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Journal of Virology, April 2003, p. 4139-4148, Vol. 77, No. 7
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.7.4139-4148.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

A Positive Autoregulatory Loop of LMP1 Expression and STAT Activation in Epithelial Cells Latently Infected with Epstein-Barr Virus

Honglin Chen,1 Lindsey Hutt-Fletcher,2 Liang Cao,3,{dagger} and S. Diane Hayward1,4*

Sidney Kimmel Comprehensive Cancer Center,1 Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21231,4 School of Biological Sciences, University of Missouri—Kansas City, Kansas City, Missouri 64110,2 University of Hong Kong, Hong Kong, People's Republic of China3

Received 18 October 2002/ Accepted 8 January 2003


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ABSTRACT
 
STAT3 and STAT5 are constitutively activated and nuclear in nasopharyngeal carcinoma (NPC) cells. In normal signaling, STATs are only transiently activated. To investigate whether Epstein-Barr virus (EBV), and in particular the protein LMP1, contributes to sustained STAT phosphorylation and activation in epithelial cells, we examined STAT activity in two sets of paired cell lines, HeLa, an EBV-converted HeLa cell line, HeLa-Bx1, the NPC-derived cell line CNE2-LNSX, and an LMP1-expressing derivative, CNE2-LMP1. EBV infection was associated with a significant increase in the tyrosine-phosphorylated forms of STAT3 and STAT5 in HeLa-Bx1 cells. This effect correlated with LMP1 expression, since phosphorylated STAT3 and STAT5 levels were also increased in CNE2-LMP1 cells relative to the control CNE2-LNSX cells. No change was observed in STAT1 or STAT6 phosphorylation in these cell lines, nor was there a significant change in the levels of total STAT3, STAT5, STAT1, or STAT6 protein. Tyrosine phosphorylation allows the normally cytoplasmic STAT proteins to enter the nucleus and bind to their recognition sequences in responsive promoters. The ability of LMP1 to activate STAT3 was further established by immunofluorescence assays in which coexpression of LMP1 in transfected cells was sufficient to mediate nuclear relocalization of Flag-STAT3 and by an electrophoretic mobility shift assay which showed that LMP1 expression in CNE2-LNSX cells was associated with increased endogenous STAT3 DNA binding activity. In addition, the activity of a downstream target of STAT3, c-Myc, was upregulated in HeLa-Bx1 and CNE2-LMP1 cells. A linkage was established between interleukin-6 (IL-6)- and LMP1-mediated STAT3 activation. Treatment with IL-6 increased phosphorylated STAT3 levels in CNE2-LNSX cells, and conversely, treatment of CNE2-LMP1 cells with IL-6 neutralizing antibody ablated STAT3 activation and c-Myc upregulation. The previous observation that STAT3 activated the LMP1 terminal repeat promoter in reporter assays was extended to show upregulated expression of endogenous LMP1 mRNA and protein in HeLa-Bx1 cells transfected with a constitutively activated STAT3. A model is proposed in which EBV infection of an epithelial cell containing activated STATs would permit LMP1 expression. This in turn would establish a positive feedback loop of IL-6-induced STAT activation, LMP1 and Qp-EBNA1 expression, and viral genome persistence.


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INTRODUCTION
 
Epstein-Barr virus (EBV) is associated with a variety of human malignancies (62). In settings such as posttransplant lymphoproliferative disease, where the full latency III program is expressed, EBV nuclear-associated protein 2 (EBNA2) and LMP1 make critical contributions (10, 41). The Cp promoter that drives EBNA2 expression along with that of EBNA-LP and EBNA3A, -3B, and -3C is regulated by EBNA2 (39, 52, 72), as are the promoters for LMP1 and LMP2A (40, 49, 85, 91). EBNA2 also contributes to dysregulated cellular growth proliferation and provides a cell survival function. EBNA2 is a transcriptional activator that targets responsive promoters through interactions with the cell DNA-binding proteins Pu.1 and CBF1 (RBPJ-{kappa}) (20, 26, 31, 48, 83, 92).

In targeting CBF1, EBNA2 mimics activated Notch, NotchIC, and thus EBNA2 can modify cellular gene transcription in a manner that resembles constitutively activated Notch signaling (24, 32, 37, 71, 90). NotchIC has a separate antiapoptotic activity mediated through targeting of the immediate-early response factor Nur77 (38), and this activity is also demonstrated by EBNA2 (50).

