Roles of TRAF2 and TRAF3 in Epstein-Barr virus latent membrane protein 1-induced alternative NF-κB activation

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1)-induced NF-κB activation is essential for EBV-transformed B cell survival. LMP1 has two C-terminal cytoplasmic domains referred to as C-Terminal Activation Regions (CTAR) 1 and 2 that activate the alternative and canonical NF-κB pathways, respectively. While CTAR2 activates TRAF6, IKKβ and IKKγ-dependent canonical NF-κB pathway, CTAR1 interacts with TRAF2 and TRAF3 and activates NIK and IKKα-dependent alternative NF-κB pathway involving p100 processing into functional p52. Using IKKα^−/−, IKKβ^−/−, IKKγ^−/−, TRAF2^−/−, TRAF3^−/−, TRAF6^−/−, and NIK^aly/aly mouse embryonic fibroblasts (MEFs), potential roles of these proteins in LMP1-induced alternative NF-κB activation were investigated. Deficiency in IKKα or functional NIK, but not in IKKβ, IKKγ, or TRAF6, severely impaired LMP1-induced p100 processing. Notably, p100 was constitutively processed in TRAF2^−/− or TRAF3^−/− MEFs independently of LMP1 suggesting that TRAF2 or TRAF3 may play a regulatory role in p100 processing. Subsequently, TRAF2 or TRAF3 over-expression in HEK293 cells significantly blocked LMP1-induced p100 processing. The LMP1 CTAR1 expression in 293HEK cells activated the alternative p65/p52 complex while CTAR2 failed to do so. Taken together, LMP1 activates alternative NF-κB pathway through functional NIK and IKKα that is regulated by TRAF2 or TRAF3.     

Epstein-Barr virus encoded latent membrane protein 1 (LMP1) and TNF receptor associated factors (TRAF): colocalisation of LMP1 and TRAF1 in primary EBV infection and in EBV associated Hodgkin lymphoma.

AIMS: Epstein-Barr virus (EBV) immortalises B cells in vitro and is associated with several malignancies. Most phenotypic effects of EBV are mediated by latent membrane protein 1 (LMP1), which interacts with tumour necrosis factor receptor associated factors (TRAFs) to activate NF-kappaB. This study examines TRAF1 and LMP1 expression in EBV associated lymphoproliferations. METHODS: TRAF1 expression was investigated in 26 Hodgkin lymphomas (HL; 18 EBV+, eight EBV-), seven EBV+ Burkitt lymphomas (BL), two infectious mononucleosis (IM) tonsils, and lymphoreticular tissue from eight chronic virus carriers. Seven anaplastic large cell lymphomas and 10 follicular B cell lymphomas were also studied. Colocalisation of TRAF1 and LMP1 was studied by immunofluorescent double labelling and confocal laser microscopy. RESULTS: TRAF1 colocalises with LMP1 in EBV infected cells in IM. EBV positive lymphocytes from chronic virus carriers were negative for TRAF1 and LMP1. In HL biopsies, TRAF1 was strongly expressed independently of EBV status, whereas all BL cases were TRAF1-. In EBV+ HL cases, TRAF1 colocalised with LMP1. Eight of 10 follicular lymphomas expressed TRAF1 in centroblast-like cells. Four of seven anaplastic large cell lymphomas weakly expressed TRAF1. CONCLUSIONS: These results suggest that in non-neoplastic lymphocytes, TRAF1 expression is dependent on the presence of LMP1, and that in IM B cells in vivo, LMP1 associated signalling pathways are active. In HL, TRAF1 is expressed independently of EBV status, probably because of constitutive NF-kappaB activation. The function of TRAF1 in HL remains to be determined.     

Hepatitis C virus core protein enhances FADD-mediated apoptosis and suppresses TRADD signaling of tumor necrosis factor receptor

Hepatitis C virus (HCV) core protein has been shown to interact with the death domain (DD) of tumor necrosis factor receptor-1 (TNFR1). In this study, we further examined the interaction of the core protein with the signaling molecules of TNFR1, including FADD, TRADD, and TRAF2, in a human embryonic kidney cell line, HEK-293, that overexpresses the HCV core protein. This core protein-expressing cell line exhibited enhanced sensitivity to TNF-induced apoptosis. By in vitro binding and in vivo coimmunoprecipitation assays, we showed that the HCV core protein interacted with the DD of FADD and enhanced apoptosis induced by FADD overexpression. This enhancement could be blocked by a dominant-negative mutant of FADD. In contrast, the core protein did not directly interact with the DD of TRADD, but could disrupt the binding of TRADD to TNFR1. TRAF2 recruitment to the TNFR1 signaling complex was also disrupted by the core protein. Correspondingly, TRAF2-dependent activation of the protein kinase JNK was suppressed in the core protein-expressing cells. However, NF kappa B activation by TNF was not significantly altered by the HCV core protein, suggesting the existence of TRAF2-independent pathways for NF kappa B activation. These results combined indicate that the HCV core protein sensitizes cells to TNF-induced apoptosis primarily by facilitating FADD recruitment to TNFR1. The inhibition of JNK activation by the HCV core protein may also contribute to the increased propensity of cells for apoptosis. These results, in comparison with other published studies, suggest that the effects of the HCV core protein and their underlying mechanisms vary significantly among cells of different origins.     

