospho-p38 MAPK (1:2000 dilution; Cell Signaling Technology, Inc., Beverly, MA) overnight at 4°C. After thorough washes with PBS, the cells were visualized with alkaline phosphatase-conjugated goat antirabbit secondary antibody and nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate substrates. Photographs were taken using an inverse light microscope (Olympus).

Western blotting
Molt-4 and U937 cells maintained for 12 h in the serum-free medium were stimulated for 15 min at 37°C with bovine serum albumin (BSA) or HCV E2 protein at a concentration of 1 µg/ml. In addition, HCV E2 protein reacted with the E2 mAb for 1 h and was allowed to treat the cells as above. For competition assay, Molt-4 and U937 cells were preincubated for 1 h with CD81 mAb (JS81, PharMingen, San Diego, CA) or LDLR mAb (15C8, Oncogene, Cambridge, MA) at a final concentration of 4 µg/ml, washed twice with PBS, and stimulated for another 15 min with 1 µg/ml E2 protein or BSA at 37°C. The cells were then lysed in cold sodium dodecyl sulfate (SDS) sample buffer. The cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes, followed by treatment with blocking buffer containing 5% nonfat dry milk in Tris-buffered saline/Tween 20 [20 mM Tris (pH 8.0), 150 mM NaCl, 0.1% Tween-20]. The membranes were incubated overnight at 4°C with rabbit antibodies against total ERK, total p38 MAPK, phospho-ERK, or phospho-p38 MAPK (Cell Signaling Technology, Inc.) at 1:1000 dilution. The signal was developed with alkaline phosphatase-conjugated goat antirabbit antibody and nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate substrates.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay
To evaluate the effect of HCV E2 protein on cell proliferation, Molt-4 and U937 cells were seeded at a density of 2 x 104 cells per well in a 96-well tissue-culture plate and grown in the medium with 1% FBS plus various concentrations of HCV E2 protein (ranging from 1 µg/ml to 15 µg/ml) and 15 µg/ml BSA, respectively. Up to 48 h, cell proliferation was measured by MTT assay as recommended by the manufacturer (Chemicon, El Segundo, CA). Optical density (OD) was determined on an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Rad, Hercules, CA) with a test wavelength of 570 nm. The data represented the mean OD and standard deviation of triplicate cultures for three independent experiments.

ELISA assessment
Molt-4 cells were cultured in the 10% FBS medium containing HCV E2 protein at indicated concentrations or the complex of HCV E2-E2 mAb in a 96-well tissue-culture plate. After 48 h of culture, supernatants were collected and analyzed for cytokine production. ELISA was carried out for detection of IFN-{gamma} and IL-10 according to the manufacturer’s instructions (Genzyme, Inc., Cambridge, MA). Briefly, the culture supernatants were transferred into an ELISA plate coated with IFN-{gamma} mAb or IL-10 mAb as triplicate cultures, developed with the biotinylated antibodies, avidin-conjugated horseradish peroxidase, and chromogenic substrates of o-phenylenediamine dihydrochloride and hydrogen peroxide. The OD was determined with a test wavelength of 450 nm on the ELISA reader. Concentrations of IFN-{gamma} and IL-10 were determined by comparison with standard curves. The data were expressed as the mean and standard deviation of triplicate cultures for three independent experiments.

Statistical analysis
Student’s t-test was performed to determine statistical significance. Values of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HCV E2 binds to target cells
Direct interaction of HCV E2 protein with susceptible cells is required for recognition and internalization of the E2, followed by transmitting the E2 stimulation to intracellular signal pathways. It is known that HCV E2 protein mediates the viral attachment to susceptible cells through engagement with relevant cellular receptors. Proof was provided that HCV E2 protein bound to a number of cell lines as a result of its high affinity with human CD81 on the surface of cells [24 ]. Moreover, binding of HCV E2 protein to CD81 on human hepatic stellate cells was suggested to induce ERK activation [25 ]. We previously documented that HCV E2 protein expressed in CHO cells was able to bind to human hepatoma Huh-7 cells and thereby induced the activation of an ERK signaling pathway [23 ]. In this study, functional interaction of this highly purified HCV E2 protein with human T lymphoma Molt-4 cells was tested to evaluate the effect of E2 on MAPK signaling pathways. Human CD81-deficient, histiocytic lymphoma U937 cells served as a control cell line. We initially studied whether the HCV E2 protein could bind to the target cells. For this purpose, Molt-4 and U937 cells were incubated with 6 µg/ml E2 protein. E2 binding was then detected with the E2 mAb and FITC-conjugated goat antimouse IgG and analyzed by FACS analysis. Binding of the E2 protein to the cells was observed in the groups incubated with E2 protein at a different level. As shown in Figure 1A , there were no Molt-4 cells with E2 binding in the control group incubated with no antigen, and the percentage of cell-bound E2 was 95.3% in the group with E2 incubation. Similarly, U937 cell-bound E2 was undetectable in the control group, which received no antigen, and the positive rate of cells with E2 binding was 26% in the group with E2 incubation (Fig. 1B) . These data demonstrated the interaction of HCV E2 protein with Molt-4 as well as U937 cells.