LMP1 functions as a constitutively active tumor necrosis factor receptor and mimics aspects of CD40 signaling (15, 23, 58, 81). The cytoplasmic carboxy terminus of LMP1 contains two effector domains, CTAR1 and CTAR2, that interact with tumor necrosis factor receptor-associated factors and with tumor necrosis factor receptor-associated death domain and receptor interacting protein, respectively, to activate NF-{kappa}B, p38 mitogen-activated protein kinase, and JNK pathways (12, 14, 16, 22, 33, 35, 36, 43, 56, 73). As a downstream consequence of these pathways, LMP1 provides a cell survival function through upregulation of antiapoptotic proteins such as Bcl-2, Mcl-1, Bfl-1, and A20 (13, 18, 25, 47, 86) and alters cell growth through induction of epidermal growth factor receptor and cytokines such as interleukin-6 (IL-6) (16, 17, 28, 55). Another way in which LMP1 may contribute to altered cell growth is through inhibition of p16 to counter cellular senescence (87). LMP1 signaling also leads to tumorigenic growth, as demonstrated originally by the ability of rodent fibroblasts expressing LMP1 to grow in an anchorage-independent manner and form tumors in nude mice (84).

In Hodgkin's disease and in most EBV-associated epithelial tumors, a more limited type II latency program occurs in which EBNA1 is expressed from the Qp promoter and LMP1 is expressed but EBNA2 is not. An evaluation of the factors regulating latent gene expression in these cells in the absence of EBNA2 revealed a role for the Janus-associated kinase (JAK)-STAT pathway in both Qp-EBNA1 and LMP1 expression (6). LMP1 is transcribed from two promoters, the well-characterized ED-L1 (51, 68, 69, 77, 89) and a second promoter located within the terminal repeats, TR-L1 (63, 76). Promoter-reporter assays revealed that the TR-LMP1 promoter is positively regulated by STATs. Activation of the TR-LMP1 promoter by v-Src and the abolition of this effect by a dominant negative STAT3 construction implicated STAT3 as the potentially relevant STAT family member (7).

Additional evidence linking STATs and EBV-associated tumorigenesis was provided by immunohistochemical studies which found nuclear activated STAT3 and STAT5 in nasopharyngeal carcinoma (NPC) (7) and STAT3 and STAT6 in Hodgkin's disease tissues (7, 46, 70). STAT proteins are normally cytoplasmic, and their nuclear translocation is activated transiently by tyrosine phosphorylation mediated by JAK family kinases associated with cell surface receptors (61). The transient nature of STAT signaling is controlled by multiple negative regulatory steps that include dephosphorylation of JAKs by the phosphatases SHP1, SHP2, and CD45, antagonism of STAT activation by the suppressor of cytokine signaling family proteins, and prevention of DNA binding by activated STATs through interaction with protein inhibitor of activated STATs proteins that act as E3-like ligases for sumoylation (44).

Constitutive STAT activation can be induced through genetic mutation or through persistent cytokine or growth factor signaling. Constitutive activation of STATs, in particular STAT3 and STAT5, has been found in a variety of cancers (1, 4). Activated STAT3 and STAT5 have also been shown to contribute directly to oncogenesis. STAT3 is required for oncogenic transformation by v-Src (2, 78, 88), and a constitutively active form of STAT3, STAT3C, induces rodent fibroblasts to form colonies in soft agar and tumors in nude mice (3). STAT3 may also promote tumor angiogenesis through upregulation of vascular epidermal growth factor expression (60). The Bcr-Abl fusion protein found in chronic myelogenous leukemia patients leads to constitutive STAT5 activity, and a dominant negative STAT5 abolishes Bcr-Abl-induced cellular transformation (11, 59, 66).

Viruses are expert at manipulating cell signaling to their advantage. We wondered if EBV might be contributing to STAT activation in epithelial tumors such as NPC. LMP1 has been found to associate with JAK3 and upregulate STAT1 DNA binding activity (19, 29), and LMP1 is also known to upregulate expression of IL-6 (17, 75), a cytokine that activates JAK-STAT signaling predominantly through STAT3 (30). In this study, evidence is provided for a self-reinforcing cycle of LMP1-driven STAT3 activation that is mediated by IL-6 and can operate in epithelial cells. STAT activation may be one of the factors that favor the establishment of a latent infection in epithelial cells by stimulating Qp-EBNA1 and LMP1 expression. The data suggest that in situations in which the initial STAT activation occurred through normal ligand-mediated signaling, LMP1 expression could instigate an IL-6-mediated signaling loop that would render the cell independent of the initial stimulus and provide continuous STAT3 activation.


