A positive autoregulatory loop of LMP1 expression and STAT activation in epithelial cells latently infected with Epstein-Barr virus

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.     

Proteome analysis of the transformation potential of the Epstein–Barr virus-encoded latent membrane protein 1 in nasopharyngeal epithelial cells NP69

Latent membrane protein 1 (LMP1) of Epstein-Barr virus has been identified to be crucial in inducing cell transformation. However, the mechanism of LMP1-mediated epithelial cell transformation remains unclear. In this study, nasopharyngeal epithelial cells NP69 were infected with retrovirus with gene encoding wild type LMP1 or mutational LMP1 defective in binding to tumor necrosis factor receptor-associated death domain (TRADD). The NP69-LMP1(TRADD) lost some malignant phenotypes compared with the NP69-LMP1(WT). We performed proteomic approach to gain the differential protein expression profile associated with LMP1-mediated epithelial cell transformation. Furthermore, the differential expressional levels of partial identified proteins were confirmed by Western blot and real-time RT-PCR. Some were known to be related to the development of LMP1-induced transformation, and some were new LMP1-associated proteins. These data are valuable for further study of the mechanism of LMP1 in human nasopharyngeal carcinoma and provide some new clues for investigating other LMP1-associated tumors.     

The 30-Base-Pair Deletion in Chinese Variants of the Epstein-Barr Virus LMP1 Gene Is Not the Major Effector of Functional Differences between Variant LMP1 Genes in Human Lymphocytes

One group of sequence variants of Epstein-Barr virus is characterized by a 10-amino-acid deletion within the CTAR-2 functional domain of the latent membrane protein, LMP1. A role for this deletion in enhancing the tumorigenicity of the viral oncogene in rodent fibroblasts was recently demonstrated. We examined the effect of this deletion upon LMP1 function in four human lymphoid cell lines by using three natural variants of LMP1: the prototype B95.8 gene and the CAO and AG876 genes, both of which have codons 343 to 352 of the B95.8-LMP1 deleted. These experiments revealed that LMP1-mediated upregulation of CD40 and CD54 was markedly impaired (by 60 to 90%) with CAO-LMP1 compared with B95.8-LMP1. In contrast, the function of AG876-LMP1 was indistinguishable from that of B95.8-LMP1 in two lines and was only slightly impaired in the other two lines. Activation of NF-κB by CAO-LMP1 was not impaired in any of the lines; rather, activation of an NF-κB reporter by CAO-LMP1 was consistently about twofold greater than the activation with B95.8- or AG876-LMP1. Therefore, while the CAO-LMP1 is functionally distinct from the prototype B95.8-LMP1 in human lymphocytes, the 10-amino-acid deletion appears not to be directly responsible. This conclusion was confirmed by using a B95.8-LMP1 mutant with codons 343 to 352 deleted and chimerae of CAO- and B95.8-LMP1 in which the CTAR-2 domains of these genes were exchanged. Sequences outside the CTAR-2 domain were implicated in the distinct functional characteristics of CAO-LMP1 in human lymphoid cells.     

EB病毒潜伏膜蛋白1在鼻咽癌中结合磷酸化的TRAFs

 对鼻咽癌中潜伏膜蛋白 1 (LMP1 )是否结合磷酸化的肿瘤坏死因子受体相关因子 (tumornecrosis factor receptor- associated factors,TRAFs)信号分子进行探讨 .首先应用 CSA/SP双染色法在 30例鼻咽癌活检组织中发现 1 6例 (52 % ) LMP1与 TRAF1、TRAF2和 TRAF3共表达于癌细胞胞膜及胞浆同一部位 .这提示 EB病毒 LMP1在鼻咽癌中可能结合 TRAF1、TRAF2或TRAF3发挥作用 .进一步以导入载体 p SG5的鼻咽癌细胞系 HNE2 - p SG5为对照 ,建立了稳定表达 LMP1的鼻咽癌细胞系 HNE2 - LMP1 ,利用这两种细胞系 ,以免疫共沉淀 - Western印迹方法 ,证实了 LMP1可与磷酸化的 TRAF1、TRAF2或 TRAF3直接或间接作用形成免疫共沉淀复合物 .     