The Epstein-Barr virus latent membrane protein 1 (LMP1) enhances TNF alpha-induced apoptosis of intestine 407 epithelial cells: the role of LMP1 C-terminal activation regions 1 and 2

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) can protect some kinds of lymphocytes from apoptotic cell death. In contrast, the present study showed that the expression of LMP1 induced high susceptibility to tumor necrosis factor alpha (TNFalpha)-induced apoptosis in intestine 407 epithelial cells, without affecting expression of TNF receptors I and II. LMP1-deletion mutants lacking either C-terminal activation region (CTAR)-1 or CTAR-2 had ability to enhance TNFalpha-induced apoptosis, whereas the deletion of both activation regions completely abolished the induction of high susceptibility to TNFalpha. Phosphorylation of the NFkB-inhibitory molecule IkB-alpha, another biological activity of TNFalpha, was not enhanced by LMP1-expression. LMP1 upregulated antiapoptotic gene A20 expression, suggesting that A20 can not block TNFalpha-induced apoptosis in this cell system. Apoptosis triggered by TNFalpha in LMP1-expressing intestine 407 cells was blocked by inhibitors of caspases-8 and -3. It is therefore concluded that in intestine 407 epithelial cells, LMP1 enhances primarily signal cascade responsible for TNFalpha-induced apoptosis, which occurs at a level upstream of acting site of caspases-8 and -3 and that CTAR-1 and CTAR-2 are involved in enhancement of TNFalpha-induced apoptosis.     

The Epstein-Barr virus latent membrane protein 1 induces interleukin-10 in Burkitt's lymphoma cells but not in Hodgkin's cells involving the p38/SAPK2 pathway

Infection of B cells with Epstein-Barr Virus (EBV) induces interleukin-10 (IL-10) production, which may contribute to transformation. IL-10 can modulate the immune response at certain levels, playing a crucial role in balancing humoral and cellular responses. Moreover, it can function as a growth and differentiation factor for B cells. However, the mechanism of IL-10 induction is still unclear. Here we demonstrate that IL-10 was specifically induced by the EBV-latent membrane protein 1 (LMP1) in Burkitt's lymphoma (BL) cell lines BL2 and BL41. In two T cell lines (Jurkat, MOLT3), two NHL cell lines (U266, MHH-PREB1), or three Hodgkin's disease (HD) cell lines (L428, L540, and KMH2), LMP1 did not induce IL-10 expression. In contrast, LMP1 activated CD40 or CD54 (ICAM1) expression in the analyzed cell lines. LMP1 derivatives lacking the C-terminal activation regions (CTAR), by deletion of the amino acids between 187 and 351 (Delta CTAR1) or 232 and 386 (Delta CTAR2), alone, or together induced IL-10 at very low amounts compared to wild-type LMP1. Inhibition of LMP1-mediated NF kappa B activation by constitutive repressive I kappa B-alpha only marginally impaired IL-10 expression in BL2 cells, while SB2035080 at 5 microM (a specific p38/SAPK2 inhibitor) led to reduced IL-10 expression. Our findings confirm the role of LMP1 in transactivation of cellular genes possibly important for tumor immunoescape but show that more than one signaling pathway is involved in this activation and suggests the necessity of a defined conformation of CTARs to activate IL-10 involving p38/SAPK2.     

Identification of functional differences between prototype Epstein-Barr virus-encoded LMP1 and a nasopharyngeal carcinoma-derived LMP1 in human epithelial cells

The contribution of Epstein-Barr virus (EBV) strain variation to the pathogenesis of virus-associated tumours remains unknown. Given the central role of LMP1 in EBV-induced transformation, much interest has focused on the influence of LMP1 sequence variation on the signaling pathways and multiple downstream phenotypic consequences of LMP1 expression. The identification of LMP1 variants with a common 10-amino-acid deletion and additional point mutations (typified by the CAO-LMP1 isolate) in EBV strains associated with nasopharyngeal carcinoma prompted us to examine the effect of stable prototype B95.8-LMP1 and CAO-LMP1 expression on the phenotype and differentiation of SCC12F human epithelial cells. Both forms of LMP1 were able to induce expression of the antiapoptotic A20 protein and provide protection from tumour necrosis factor-alpha-induced cytotoxicity. Although B95.8-LMP1 induced growth inhibition, expression of certain cell surface molecules (CD40, CD44, and CD54), and secretion of interleukin-6 and -8 in SCC12F cells, stable CAO-LMP1 expression failed to elicit these effects. Furthermore, B95. 8-LMP1, but not CAO-LMP1, induced alterations in cell morphology and blocked epithelial cell differentiation. Both B95.8-LMP1 and CAO-LMP1 induced similar levels of nuclear factor-kappaB activation, but the ability of CAO-LMP1 to activate the AP-1 pathway was relatively impaired. These data highlight significant functional differences between the prototype B95.8-LMP1 and the CAO-LMP1 variant when stably expressed in human epithelial cells and suggest that continued analysis of LMP1 variants will help to further dissect the signaling pathways activated by LMP1 as well as provide insights into the contribution of LMP1 sequence variation to the pathogenesis of EBV-associated tumours.     