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MATERIALS AND METHODS
 
Cell lines. The CNE-2 cell line was derived from a poorly differentiated nasopharyngeal carcinoma (67). CNE2-LMP1 was established by infecting CNE2 cells with a recombinant retrovirus carrying the LMP1 cDNA (pLNSX-LMP1) (87). Stable LMP1-expressing cells and control retrovirus vector-transduced cells (CNE2-LNSX) were selected with 500 µg of geneticin (G418) per ml in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. HeLa-Bx1 was established by infecting HeLa cells with a recombinant EBV, Bx1 (57). EBV-positive cells were selected with G418 (600 µg/ml). Stable HeLa-Bx1 green fluorescent protein (GFP)-expressing cells were maintained in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum and 500 µg of G418 per ml. C-666-1, an EBV-positive NPC cell line (9), was maintained in RPMI 1640 plus 10% fetal bovine serum. NPC-KT, an EBV-positive epithelial cell line (74), was cultured in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. Daudi and B95-8 (EBV positive) and CA46 (EBV negative) B-cell lines were cultured in RPMI 1640 plus 10% fetal bovine serum.

Plasmids. STAT3-C was a generous gift from J. Darnell (3). Flag-tagged STAT3 (pHC79) was constructed by PCR amplification of a mouse STAT3 cDNA and insertion into a 5'-Flag-tagged pSG5 vector. SG5-LMP1 (pHC76) was constructed by inserting an LMP1 genomic fragment into the pSG5 vector at the EcoRI site. STAT6 was obtained from Tularik Inc. (California) (54).

Western blotting. To study STAT3 phosphorylation, CNE2-LNSX cells were treated with human recombinant IL-6 (50 ng/ml) (R&D, Minneapolis, Minn.) for 30 min before harvesting. To neutralize IL-6, monoclonal IL-6 antibody (Immunotech) was added to the medium at 4 µg/ml for 36 to 40 h before harvesting. Nuclear extracts were prepared by swelling cells in hypotonic buffer (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM NaF, 0.5 mM Na3VO4, 1 µg of leupeptin per ml, and 1 µg of pepstatin per ml) on ice for 30 min, and after the addition at 0.6% NP-40, the mixture was vortexed vigorously for 1 min to release the nuclei. Nuclei were collected by microcentrifugation at maximum speed for 15 s. After removal of the supernatant, nuclei were lysed in sample buffer (0.5 M Tris [pH 6.8], 10% glycerol, and 2% sodium dodecyl sulfate) and incubated at 100°C for 5 min.

Whole-cell lysates were prepared by sonicating cell pellets in sample buffer and boiling for 5 min before loading onto the gel. Proteins were fractioned on 10 to 12% polyacrylamide gels and blotted onto a nitrocellulose membrane (Bio-Rad), following the manufacturer's protocol. Antibodies for detecting phosphorylated STATs were purchased from Cell Signaling Inc. (New England BioLab) and used at a 1:1,000 dilution in phosphate-buffered saline with 5% skim milk. LMP1 antibody-expressing hybridoma cells (S12) were obtained from D. Thorley-Lawson (53). Antibodies against STAT3 were purchased from Upstate, and STATI, STAT5, and STAT6 antibodies were from Santa Cruz. Antibodies against c-Myc and Flag were obtained from Sigma, St. Louis, Mo. IL-6 antibody was obtained from R&D. Horseradish peroxidase-conjugated secondary antibody and the ECL color developing kit were purchased from Pharmacia-Amersham and used according to the manufacturer's instructions.

Reverse transcription-PCR. RNA was isolated with a Microprep kit (Pharmacia-Amersham). First-strand cDNA was prepared by reverse transcription at 42°C for 1 h with random priming. Then 1 to 2 µl of the cDNA reaction was used for the PCR. PCRs were carried out for 35 cycles of 94°C for 1 min, 57°C for 1 min, and extension at 72°C for 1 min. Real-time PCR was performed in duplicate with Sybr Green I and calibrated with ß-actin as the control. Data were analyzed with sequence detector system software version 1.7a (PE Applied Biosystems). The relative quantitation value was determined with the {Delta}{Delta}CT method. The primers used for reverse transcription-PCR were IL-6R (5'-CAGTATTCCCAGGAGTCCCAGAAG-3') and 5'-CATCCATGTTGTGAATGTCTTT G-3'), gp130, (5'-CACCTTCCAAAGGACCTACTG-3' and 5'-GTGAATTCTGGACATCCTTC-3'), and LMP1-TR (5'-CTAACACAAACACACGCTTTCTAC-3' and 5'-GAGAGCAATAATGAGCAGGATC-3'). Primers for EBV BARF0, RPMS1, Qp-EBNA1, LMP1(ED-L1+TR), and cellular actin have been described (7, 8).

Electrophoretic mobility shift assay. The oligonucleotide probe containing the STAT3 binding site (AGC TTC ATT CCC GTA AAT CCC TA) has been described previously (5). Double-stranded probe was labeled with Klenow polymerase and purified by centrifugation through a Sephadex G25 column. The electrophoretic mobility shift assay was performed as described before (6).