A mutation in Epstein-Barr virus LMP2A reveals a role for phospholipase D in B-Cell antigen receptor trafficking

Epstein-Barr virus (EBV) latent infection of B cells blocks the interrelated signaling and antigen-trafficking functions of the BCR through the activity of its latent membrane protein 2A (LMP2A). At present, the molecular mechanisms by which LMP2A exerts its control of BCR functions are only poorly understood. Earlier studies showed that in B cells expressing LMP2A containing a tyrosine mutation at position 112 in its cytoplasmic domain (Y112-LMP2A), the BCR could initiate signaling but could not properly traffic antigen for processing. Here, we show that BCR signaling in Y112-LMP2A-expressing cells is attenuated with a reduction in both the degree and duration of phosphorylation of key components of the BCR signaling cascade including Syk, BLNK, PI3K, and Btk. Notably, Y112-LMP2A expression completely blocked the BCR-induced activation of phospholipase D (PLD), a lipase implicated in the intracellular trafficking of a variety of surface receptors. We show that blocking PLD activity, by expressing Y112-LMP2A, treating cells with the PLD inhibitor 1-butanol or reducing PLD expression by siRNA, blocked BCR trafficking to class II-containing compartments. Moreover, Y112-LMP2A expression blocked the recruitment of phosphorylated forms of the downstream BCR signaling components, Erk and JNK, through both PLD-dependent and PLD-independent mechanisms. Thus, the investigation of the mechanism by which Y112-LMP2A blocks BCR function revealed an essential role for PLD in BCR trafficking for antigen processing.     

Interferon regulatory factor 5 represses expression of the Epstein-Barr virus oncoprotein LMP1: braking of the IRF7/LMP1 regulatory circuit

We have reported evidence for a positive regulatory circuit between interferon regulatory factor 7 (IRF7) and the Epstein-Barr virus (EBV) oncoprotein 1 (LMP1) (S. Ning, A. M. Hahn, and J. S. Pagano, J. Virol. 77:9359-9368, 2003). To explore a possible braking mechanism for this circuit, several type II EBV-infected cell lines that express different levels of LMP1 and IRF7 proteins and therefore are convenient for studying modulation of expression of LMP1 were analyzed. Endogenous levels of IRF7 and LMP1 were directly correlated. Transient expression of an IRF7 dominant-negative mutant decreased LMP1 levels. Endogenous IRF5 and IRF7 proteins were shown to physically associate in EBV-positive cells. Transient expression of IRF5 decreased activation of the LMP1 promoter by IRF7 in a dose-dependent manner. Finally, transfection of either an IRF5 dominant-negative construct or IRF5 small interfering RNA in these cells resulted in increases in endogenous levels of LMP1. These results indicate that IRF5 can downregulate IRF7's induction of expression of LMP1 most likely by interacting with IRF7 and provide a means of modulating a regulatory circuit between IRF7 and LMP1.     

鼻咽癌中EB病毒LMP1激活TRAFs的初步研究

在EB病毒潜伏膜蛋白LMP1介导的信号传导通路中,TRAFs作为LMP1活化的第一位信号分子,可能扮演着重要的分子开关角色。令人关注的是,在上皮性肿瘤NPC的发生中,EB病毒LMP1能否激活重要的TRAFs信号分子?究竟激活何种TRAFs信号分子,激活的机制何在?将LMP1cDNA导入LMP1表达阴性的HNE2中,建立稳定表达LMP1的鼻咽癌细胞系HNE2-LMP1。以此为材料,应用差异RT-PCR和Western blotting法证实,无论在RNA水平,还是蛋白水平上,TRAF1在HNE2-LMP1中表达较HNE2强,而TRAF2及TRAF3在HNE2-LMP1与HNE2细胞中表达无明显差异;进一步用免疫共沉淀-Western blotting证实LMP1可使TRAF1、TRAF2、TRAF3磷酸化而被活化。这些结果提示在鼻咽癌中,LMP1可能诱导TRAF1表达,而对TRAF2及TRAF3并不影响,但LMP1可磷酸化TRAF1、TRAF2、TRAF3而使其功能性活化。q     

Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells.