Figure 1
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  		Figure 1. Binding of the HCV E2 protein to target cells. Molt-4 and U937 cells were incubated with no antigen or E2 protein. Cell-bound E2 protein was detected with the E2 mAb and FITC-conjugated goat antimouse IgG. Binding of E2 protein to Molt-4 cells (A) or U937 cells (B) was determined by FACS analysis. The control indicates no antigen incubation.

 
HCV E2 activates ERK and p38 MAPK
Being serine/threonine protein kinases, MAPKs, are activated through dual phosphorylation of threonine and tyrosine (Thr202 and Tyr204 of ERK; Thr180 and Tyr182 of p38 MAPK). In human hepatoma HepG2 and Huh-7 cells, activation of ERK was induced by HCV E2 protein in the absence of additional stimuli [23 , 26 ]. Here, regulation of ERK and p38 MAPK by HCV E2 protein was analyzed further in Molt-4 cells. Based on the E2-target cell interaction, immunocytochemical staining was carried out to detect phosphorylation and activation of the MAPKs. The positive signal (phosphorylated ERK and p38 MAPK) was found to localize to the cytoplasm of Molt-4 cells, with or without HCV E2 treatment. As shown in Figure 2A , ERK and p38 MAPK were obviously phosphorylated after exposure to 1 µg/ml E2 protein, and the phosphorylation levels of ERK and p38 MAPK in untreated Molt-4 cells maintained in the serum-free medium were low as compared with E2 treatment. BSA, an activator for ERK [27 ], was chosen to be a control antigen and tested for its influence on the MAPK activities and cell proliferation. Levels of ERK and p38 MAPK phosphorylation in Molt-4 and U937 cells were additionally detected by Western blot analysis. Consistent with the results achieved by immunocytochemistry, strong phosphorylation of the MAPKs was detectable in Molt-4 cells treated with E2 protein. Figure 2B showed that phosphorylation of ERK and p38 MAPK was weak in untreated Molt-4 cells, which likely reflect constitutive activities of the kinases. Levels of ERK and p38 MAPK phosphorylation were high in Molt-4 cells stimulated with 1 µg/ml BSA compared with the constitutive activities of kinases, and such levels were elevated in response to 1 µg/ml E2. These data indicated the activation of ERK and p38 MAPK induced by E2 protein. To define specific regulation of E2 protein, the effect of the E2 mAb on the kinase activation was then examined. An apparent decrease in the activation of ERK and p38 MAPK was detectable in Molt-4 cells treated with the E2-E2 mAb complex, which suggested that the E2 mAb at a concentration of 2 µg/ml inhibited the activation of MAPKs induced by 1 µg/ml E2 protein. Under the same condition, status of the MAPKs was studied in U937 cells, and similar results were obtained. Increased phosphorylation of ERK and p38 MAPK was detectable in U937 cells exposed to 1 µg/ml E2 protein, and such phosphorylation was impaired after treatment with the E2 mAb (Fig. 2C) . We noticed that the levels of ERK and p38 MAPK activation induced by E2 protein were higher in Molt-4 cells than those in U937 cells. The constant amounts of total ERK and p38 MAPK were monitored to ensure reliability of the tests. HCV E2 protein had an active effect on up-regulation of ERK and p38 MAPK activities.