Indirect immunofluorescence assay. Transient transfection of Flag-STAT3, STAT6, and LMP1 into HeLa cells was carried out with the calcium-phosphate procedure and 1 µg of each plasmid DNA. To induce STAT6 nuclear translocation, STAT6-transfected cells were treated with alpha interferon for 30 min before harvesting. At 36 to 40 h posttransfection, cells were fixed with methanol and immunostained by standard procedures. Antibodies against STAT6 were obtained from Santa Cruz, Flag antibody was from Sigma, and antibody for detecting LMP1 (S12) has been described previously (53). Images were captured with a Leitz fluorescence microscope and Image-Pro software (Media Cybernetics, Silver Spring, Md.).


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RESULTS
 
EBV infection activates STAT3 and STAT5. We previously reported that JAK-STAT signaling activated the EBV Qp-EBNA1 and LMP1 in reporter assays (6, 7). Experiments are now presented to strengthen the case for STAT regulation of these latency genes and to provide evidence for a model (Fig. 1) in which EBV infection itself contributes to constitutive JAK-STAT signaling through LMP1 activation of STATs mediated by induction of IL-6.



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  		FIG. 1. Model illustrating a potential positive feedback loop connecting LMP1 signaling to IL-6 induction, STAT activation, and reinforced LMP1 expression.

An EBV-infected HeLa cell line, HeLa-Bx1, was established as a tool for testing the individual steps of the model. HeLa cells were infected with recombinant Bx1 virus, which carries both a GFP gene and a neomycin resistance marker (57), and cells carrying the EBV genome were selected in G418. The resulting HeLa-Bx1 cell line was GFP positive (Fig. 2A). A Southern blot with an EBV BamHI N-het fragment as the probe detected a single terminal repeat fragment, indicating that HeLa-Bx1 cells carry the EBV genome as an episome (Fig. 2B). Analysis of EBV gene expression in HeLa-Bx1 cells revealed a typical latency II program. Reverse transcription-PCR assays detected Qp-EBNA1, LMP1, BamHI-A rightward transcripts, and LMP2A (Fig. 2C). No Cp promoter usage was detected, nor was atypical latency promoter usage (42) detected (data not shown). Expression of LMP2A (TP1) is also consistent with the presence of circular as opposed to linear integrated forms of the EBV genome in HeLa-Bx1 cells.



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  		FIG. 2. Characterization of the EBV-converted HeLa-Bx1 cell line. (A) Immunofluorescence micrograph showing GFP expression in HeLa-Bx1 cells. (B) Southern blot of HeLa-Bx1 DNA probed with an EBV BamHI N-het fragment. The single band indicates the presence of episomal EBV DNA. (C) Ethidium bromide-stained reverse transcription-PCR products illustrating latency II EBV gene expression in HeLa-Bx1 cells. BART, BamHI-A rightward transcript.

Type II latency is the program found in EBV-associated epithelial malignancies such as nasopharyngeal carcinoma. The HeLa-Bx1 cell line therefore provides a suitable cell system for examining interactions between latency II genes such as LMP1 and the JAK-STAT pathway. Among the seven STAT family members, the ones that are associated with cell proliferation and are most frequently activated in human cancers are STAT3 and STAT5. These two STATs were also previously found to be activated in nasopharyngeal carcinoma tissues (7). STATs are activated by tyrosine phosphorylation. The phosphorylation status of STAT3 and STAT5 was examined by Western blotting with antibodies specific for tyrosine-phosphorylated STAT3 (Y705) and STAT5 (Y694). A comparison of HeLa and HeLa-Bx1 cells found no change in total STAT3 protein but an increase in phosphorylated STAT3 in HeLa-Bx1 cells (Fig. 3A). Phosphorylated STAT5 was also increased in HeLa-Bx1 cells, again in the absence of a change in total STAT5 protein (Fig. 3B).



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  		FIG. 3. STAT3 and STAT5 are activated in HeLa-Bx1 cells. Western blot analysis of nuclear extracts from HeLa-Bx1 and parental HeLa cells comparing the levels of (A) phosphorylated STAT3 (upper) and total STAT3 (lower) and (B) phosphorylated STAT5 (upper) and total STAT5 (lower). Antibodies used were anti-STAT3 (Y705), anti-STAT3, anti-STAT5 (Y694), and anti-STAT5. NS, nonspecific protein band used as a loading control.