Starting with mRNA derived from Staphylococcal enterotoxin A induced human splenocytes, dsDNA was synthesized and inserted into unique BamHI site of the eukaryotic expression vector pSV529 (1). A recombinant plasmid containing human immune interferon (IFN-gamma) cDNA was identified by hybridization of plasmid inserted DNA bound onto nitrocellulose filters with mRNA derived from SEA-induced splenocytes, translation of the eluted RNA in Xenopus laevis oocytes and assaying for IFN activity. Plasmids containing the entire human IFN-gamma cDNA sequence were identified by colony hybridization and were sequenced. A unique coding region was identified which predicted a protein of 166 amino acids, the 20 N-terminal amino acids of which presumably represent a signal peptide. After transfection of monkey cells with plasmid DNA isolated from one of the recombinant clones (pHIIF-SV-gamma 1), IFN was excreted into the culture medium. This IFN was not distinguishable from human IFN-gamma by serological criteria or by cell target species specificity.     

Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) and LMP2A function cooperatively to promote carcinoma development in a mouse carcinogenesis model

The Epstein-Barr virus (EBV) proteins latent membrane proteins 1 and 2 (LMP1 and LMP2) are frequently expressed in EBV-associated lymphoid and epithelial cancers and have complex effects on cell signaling and growth. The effects of these proteins on epithelial cell growth were assessed in vivo using transgenic mice driven by the keratin 14 promoter (K14). The development of papillomas and carcinomas was determined in the tumor initiator and promoter model using dimethyl benzanthracene (DMBA), followed by repeated treatments of 12-O-tetradecanoyl phorbol 13-acetate (TPA). In these assays, LMP1 functioned as a weak tumor promoter and increased papilloma formation. In contrast, mice expressing LMP2A did not induce or promote papilloma formation. Transgenic LMP1 mice had slightly increased development of squamous cell carcinoma; however, the development of carcinoma was significantly increased in the doubly transgenic mice expressing both LMP1 and LMP2A. DMBA treatment induces an activating mutation in the Harvey-ras (H-ras(61)) oncogene, and this mutation was identified in most papillomas and carcinomas although several papillomas and carcinomas in K14-LMP1 and K14-LMP1/LMP2A mice lacked the mutation. Analysis of signaling pathways that are known to be activated by LMP1 and/or LMP2 indicated that all genotypes had high levels of activated extracellular signal-regulated kinase (ERK) and Stat3 in carcinomas with significantly higher activation in the doubly transgenic carcinomas. These findings suggest that, in combination, LMP1 and LMP2 contribute to carcinoma progression and that this may reflect the combined effects of the proteins on activation of multiple signaling pathways. This study is the first to characterize the effects of LMP2 on tumor initiation and promotion and to identify an effect of the combined expression of LMP1 and LMP2 on the increase of carcinoma development.     

Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1.