Figure 2
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  		Figure 2. Activation of ERK and p38 MAPK induced by the HCV E2 protein. Phosphorylation of ERK and p38 MAPK was examined in Molt-4 cells, with or without E2 stimulation. The phosphorylated MAPKs were developed with rabbit antibodies against phospho-ERK or phospho-p38 MAPK using immunocytochemical staining (A, original magnification x200). Molt-4 cells (B) and U937 cells (C) were treated with BSA, E2 protein, or the E2-E2 mAb complex. Cell lysates were analyzed for phosphorylation of ERK and p38 MAPK by Western blotting with the relative antibodies. T-ERK/p38 MAPK, Total ERK or p38 MAPK; P-ERK/p38 MAPK, phosphorylated ERK or p38 MAPK. The control indicates no antigen treatment.

 
CD81 and LDLR transmit HCV E2 signal to MAPK pathways
Cellular receptors perform their functions by interplaying and carrying viruses from the circulation to target cells. We were interested in roles of CD81 and LDLR, proposed cellular receptors for HCV, in E2-induced ERK and p38 MAPK activation. First, levels of CD81 and LDLR on the surface of Molt-4 and U937 cells were detected with CD81 mAb or LDLR mAb and FITC-conjugated secondary antibody by FACS analysis. Treatment with an isotype control mouse IgG1 was used to determine background fluorescence. Our data showed that CD81 and LDLR were expressed naturally on Molt-4 cells, and only LDLR expression was detected on U937 cells. As shown in Figure 3A , CD81 was expressed on Molt-4 cells at a level of 98.7%, and LDLR was expressed at a level of 55.9%. As for U937 cells, the expression of CD81 was undetectable, and LDLR was expressed at a level of 40.2%.


Figure 3
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  		Figure 3. Participation of CD81 and LDLR in HCV E2-induced the activation of ERK and p38 MAPK. Molt-4 and U937 cells were incubated with CD81 mAb or LDLR mAb, followed by incubation with FITC-conjugated goat antimouse IgG. Mouse IgG1 was used to be an isotype control. The presence of CD81 and LDLR on the surface of cells was assessed by FACS analysis (A). Molt-4 and U937 cells were pretreated with CD81 mAb or LDLR mAb and then treated with E2 protein. Phosphorylated and total ERK and p38 MAPK were detected by Western blot analysis in the cell lysates of Molt-4 (B) and U937 (C). Molt-4 cells were pretreated with CD81 mAb or LDLR mAb and then received BSA treatment. Phosphorylation of ERK and p38 MAPK was analyzed by Western blotting (D). The control indicates no antigen treatment.

 
As the E2-target cell interaction and the subsequent activation of ERK and p38 MAPK were observed, we thereby wondered if CD81 and LDLR on the cells took part in transmitting an E2 signal to the MAPK pathways. To estimate roles of CD81 and LDLR in E2-induced the MAPK activation, Molt-4 and U937 cells were pretreated with 4 µg/ml CD81 mAb or LDLR mAb followed by 1 µg/ml E2 treatment, and the phosphorylated and total MAPKs were then analyzed by Western blotting. Our data showed that the phosphorylation levels of ERK and p38 MAPK in Molt-4 cells pretreated with CD81 mAb or LDLR mAb were lower than those in response to 1 µg/ml E2 treatment (Fig. 3B) . Influence of the mAb on the MAPK activation induced by E2 protein was explored as well in U937 cells. Figure 3C showed that a concentration of 4 µg/ml was sufficient for LDLR mAb to reduce the activation of ERK and p38 MAPK induced by the E2 protein. In contrast, there was little difference in levels of the ERK and p38 MAPK phosphorylation between E2 treatment and CD81 mAb pretreatment. To rule out effects of the mAb on the kinases, Molt-4 and U937 cells were treated with the CD81 mAb and LDLR mAb for comparison, and the levels of ERK and p38 MAPK phosphorylation were similar to those in untreated cells (data not shown). At the same time, pretreatment with CD81 mAb or LDLR mAb failed to decrease levels of ERK and p38 MAPK phosphorylation in Molt-4 cells stimulated with BSA (Fig. 3D) , indicating that CD81 mAb and LDLR mAb have no inhibitory effects on BSA-induced kinase activation. These data suggested that pretreatment with CD81 mAb as well as LDLR mAb prevented the activation of ERK and p38 MAPK induced by E2 protein in Molt-4 cells, and such activation was impaired as a result of the LDLR mAb pretreatment but unimpaired in U937 cells with the CD81 mAb pretreatment, which was concordant with the levels of CD81 and LDLR on the cells. Therefore, we believed that CD81 and LDLR were involved in HCV E2-induced activation of ERK and p38 MAPK.