LMP1 activates STAT3 and STAT5. The HeLa-Bx1 cells exhibit a latency II program that includes LMP1 expression. The contribution of LMP1 to STAT3 and STAT5 phosphorylation was examined with the paired NPC cell lines CNE2-LNSX (retrovirus vector converted) and CNE2-LMP1 (retrovirus-LMP1 converted) along with the EBV-positive NPC cell line C-666-1. Expression of LMP1 in the CNE2-LMP1 cells was verified by reverse transcription-PCR, Western blotting, and immunofluorescence with anti-LMP1 S12 monoclonal antibody (Fig. 4). Tyrosine-phosphorylated STAT3 was detected by Western blotting in CNE2-LNSX cells, and the level was increased by the expression of LMP1 in CNE2-LMP1 cells (Fig. 5A). Phosphorylated STAT5 was not detected in CNE2-LNSX cells but was also significantly upregulated in CNE2-LMP1 cells (Fig. 5B). The EBV-positive C-666-1 NPC cells contained constitutively activated STAT3 and STAT5 (Fig. 5A and 5B), which is consistent with the detection of nuclear STAT3 and STAT5 in immunohistochemical analyses of NPC tissues (7). In contrast, the phosphorylated form of STAT1 was not detected in C-666-1, CNE2-LNSX, or CNE2-LMP1 cells (Fig. 5C), and expression of phosphorylated STAT6 was not altered in CNE2-LMP1 cells or in HeLa-Bx1 cells compared to the parental cell lines. Neither EBV infection nor LMP1 expression significantly affected total STAT protein levels (Fig. 5A, 5B, 5C, and 5D). These results support a linkage between EBV infection and STAT3 and STAT5 activation in NPC.



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  		FIG. 4. Documentation of LMP1 expression in CNE2-LMP1 cells. (A) Ethidium bromide-stained gel showing the reverse transcription-PCR product amplified with LMP1-specific primers. (B) Western blot probed with anti-LMP1 S12 monoclonal antibody, showing LMP1 protein expression. (C) Immunofluorescence assay with S12 monoclonal antibody and fluorescein isothiocyanate-conjugated secondary antibody, verifying LMP1 expression and membrane localization in CNE2-LMP1 cells.



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  		FIG. 5. LMP1 induces STAT3 and STAT5 phosphorylation. Western blot analysis of nuclear extracts of the indicated cell lines probed with anti-STAT antibodies or with phosphorylation specific antibodies for: (A) STAT3, (B) STAT5, (C) STAT1 and (D) STAT6. Equal protein loading was established with anti-ß-actin antibody (A and D) or nonspecific (NS) protein staining.

LMP1 facilitates STAT3 nuclear localization. Normally, STAT proteins exist in a latent form and reside in the cytoplasm. Tyrosine phosphorylation is an essential step for STAT dimerization and subsequent translocation into the nucleus, where STAT proteins bind to their cognate DNA sequences and activate transcription. We tested to see whether LMP1-mediated phosphorylation of STAT on tyrosines also resulted in enhanced nuclear localization of STAT3. Flag-tagged STAT3 was transfected alone or in the presence of an LMP1 expression vector into HeLa cells, and the intracellular localization of the Flag-STAT3 protein was determined by immunofluorescence with anti-Flag antibody and rhodamine-conjugated secondary antibody. STAT3 was cytoplasmic in singly transfected cells, but mixed nuclear and cytoplasmic staining was seen in cells coexpressing LMP1 (Fig. 6A). LMP1 did not result in increased phosphorylation of STAT6 (Fig. 5D), and when STAT6 was examined in a parallel transfection assay, LMP1 had no effect on the cytoplasmic localization of STAT6 protein (Fig. 6B).



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  		FIG. 6. LMP1 relocalizes STAT3 to the nucleus. Immunofluorescence assays performed on HeLa cells transfected with (A) Flag-STAT3 or Flag-STAT3 plus LMP1 or (B) STAT6, STAT6 plus LMP1, or STAT6 in the presence of alpha interferon (300 IU/ml), added for 30 min prior to fixation. Cells were stained with anti-Flag (Flag-STAT3) or anti-STAT6 antibody and rhodamine-conjugated secondary antibody (red) or with S12 anti-LMP1 antibody and fluorescein isothiocyanate-conjugated secondary antibody (green). Nuclei were stained with 4',6'-diamidino-2-phenylindole (DAPI) (blue). Merge, overlay of the DAPI image with the anti-Flag (A) or anti-STAT6 (B) image.

To further demonstrate the specificity of the activation, STAT6 was shown to translocate into the nucleus in the presence of an appropriate physiological stimulus, alpha interferon (21) (Fig. 6B). Finally, to demonstrate that STAT nuclear translocation was associated with functional DNA binding activity, an electrophoretic mobility shift assay was performed with nuclear extracts from control CNE2-LNSX and CNE2-LMP1 cells and a STAT3 binding site oligonucleotide probe. An increase in STAT3 DNA binding activity was clearly apparent in the CNE2-LMP1 extracts (Fig. 7).