Several lines of evidence are compatible with the hypothesis that Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) or leader protein (EBNA-LP) affects expression of the EBV latent infection membrane protein LMP1. We now demonstrate the following. (i) Acute transfection and expression of EBNA-2 under control of simian virus 40 or Moloney murine leukemia virus promoters resulted in increased LMP1 expression in P3HR-1-infected Burkitt's lymphoma cells and the P3HR-1 or Daudi cell line. (ii) Transfection and expression of EBNA-LP alone had no effect on LMP1 expression and did not act synergistically with EBNA-2 to affect LMP1 expression. (iii) LMP1 expression in Daudi and P3HR-1-infected cells was controlled at the mRNA level, and EBNA-2 expression in Daudi cells increased LMP1 mRNA. (iv) No other EBV genes were required for EBNA-2 transactivation of LMP1 since cotransfection of recombinant EBNA-2 expression vectors and genomic LMP1 DNA fragments enhanced LMP1 expression in the EBV-negative B-lymphoma cell lines BJAB, Louckes, and BL30. (v) An EBNA-2-responsive element was found within the -512 to +40 LMP1 DNA since this DNA linked to a chloramphenicol acetyltransferase reporter gene was transactivated by cotransfection with an EBNA-2 expression vector. (vi) The EBV type 2 EBNA-2 transactivated LMP1 as well as the EBV type 1 EBNA-2. (vii) Two deletions within the EBNA-2 gene which rendered EBV transformation incompetent did not transactivate LMP1, whereas a transformation-competent EBNA-2 deletion mutant did transactivate LMP1. LMP1 is a potent effector of B-lymphocyte activation and can act synergistically with EBNA-2 to induce cellular CD23 gene expression. Thus, EBNA-2 transactivation of LMP1 amplifies the biological impact of EBNA-2 and underscores its central role in EBV-induced growth transformation.     

IFN-beta mediates coordinate expression of antigen-processing genes in RSV-infected pulmonary epithelial cells

Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs) clear respiratory tract infections caused by the pneumovirus respiratory syncytial virus (RSV) and also mediate vaccine-induced pulmonary injury. Herein we examined the mechanism for RSV-induced MHC class I presentation. Like infectious viruses, conditioned medium from RSV-infected cells (RSV-CM) induces naive cells to coordinately express a gene cluster encoding the transporter associated with antigen presentation 1 (TAP1) and low molecular mass protein (LMP) 2 and LMP7. Neutralization of RSV-CM with antibodies to interferon (IFN)-beta largely blocked TAP1/LMP2/LMP7 expression, whereas anti-interleukin-1 antibodies were without effect, and recombinant IFN-beta increased TAP1/LMP2/LMP7 expression to levels produced by RSV-CM. LMP2, LMP7, and TAP1 expression were required for MHC class I upregulation because the irreversible proteasome inhibitor lactacystin or transfection with a competitive TAP1 inhibitor blocked inducible class I expression. We conclude that RSV infection coordinately increases MHC class I expression and proteasome activity through the paracrine action of IFN-beta to induce expression of the TAP1/LMP2/LMP7 locus, an event that may be important in the initiation of CTL-mediated lung injury.     

Honokiol, a natural plant product, inhibits inflammatory signals and alleviates inflammatory arthritis

Honokiol (HNK), a phenolic compound isolated and purified from magnolia, has been found to have a number of pharmacologic benefits, including anti-angiogenic and anti-inflammatory properties. HNK has long been used in traditional Asian medicine without toxic side effects. We and others have extensively studied signaling to B cells by CD40 and its Epstein Barr viral mimic, latent membrane protein 1 (LMP1), which has been implicated in exacerbation of chronic autoimmune disease. We asked whether HNK could inhibit CD40 and LMP1 inflammatory signaling mechanisms. In vivo, HNK stabilized the severity of symptomatic collagen-induced arthritis in both CD40-LMP1 transgenic mice and their congenic C57BL/6 counterparts. Ex vivo studies, including collagen-specific serum Ab and Ag recall responses, as well as CD40 or LMP1-mediated activation of splenic B cells, supported the anti-inflammatory effects of HNK. In mouse B cell lines expressing the human CD40-LMP1 chimeric receptor, CD40- and LMP1-mediated NF-kappaB and AP-1 activation were abrogated in a dose-dependent manner, with a concomitant decrease in TNF-alpha and IL-6. These promising findings suggest that the nontoxic anti-inflammatory properties of HNK could be valuable for blocking the autoimmune response.     

Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells

Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membraneprotein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies,such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma.However,the therapeutic amount ofCTLs is often hampered by the limited supply of antigen-presenting cells.To address this issue,an artificialantigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetramericcomplex,anti-CD28 antibody and CD54 molecule to a cell-sized latex bead,which provided the dual signalsrequired for T cell activation.By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral bloodmononuclear cells from HLA-A2 positive healthy donors,LMP2 antigen-specific CTLs were induced andexpanded in vitro.The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell,the cytotoxicity was inhibited bythe anti-HLA class Ⅰ antibody (W6/32).These results showed that LMP2 antigen-specific CTLs could beinduced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC.Thus,aAPCs coated with an HLA-pLMP2 complex,anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specificCTLs for adoptive immunotherapy.     

EB病毒潜伏膜蛋白LMP1编码基因沉默对NF—kB表达的影响

目的：探讨EBV阳性胃上皮细胞中潜伏膜蛋白1（latentmembraneprotein-1,LMP1）基因沉默对NF-kB转录表达的影响。方法：以稳定表达LMP1的EBV阳性胃上皮细胞系作为靶细胞,采用化学合成的siRNA特异性沉默LMP1,用RT-PCR和Westem-blotting分别检测其在不同时间段mRNA和蛋白水平特异性沉默效果;Westernblotting及免疫酶染色法检测LMP1基因沉默对NF—kB转录表达及核转移的影响。结果：siRNA在mRNA和蛋白水平可特异性沉默LMP1的表达,且有时间效应关系;Westem blotting及免疫酶染色法检测结果显示LMP1沉默可干扰NF—KB表达,导致靶细胞核内NF-kB水平下降,细胞浆内水平升高,而NF—kB总蛋白有下降趋势。结论：LMP1基因特异性沉默能够影响NF-kB表达,抑制其核转移。     

Proteasome subunit LMP2 is required for matrix metalloproteinase-2 and -9 expression and activities in human invasive extravillous trophoblast cell line

The ubiquitin-proteasome pathway (UPP) is involved in the degradation of the extracellular matrix (ECM) and trophoblastic invasion during early pregnancy. Our previous studies demonstrated that inhibition of UPP suppresses expression of matrix metalloproteinase (MMP)-2 and -9. LMP2 is an important proteasome subunit that is critical for proteasome activity. This study investigated the regulatory mechanism of LMP2 on the expression and activities of MMP-2 and MMP-9. Our results showed that transfection of LMP2 siRNA plasmid into the human invasive extravillous trophoblast cell line (HTR8/Svneo) could significantly suppress expression of LMP2 mRNA and protein. The mRNA expression of MMP-2 and MMP-9 and their activities were markedly decreased in the LMP2-inhibited cells. Inhibition of LMP2 could also reduce IkappaBalpha mRNA level, although the expression of phosphorylated IkappaBalpha was increased. In the LMP2-inhibited cells, expression of mRNA encoding NF-kappaB subunits p50 and p65 remained normal, but the p50 protein level was significantly decreased in the cytosolic and nuclear extracts, while p65 protein was markedly reduced only in the nuclear extract. We also demonstrated that blockage of the NF-kappaB pathway by the NF-kappaB translocation inhibitor SN50 markedly reduced the expression of MMP-2 and MMP-9 in HTR8/Svneo cells, a result that is fully consistent with the results from the LMP2-inhibited HTR8/Svneo cells. These data suggest that LMP2 contributes to IkappaBalpha degradation and p50 generation, and that inhibition of LMP2 suppresses expression and activities of MMP-2 and MMP-9 by blocking the transfer of active NF-kappaB heterodimers into the nucleus.     