HCV E2 enhances cell proliferation
HCV core protein was described to promote proliferation of HepG2 cells by activating MAPK pathways [28 ]. In Molt-4 and U937 cells, the ERK and p38 MAPK were up-regulated differently by HCV E2 protein. MTT assay was thus carried out to assess proliferative response of the cells to E2 protein. For this purpose, Molt-4 and U937 cells were incubated for 48 h with various concentrations of E2 protein or 15 µg/ml BSA. Levels of cell proliferation were evaluated. As shown in Figure 4 , the two types of cell proliferation were enhanced by the E2 protein in a concentration-dependent manner, and the levels of Molt-4 cells were higher than those of U937 cells. The proliferation level in the control group without E2 or BSA incubation represented basal level of cells grown in the medium with 1% FBS. We observed that the proliferation of Molt-4 cells reached a peak following 1 µg/ml E2 incubation, which was statistically significant as compared with the control group (P<0.05). After incubation with the E2 protein at a higher concentration of 5, 10, or 15 µg/ml, proliferation of Molt-4 cells was increased, but the levels were lower than those of 1 µg/ml E2 protein. In comparison with the E2 protein, 15 µg/ml BSA incubation caused a slight increase of cell proliferation. The data paralleled the result that treatment of Molt-4 cells with 1 µg/ml E2 protein resulted in the increased phosphorylation of ERK and p38 MAPK as compared with BSA stimulation. Furthermore, the levels of U937 cell proliferation were also consistent with the apparent levels of ERK and p38 MAPK phosphorylation induced by E2 protein. These results implied that the cell proliferation might be enhanced by HCV E2 protein through the MAPK signaling pathways.


Figure 4
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  		Figure 4. Enhancement of cell proliferation by HCV E2 protein. Molt-4 and U937 cells were grown in the 1% FBS medium with different concentrations of E2 protein or 15 µg/ml BSA. Up to 48 h, the cell proliferation was evaluated by MTT assay. The control indicates cells cultured in the medium without E2 incubation. Open bars, Molt-4 cells; solid bars, U937 cells. The results are expressed as the mean OD and standard deviation of triplicate cultures for three independent experiments.

 
HCV E2 promotes cytokine release
T cell responses are thought to be important for the control of HCV infection. A previous study reported that production of IFN-{gamma} and IL-4 was enhanced by HCV E2 protein through engagement of CD81 on human blood T cells [29 ]. Recently, IFN-{gamma} has been shown to exert an anti-HCV effect through the Ras-MAPK signaling pathway [30 ]. Moreover, blocking IL-10 activity has been suggested to be a useful immunotherapy approach to enhance the efficacy of anti-HCV treatment [31 ]. We investigated production of IFN-{gamma} and IL-10 secreted by Molt-4 cells incubated with HCV E2 protein. ELISA assessment showed that IFN-{gamma} and IL-10 were measurable at a low level (IFN-{gamma}: 11.7 pg/ml; IL-10:3.2 pg/ml) in the supernatants of control group without E2 incubation. After incubation with the E2 protein at indicated concentrations for 48 h, levels of IFN-{gamma} and IL-10 were elevated in a dose-dependent manner. As shown in Figure 5 , the levels of IFN-{gamma} and IL-10 were elevated in the group incubated with 1 µg/ml E2 protein and significantly increased in response to 15 µg/ml E2 incubation (IFN-{gamma}: 58.8 pg/ml; IL-10: 36.7 pg/ml) as compared with the levels in the control group (P<0.05). To further define specificity of the cytokine increase, 15 µg/ml E2 protein reacted with 10 µg/ml E2 mAb for 1 h and then was allowed to incubate Molt-4 cells for another 48 h. Our data showed that the levels of IFN-{gamma} and IL-10 were diminished markedly as a result of incubation with the E2-E2 mAb as compared with 15 µg/ml E2 incubation (P<0.05). An approximate two- or threefold decrease was observable in IFN-{gamma} and IL-10 levels of the group incubated with the E2-E2 mAb, respectively, which indicated a specific effect of E2 protein on the cytokine secretion. In our experiments, the impact of the E2 mAb on cytokine release was ruled out by incubating the cells with the mAb alone. It is thus conceivable to conclude that the HCV E2 protein is required for stimulation of IFN-{gamma} and IL-10 release by Molt-4 cells.