Characterization of the Cyno-EBV LMP1 homologue and comparison with LMP1s of EBV and other EBV-like viruses

EBV latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation and has been associated with several cases of malignancies. EBV-like viruses in Cynomolgus monkeys (Macaca fascicularis) have been associated with high lymphoma rates in immunosuppressed monkeys. In the study, the entire coding region of the Cyno-EBV LMP1 gene was cloned, sequenced and expressed in human embryonic kidney (HEK) cells 293. The Cyno-EBV LMP1 homologue sequence predicted a 588 amino acid (a.a.) protein with a short 19 a.a. N-terminus, six transmembrane domains and a long carboxy tail of 404 a.a. The protein contained a series of seven 9 a.a.-tandem repeats and two 20 a.a.-repeats, which harbored two potential TRAF binding motifs, PxQxT/S. These repeats shared no homology with the repeats in any other LMP1. However, the proline-rich sequence GPxxPx₆ found within the 11 a.a.-repeats of EBV LMP1 was conserved in Cyno-EBV carboxy tail and contained two consensus JAK/STAT sequences PxxPxP. A cluster of eight histidine residues was found in proximity to the last transmembrane domain of Cyno-EBV LMP1 and was exploited as a natural protein tag in expression studies. Western blot analysis revealed a major polypeptide of 110 kDa. Comparative functional studies showed that Cyno-EBV LMP1 expressed in HEK 293 cells shares the same ability as EBV LMP1 to induce NFκB driven CAT activity.     

Nonphagocytic oxidase 1 causes death in lung epithelial cells via a TNF-RI-JNK signaling axis

Airway epithelial cells are simultaneously exposed to and produce cytokines and reactive oxygen species (ROS) in inflammatory settings. The signaling events and the physiologic outcomes of exposure to these inflammatory mediators remain to be elucidated. Previously we demonstrated that in cultured mouse lung epithelial cells exposed to bolus administration of H(2)O(2), TNF-alpha-induced NF-kappaB activity was inhibited, whereas c-Jun-N-terminal kinase (JNK) activation was enhanced via a mechanism involving TNF receptor-1 (TNF-RI). In this study we used the nonphagocytic NADPH oxidase (Nox1) to study the effects of endogenously produced ROS on a line of mouse alveolar type II epithelial cells. Nox1 expression and activation inhibited TNF-alpha-induced inhibitor of kappaB kinase (IKK), and NF-kappaB while promoting JNK activation and cell death. Nox1-induced JNK activation and cell death were attenuated through expression of a dominant-negative TNF-RI construct, implicating a role for TNF-RI in Nox1 signaling. Furthermore, Nox1 used the TNF-RI adaptor protein TNF-receptor-associated factor-2 (TRAF2), and the redox-regulated JNK MAP3K, apoptosis signal kinase-1 (ASK1), to activate JNK. In addition, ASK1 siRNA attenuated both Nox1-induced JNK activity and cell death. Collectively, these studies suggest a mechanism by which ROS produced in lung epithelial cells activate JNK and cause cell death using TNF-RI and the TRAF2-ASK1 signaling axis.     

TAK1-binding protein 2 facilitates ubiquitination of TRAF6 and assembly of TRAF6 with IKK in the IL-1 signaling pathway

TAK1 mitogen-activated protein kinase kinase kinase participates in the Interleukin-1 (IL-1) signaling pathway by mediating activation of JNK, p38, and NF-kappaB. TAK1-binding protein 2 (TAB2) was previously identified as an adaptor that links TAK1 to an upstream signaling intermediate, tumor necrosis factor receptor-associated factor 6 (TRAF6). Recently, ubiquitination of TRAF6 was shown to play an essential role in the activation of TAK1. However, the mechanism by which IL-1 induces TRAF6 ubiquitination remains to be elucidated. Here we report that TAB2 functions to facilitate TRAF6 ubiquitination and thereby mediates IL-1-induced cellular events. A conserved ubiquitin binding domain in TAB2, the CUE domain, is important for this function. We also found that TAB2 promotes the assembly of TRAF6 with a downstream kinase, IkappaB kinase (IKK). These results show that TAB2 acts as a multifunctional signaling molecule, facilitating both IL-1-dependent TRAF6 ubiquitination and assembly of the IL-1 signaling complex.     