Gene hACR-1 suppresses apoptosis of striated muscle fibres of m. quadriceps femoris after ballistic transfection of mdx mice

Human minidystrophin gene (pSG5dys plasmid) and hACR-1 gene (pRc-CMV-10.1 plasmid) were cotransfected by means of "gene-gun" to M. quadriceps femoris of mdx mice. Effects of transfection on dystrophin expression and survival of striated muscle fibres (SMF) were studied on the 21st day after shots. In the control mdx dystrophin-positive muscular fibers [D(+)] SMF and destroyed SMF made 2.1 +/- 0.1 and 2.1 +/- 0.3%, respectively. In mice transfected with pSG5dys plasmid (20 mkg of DNA per mouse), the shares of D(+) SMF and dead SMF raised, respectively, up to 5.6 +/- 1.4 and 4.5 +/- 0.9%. Transfection of mice with pRc-CMV-10.1 (DNA dose is 20 mkg per mouse) reduced the levels of apoptosis in SMF and D(+) SMF level to 1.6 +/- 0.6 and 1.1 +/- 0.4%, respectively. Cotransfection by pSG5dys and pRc-CMV-10.1 plasmids (10 and 10 mkg of each plasmids DNA per mouse) reduced the share of D(+) SMF to 1.1 +/- 0.5% and SMF destruction to 0.9 +/- 0.3%. pSG5dys transfection considerably reduced the share of SMF having peripherally located nuclei, thus indicating a decrease in SMF differentiation level after transfection. Cotransfection of ACR-1 gene and a dystrophin minigene did not suppress further cytodifferentiation of mdx muscle fibers. A conclusion is made that ballistic transfection by hACR-1 gene reduces the level of apoptosis in mdx mice SMF without changing the level of SMF differentiation. The cotransfection of mdx mice muscle by hACR-1 and human minidystrophin gene reduces SMF destruction and supports SMF differentiation, too.     

Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cepsilon

ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFalpha (tumour necrosis factor alpha). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cepsilon (protein phosphatase 2Cepsilon), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cepsilon inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cepsilon mutant enhanced AP-1 activity. Exogenous PP2Cepsilon associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cepsilon and ASK1 was also observed in mouse brain extracts. PP2Cepsilon directly dephosphorylated ASK1 at Thr845 in vitro. In contrast with PP5, PP2Cepsilon transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cepsilon maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cepsilon and PP5 play different roles in H2O2-induced regulation of ASK1 activity.     

The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor.

The Epstein-Barr virus (EBV)-encoded LMP1 protein is an important component of the process of transformation by EBV. LMP1 is essential for transformation of B lymphocytes, most likely because of its profound effects on cellular gene expression. Although LMP1 is expressed in the majority of nasopharyngeal carcinoma (NPC) tumors, the effect of LMP1 on cellular gene expression and its contribution to the development of malignancy in epithelial cells is largely unknown. In this study the effects of LMP1 on the expression and tyrosine kinase activity of the epidermal growth factor receptor (EGFR) were investigated in C33A human epithelial cells. Stable or transient expression of LMP1 in C33A cells increased expression of the EGFR at both the protein and mRNA levels. In contrast, expression of the EGFR was not induced by LMP1 in EBV-infected B lymphocytes. Stimulation of LMP1-expressing C33A cells with epidermal growth factor (EGF) caused rapid tyrosine phosphorylation of the EGFR (pp170) as well as several other proteins, including pp120, pp85, pp75, and pp55, indicating that the EGFR induced by LMP1 is functional. LMP1 also induced expression of the A20 gene in C33A epithelial cells. In C33A cells, LMP1 expression increased the proliferative response to EGF, as LMP1-expressing C33A cells continued to increase in number when plated in serum-free media supplemented with EGF, while the neo control cells exhibited very low levels of viability and did not proliferate. Immunoblot analysis of protein extracts from nude mouse-passaged NPC tumors also demonstrated that the EGFR is overexpressed in primary NPC tumors as well as those passaged in nude mice. This study suggests that the alteration in the growth patterns of C33A cells expressing LMP1 is a result of increased proliferative signals due to enhanced EGFR expression, as well as protection from cell death due to LMP1-induced A20 expression. The induction of EGFR and A20 by LMP1 may be an important component of EBV infection in epithelial cells and could contribute to the development of epithelial malignancies such as NPC.