Figure 5
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  		Figure 5. Promotion of IFN-{gamma} and IL-10 production by the HCV E2 protein. Molt-4 cells were cultured for 48 h in the 10% FBS medium plus E2 protein at indicated concentrations or the E2-E2 mAb complex. Concentrations of IFN-{gamma} and IL-10 secreted by Molt-4 cells were determined by ELISA assessment. The control indicates Molt-4 cells without E2 incubation. Open bars represent IL-10, and solid bars correspond to IFN-{gamma}. The results are expressed as the mean value and standard deviation of triplicate cultures for three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Human hepatocytes and lymphocytes are susceptive cells for HCV infection, whereas the mechanisms by which HCV causes human liver damage and extrahepatic autoimmune phenomena characterized by abnormal activation and proliferation of lymphocytes remain obscure and need to be elucidated further. In recent decades, disturbance of cellular signaling cascades has been proven to be a molecular basis for the development and progression of various human diseases [32 33 34 35 ]. By the use of the HCV E2 protein expressed in eukaryotic cells, we investigated MAPK signaling pathways and cell biological behavior in response to the E2 protein. The signaling system, comprised of the HCV E2 protein (extracellular signal), human T lymphoma Molt-4 and histiocytic lymphoma U937 cells (target cells), CD81 and LDLR (cellular receptors), ERK and p38 MAPK (signaling pathways), cell proliferation, and cytokine release (biological behavior), was explored in this study.

HCV core protein and nonstructural protein 5A were shown to perturb some signaling cascades involved in the viral pathogenesis. We focused on regulation of cellular signal transduction by HCV E2 protein in the absence of assistant stimuli. Experiments with HCV E2 protein in a native form are difficult. Approaches for studying structures and functions of HCV envelope proteins have been developed, such as E2 glycoprotein expressed in Escherichia coli or mammalian cells, HCV-like particles, pseudotyped murine retrovirus bearing the envelope glycoproteins, and HCV pseudotype particles containing envelope glycoproteins [36 37 38 39 40 ]. Particularly, the soluble E2 protein from HCV subtype 1a (amino acids 383–715 of the polyprotein with a purity of 98%–99%) derived from CHO cells was suggested to possess highly biological activities and proposed to be an effective tool for elucidation of E2 features [36 ]. We previously documented that the HCV E2 protein was capable of up-regulating the ERK pathway in human hepatoma cell lines [23 , 26 ]. ERK (also named p44/42 MAPK), p38 MAPK, and stress-activated protein kinase (SAPK)/Jun N-terminal kinase are three subfamilies of MAPK signaling pathways, which respond to survival, proliferation, and stress signals and modulate a broad array of cellular functions [41 ]. In HepG2 cells, the activation of p38 MAPK was induced cooperatively by the HCV E2 protein and human immunodeficiency virus envelope glycoprotein gp120 [42 ]. Here, investigation of ERK and p38 MAPK signaling pathways triggered by the HCV E2 protein was extended in different human cell lines. Detection of phosphorylated ERK and p38 MAPK was thus performed by immunocytochemistry and Western blot analyses in Molt-4 cells following stimulation with the HCV E2 protein. Our data demonstrated that the phosphorylation levels of ERK and p38 MAPK were much higher after exposure to the E2 protein compared with those in the cells without E2 treatment, as well as with those in response to control BSA stimulation. Furthermore, the levels of phosphorylated MAPKs were obviously decreased in the cells treated with the HCV E2-E2 mAb. U937 cells served as a control cell line and were tested for their response to the E2 protein. Similar results were also obtained in U937 cells. The phosphorylation and activation of ERK and p38 MAPK were suppressed by the specific E2 mAb, indicating that such activation indeed results from the HCV E2 protein without synergetic stimulation.