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  		FIG. 7. LMP1 increases STAT3 DNA binding activity. Electrophoretic mobility shift assay performed with a 32P-labeled STAT3 oligonucleotide probe and nuclear extracts from CNE2-LNSX and CNE2-LMP1 cells. NS, nonspecific bands.

IL-6 signaling is required for activation of STAT3 by LMP1. LMP1 signals through tumor necrosis factor receptor-associated factors (TRAFs) to activate NF-{kappa}B-inducing kinase and NF-{kappa}B. LMP1 may also interact with JAK3 to upregulate STAT1 DNA binding activity (19, 29), but phosphorylated STAT1 was not observed in the CNE2-LMP1 cells (Fig. 5C). One downstream effect of NF-{kappa}B signaling is induction of IL-6 by LMP1 (17, 75). We hypothesized that STAT activation, specifically STAT3 activation, might be mediated through IL-6. First, reverse transcription-PCR assays were performed to examine the expression of the IL-6 receptor and its partner in the receptor complex, the common IL-6 family receptor subunit gp130. With primers spanning gp130 exons 13 and 14 and IL-6 receptor exons 4 and 5, expression of both genes was detected in each of the epithelial cell lines examined, including CNE2-LNSX, CNE2-LMP1, HeLa-Bx1, and the EBV-positive NPC cell lines C-666-1 and NPC-KT (Fig. 8). Neither gp130 nor the IL-6 receptor was expressed in the Burkitt's lymphoma cell lines Daudi and CA46.



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  		FIG. 8. IL-6 receptor and gp130 common receptor subunit are expressed in HeLa-Bx1 cells and NPC-derived cell lines, including CNE2-LNSX. Ethidium bromide-stained products of reverse transcription-PCRs with primers spanning intron regions of gp130 (upper) and the IL-6 receptor (middle). Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control for RNA integrity (bottom).

The ability of LMP1 to induce expression of IL-6 was confirmed by comparing intracellular IL-6 protein levels in the CNE2-LNSX and CNE2-LMP1 cell lines (Fig. 9A). To test whether IL-6 is a key intermediate in the observed LMP1 activation of STAT3, CNE2-LNSX cells were treated transiently with recombinant human IL-6. The addition of IL-6 to the growth medium led to an increase in the phosphorylated form of STAT3 in CNE2-LNSX cells (Fig. 9B). In a complementary experiment, CNE2-LMP1 cells were grown in medium containing antibody against IL-6. Removal of IL-6 had the effect of reducing the levels of phosphorylated STAT3 to the basal level seen in the control CNE2-LNSX cells (Fig. 9B).



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  		FIG. 9. IL-6 is required for LMP1-mediated STAT3 phosphorylation. (A) Western blot showing increased IL-6 expression in CNE2-LMP1 cells compared to control CNE2-LNSX cells. The membrane was probed with anti-IL-6 antibody and then stripped and reprobed with anti-ß-actin antibody. (B) Increased phospho-STAT3 in CNE2-LNSX cells treated with IL-6 (100 ng/ml), and decreased phospho-STAT3 in CNE2-LMP1 cells treated with anti-IL-6 neutralizing antibody (4 µg/ml). The membrane was probed with an antibody specific for tyrosine-phosphorylated STAT3.

Among the cellular genes regulated by STAT3 is c-myc (45). To further confirm the functional nature of the STAT signaling induced by LMP1 and the linkage through IL-6, we examined c-Myc protein expression in the paired cell lines HeLa/HeLa-Bx1 and CNE2-LNSX/CNE2-LMP1. Western blotting experiments showed that c-Myc was upregulated twofold in HeLa-Bx1 cells relative to the parental HeLa cells and significantly upregulated in CNE2-LMP1 cells relative to parental CNE2-LNSX cells (Fig. 10). As was the case for STAT3 activation, the increase in c-Myc expression in CNE2-LMP1 cells was minimized by the addition of neutralizing antibody against IL-6 to the culture medium (Fig. 10). These experiments provide evidence for a direct linkage between IL-6 induction by LMP1 and LMP1-mediated STAT3 activation in epithelial cells.



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  		FIG. 10. Modulation of c-Myc expression via the LMP1-IL-6 pathway. Western blot analysis of c-Myc expression with anti-Myc antibody and control anti-ß-actin antibody. The CNE2-LMP1 + {alpha}-IL-6 cultures were incubated with antibody against IL-6 (4 µg/ml) for 36 h prior to harvesting. As illustrated in the lower panel, the c-myc promoter is positively regulated by STATs (45) through interferon-stimulated response (ISRE)/gamma interferon activation site (GAS) elements.

STAT3 induction of endogenous LMP1 and EBNA1 genes in HeLa-Bx1 cells. To examine the STAT responsiveness of EBV latency promoters in the background of the EBV genome, an expression vector for constitutively active STAT3, STAT3-C (3), was transfected into HeLa-Bx1 cells. Reverse transcription-PCR analysis of mRNA extracted from STAT3-C- and control vector-transfected cells detected STAT3-C upregulation of total LMP1 mRNA derived from the sum of the LMP1 ED-L1 and TR promoters and from the TR promoter (Fig. 11A). Qp-EBNA1 transcripts were also increased in STAT3-C-transfected cells. No change was observed in the levels of cellular glyceraldehyde-3-phosphate dehydrogenase transcripts, and a control EBV gene, BARF0, was slightly downregulated (Fig. 11A).



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  		FIG. 11. STAT3 induces LMP1 expression in HeLa-Bx1 cells. (A) Ethidium bromide-stained reverse transcription-PCR products showing upregulation of LMP1 and Qp-EBNA1 transcripts in cells transfected with a plasmid expressing a constitutively active form of STAT3, STAT3-C. The reverse transcription-PCR products for EBV BARF0 and cell glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are included for comparison. (B) Western blot showing increased LMP1 protein in cells transfected with Flag-STAT3-C versus control vector-transfected HeLa-Bx1 cells. Antibodies used for detection were anti-LMP1 S12 monoclonal antibody, anti-Flag antibody (Flag-STAT3-C), and anti-ß-actin antibody.

The mRNAs were also subjected to real-time PCR analysis for quantitation. In this assay, LMP1 was found to be upregulated approximately sixfold in STAT3-C-transfected cells, Qp-EBNA-1 was upregulated 4.5-fold, and BARFO was found to be downregulated twofold (data not shown). An increase in the amount of LMP1 protein was also observed in Western blots of STAT3-C-expressing cells (Fig. 11B). The STAT responsiveness of the LMP-1 TR promoter provides experimental evidence for the final stage of the regulatory loop proposed in Fig. 1.


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DISCUSSION
 
Each of the individual steps depicted in Fig. 1 has been documented previously. The goals of this study were to demonstrate that the entire cycle of events could take place in an epithelial culture model for NPCs to strengthen the evidence for STAT regulation of LMP1 expression and to determine the extent to which the LMP1-mediated induction of IL-6 contributes to STAT activation in EBV-infected epithelial cells. STATs are cytoplasmic transcription factors that are tyrosine phosphorylated by JAK family kinases in response to ligand-mediated activation of growth factor receptors and cytokine receptors or in response to activation by nonreceptor tyrosine kinases such as Src. We focused on activation by the cytokine IL-6, which utilizes a receptor complex comprising the IL-6 receptor and two copies of the common receptor subunit, gp130. JAK1 is the Janus kinase most associated with the IL-6 receptor complex, and receptor signaling leads predominantly to activation of STAT3 but also in some cases to STAT1 activation.

We observed increased tyrosine phosphorylation of STAT3 and STAT5 in HeLa-Bx1 cells and in CNE2-LMP1 cells relative to the control cell lines. This suggests that LMP1 is primarily responsible for the STAT activation that occurs on EBV infection of epithelial cells. The tyrosine phosphorylation of STAT3 induced by LMP1 led to functional activation, as demonstrated by the ability of LMP1 to induce nuclear translocation of STAT3 in transfected cells and to increase STAT3 DNA binding activity in CNE2-LMP1 cells. We had previously shown binding of STATs to the LMP1 TR promoter and demonstrated activation of an LMP1-TR promoter-reporter by v-Src (7). The abolition of this activation by a dominant negative STAT3 protein implied that the STAT family member involved in the LMP1 TR promoter response was STAT3. This conclusion has now been verified with the demonstration that the LMP1 gene within the endogenous EBV genomes in HeLa-Bx1 cells is regulated by STAT3. Both LMP1 transcripts and LMP1 protein were upregulated in HeLa-Bx1 cells transfected with a constitutively active form of STAT3.

The majority of the STAT3 phosphorylation induced by LMP1 was mediated through IL-6. LMP1 is known to upregulate IL-6 synthesis as a downstream effect of NF-{kappa}B activation, and IL-6 protein was indeed found to be increased in the CNE2-LMP1 cell line. Blocking of extracellular IL-6 with anti-IL-6 neutralizing antibody completely abolished the increase in STAT3 tyrosine phosphorylation seen in CNE2-LMP1 cells. The effect of IL-6 was also seen in the complementary experiment, in which addition of IL-6 to the medium of CNE2-LNSX cells led to increased STAT3 phosphorylation. IL-6 can also lead to tyrosine phosphorylation of STAT1. However, no increase in STAT1 phosphorylation was seen in HeLa-Bx1 or CNE2-LMP1 cells compared to the control cell lines.