Properties of viral receptors are believed to account for susceptibility to viruses. Functional interplay between HCV E2 protein and its receptors may trigger cellular signaling pathways at an early stage of HCV infection. A notion was given that T cell receptor signaling was enhanced by HCV E2 protein through CD81 cross-linking on human T cells [43 ]. Binding of HCV E2 protein to CD81 on human hepatic stellate cells was also suggested to up-regulate matrix metalloproteinase 2 [25 ]. CD81 and LDLR are known to be cellular receptors for HCV. Involvement of these receptors in HCV E2-induced activation of ERK and p38 MAPK was thus studied. Our data showed that CD81 and LDLR were expressed differently on the surface of Molt-4 and U937 cells. Interaction of HCV E2 protein with the cells was revealed by FACS analysis. E2 bound to Molt-4 cells at a high level, and a low percentage of U937 cell-bound E2 was detectable, which paralleled the levels of CD81 and LDLR on the cells. We next examined whether CD81 and LDLR transmitted the E2 signal to intracellular MAPK pathways. Status of ERK and p38 MAPK was then analyzed for defining downstream signaling events following the E2-target cell interaction. We found that the phosphorylation and activation of MAPKs induced by the E2 protein correlated with the expression levels of CD81 and LDLR. By receptor competition assay, phosphorylation of the MAPKs was assessed in the cells pretreated with CD81 mAb or LDLR mAb. Blockage of these cellular receptors on Molt-4 cells with CD81 mAb or LDLR mAb led to the reduced activation of ERK and p38 MAPK induced by the E2 protein. As expected, the levels of ERK and p38 MAPK phosphorylation were increased slightly in CD81-deficient U937 cells exposed to the E2 protein, and such increase was impaired as a result of the pretreatment of LDLR mAb but not CD81 mAb. Moreover, pretreatment with CD81 mAb or LDLR mAb failed to inhibit BSA-induced activation of MAPKs. Although activation, aggregation, and recruitment of intracellular adaptors of CD81 and LDLR need to be clarified further, these results implicated that the interaction of HCV E2 protein with CD81 as well as LDLR provided a stimulatory signal for target cells and consequently triggered ERK and p38 MAPK signaling pathways. However, involvement of additional HCV receptors in such modulation is not excluded.

Alterations of MAPK signaling cascades are linked to cell growth. Proliferation and dormancy of cancer cells were suggested to be associated with the ERK/p38 (SAPK) activity ratio [44 ]. Consistent with the activation of ERK and p38 MAPK, we documented that the proliferation of Molt-4 cells was enhanced by HCV E2 protein. In contrast to Molt-4 cells, U937 cells had a tendency to a slight increase in the activation of ERK and p38 MAPK and the levels of cell proliferation after exposure to the E2 protein. These results highly implied that the proliferation but not apoptosis of susceptible cells toward E2 protein might facilitate the maintenance of HCV persistence and subsequent establishment of chronic infection. In other words, MAPK signaling pathways triggered by the E2 protein may transmit a survival signal to HCV-infected cells. Cytokines, mediators of inflammation and immunity, are known to correlate closely with the damage and recovery from HCV infection. Recently, modulation of MAPK signaling pathways by some signals has been shown to be responsible for changes of immunity such as cytokine response [45 , 46 ]. We observed that the secretion of IFN-{gamma} and IL-10 by Molt-4 cells was elevated specifically following incubation with the E2 protein. Here, a direct association between the cytokine release and the up-regulation of ERK and p38 MAPK signaling pathways is not studied. It is interesting that the elevation of IFN-{gamma} and IL-10 production was not parallel with the increase of cell proliferation toward the indicated concentrations of the E2 protein, which may be a result of a variation of cellular response. Therefore, activation, expansion, and cytokine secretion by human T lymphoma Molt-4 cells were driven by the HCV E2 protein. Similarly, the HCV E2 protein was shown to enhance human blood T cell proliferation as well as induction of IFN-{gamma} and IL-4 [29 ]. As for natural killer cells, proliferation and cytokine production were inhibited as a result of HCV E2 treatment [47 ], indicating a cell type-specific effect of E2 protein. Collectively, our results suggest that the HCV E2 protein may affect susceptible cells by targeting receptor-mediated ERK and p38 MAPK signaling pathways, which might be a clue for the explanation of clinical manifestations associated with HCV infection.


   ACKNOWLEDGEMENTS
 
This work was supported by the National Natural Science Foundation of China (No. 30500021) and the National Key Basic Research and Development (973) Program of China (No. 2002CB513006). We are greatly thankful to Drs. Michael Houghton, Christine Dong, and Shirley Wong (Chiron Corporation, Emeryville, CA) for generously providing HCV E2 protein and HCV E2 mAb. We also thank Dr. Li Li (Department of Laboratory Diagnosis, Changzheng Hospital, Shanghai, China) for technical assistance.

Received January 8, 2006; revised March 17, 2006; accepted April 5, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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