It is possible that this lack of STAT1 phosphorylation is linked to another activity of LMP1. LMP1 also induces IL-10 synthesis (27, 82), and IL-10 is known to suppress tyrosine phosphorylation of STAT1 (34). The lack of STAT1 phosphorylation also suggests that the proposed STAT1 activation mediated by LMP1-bound JAK3 does not occur in HeLa-Bx1, CNE2-LMP1, or C-666-1 NPC cells. Along with STAT3, STAT5 was tyrosine phosphorylated in HeLa-Bx1 and CNE2-LMP1 cells, implying activation of STAT5 by EBV infection and by LMP1. The mechanism of STAT5 activation was not pursued here. STAT5 is activated by IL-6-type cytokines in some cell lines, and STAT5 is also one of the STATs activated by IL-10 and by the epidermal growth factor receptor. Thus, STAT5 phosphorylation could potentially be a downstream consequence of LMP1-mediated upregulation of IL-10 or epidermal growth factor receptor.

Of the seven STAT family members, STAT3 and STAT5 are those most associated with cell proliferation and prevention of apoptosis through upregulation of the antiapoptotic proteins Bcl-xL, Bcl-2, and Mcl-1 and cell cycle regulators such as cyclin D1, cyclin D2, and c-Myc (44). STAT3 and STAT5 are also directly capable of inducing cellular transformation and are activated in a variety of human leukemias, lymphomas, and solid tumors, such as breast cancer (4, 64). In the case of EBV-associated epithelial tumors, STAT3 in particular appears to be playing a dual role by regulating EBV latent infection in addition to making a direct contribution to the tumorigenic cell phenotype. EBV infection of epithelial cells in culture is inefficient and appears to be biased towards a lytic or abortive lytic outcome. It is very difficult to establish latently EBV infected epithelial cell lines in the absence of a strong selective pressure such as that provided by the presence of an antibiotic resistance marker incorporated into the virus genome. By positively regulating the Qp-EBNA1 and LMP1-TR promoters, activated STAT3 may bias the EBV infection of epithelial cells against the lytic program and towards the establishment of latency. Once expression of LMP1 occurs, our data suggest that LMP1 could prolong STAT activation through a positive feedback loop of IL-6 induction, STAT3 phosphorylation, and reinforced LMP1 expression. In this scenario, constitutive or prolonged STAT activation in epithelial cells would be a predisposing condition for the development of EBV-associated malignancies by supporting latent EBV infection.

The association of activated STATs with human cancers has raised interest in targeting them as a therapeutic intervention strategy (4, 79). JAK family tyrosine kinase inhibitors and Src family kinase inhibitors block STAT3 activation and inhibit survival of human cancer cell lines. Peptides that block STAT3 dimerization and DNA binding activity have also been developed and shown to inhibit cell transformation by Src (80). It is possible that EBV-associated epithelial tumors such as NPC might be among the cancers that could be a target for these reagents.

In circumstances in which constitutive STAT signaling did not occur as a result of a genetic alteration but remained dependent on LMP1 stimulation, the role of IL-6 as a key intermediate in STAT3 activation in EBV-infected epithelial cells raises the possibility that targeting of IL-6 through antibody-mediated inactivation might also be considered as a therapeutic direction for treatment of these tumors. Herceptin, the monoclonal antibody against the HER2/Neu receptor tyrosine kinase that is used for the treatment of breast cancer patients with HER2/Neu-overexpressing tumors, and Gleevec, which is used for the treatment of chronic myelogenous leukemia patients with constitutively active tyrosine kinase activity driven by the Bcr-Abl translocation, are encouraging examples of the therapeutic potential of targeting receptor tyrosine kinase-mediated signaling pathways (65).


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ACKNOWLEDGMENTS
 
We are grateful to James Darnell and Tularik Inc. for the STAT3-C and STAT6 plasmids, respectively, and David Thorley-Lawson for the S12 monoclonal antibody. We thank Dolly Huang for the C-666-1 NPC cell line, Yanxing Yu for assistance with real-time PCR analysis, and Feng Chang for manuscript preparation.

This work was funded by Public Health Services grant RO1 CA30356 to S.D.H. and R01 AI20662 to L.H.-F.


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FOOTNOTES
 
* Corresponding author. Mailing address: Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Bunting-Blaustein Building CRB308, 1650 Orleans St., Baltimore, MD 21231. Phone: (410) 955-2548. Fax: (410) 502-6802. E-mail: dhayward{at}jhmi.edu. Back

{dagger} Present address: Aptus Pharmaceutical, Inc., Gaithersburg, MD 20878. Back


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Journal of Virology, April 2003, p. 4139-4148, Vol. 77, No. 7
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.7.4139-4148.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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