EB病毒潜伏膜蛋白1羧基端活化区域2蛋白特异性结合短肽的筛选

目的 利用噬菌体12肽库筛选能与EB病毒潜伏膜蛋白1(LMPl)羧基端活化区域(CTAR)2蛋白特异性结合的短肽.方法 用纯化的CTAR2蛋白筛选噬菌体12肽库.通过夹心ELISA方法分析噬菌体克隆与CTAR2蛋白的亲和力.利用硝酸纤维膜斑点印迹法进行阳性克隆鉴定,并对阳性克隆进行测序及序列分析.结果 CTAR2蛋白对噬菌体12肽库进行3轮筛选,第3轮的产率是第1轮的143倍[(3.0×10-6)/(2.1×10-8)],噬菌体克隆得到较好的富集.ELISA方法提示筛选出来的噬菌体克隆能与CTAR2蛋白高效结合.硝酸纤维膜斑点印迹法证实阳性率达到100％.测序结果显示噬菌体上的短肽有共同序列:GLKHHSPGLLLY.结论 筛选出与CTAR2蛋白特异性结合的短肽序列GLKHHSPGLLLY,为研究LMP1致瘤机制和开发拮抗LMP1蛋白的小分子短肽类药物提供实验依据.     

TRAF4 acts as a silencer in TLR-mediated signaling through the association with TRAF6 and TRIF

Toll-like receptors (TLR) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase play an essential role in intracellular eradication of engulfed pathogens. Here, we demonstrate the physical and functional association between components of the cytosolic NADPH oxidase and TLR-mediated signaling molecules. Cytosolic components of NADPH oxidase suppressed TLR-mediated NF-kappaB activation as well as IFN-beta promoter activation. We demonstrate that TNF-associated factor (TRAF) 4 associates with p47(phox), a component of cytosolic NADPH oxidase, and physically interacts and functionally counteracts with TRAF6 and Toll-IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-beta (TRIF) molecules that critically regulate TLR-mediated signaling. TRAF4 mRNA expression was elicited in RPMI 8226 cells following LPS or CpG DNA treatment. These results suggest that TRAF4 participates in the molecular mechanism underlying silencing of TLR-mediated signaling through the interaction with molecules harboring phagosome/endosome membrane.     

JAB1 determines the response of rheumatoid arthritis synovial fibroblasts to tumor necrosis factor-alpha

Fibroblast-like synoviocytes (FLSs) of patients with rheumatoid arthritis (RA FLSs) exhibit prosurvival, rather than apoptotic, response to tumor necrosis factor (TNF)-alpha stimulation. Here, we show that JAB1 is a critical regulator of the TNF-alpha-mediated anti-apo-ptosis pathways in RA FLSs. We found that knockdown of JAB1 using small interfering (si)RNA led to restoration of the TNF-alpha-induced apoptosis response, reduction of nuclear factor-kappaB activity, delayed degradation of IkappaB-alpha, and inhibited phosphorylation of JNK. Analysis of the interactions of JAB1 by reciprocal co-immunoprecipitations and confocal microscopy revealed that JAB1 interacts with TNF receptor-associated-factor 2 (TRAF2). The generation of the anti-apoptotic signal on binding of TNF-alpha to the TNF receptor (TNFR)1 has been shown to be associated with the recruitment of TRAF2 to the TNFR1 in a process that requires ubiquitination of TRAF2 with lysine-63-linked polyubiquitin chains. We found that TNF-alpha stimulation of JAB1 siRNA-transfected RA FLSs failed to stimulate ubiquitination of TRAF2. Thus, we conclude that JAB1-regulated ubiquitination of TRAF2 is a novel mechanism whereby TNF-alpha can induce anti-apoptosis signaling and production of matrix metalloproteinases through activation of nuclear factor-kappaB and JNK in RA FLSs.     

Frequent expression of the tumor necrosis factor receptor-associated factor 1 in latent membrane protein 1-positive posttransplant lymphoproliferative disease and HIV-associated lymphomas

The tumor necrosis factor receptor-associated factor 1 (TRAF1) participates in the signal transduction of various members of the tumor necrosis factor receptor (TNFR) family, including TNFR2, CD40, CD30, and the Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1). In vitro, TRAF1 is induced by LMP1, and previous studies have suggested that expression of TRAF1 is higher in EBV-associated tumors than in their EBV-negative counterparts. To determine whether this was the case in posttransplant lymphoproliferative disease (PTLD) and related disorders, we used immunohistochemistry to analyze expression of TRAF1 in a total of 42 such lesions arising in a variety of immunosuppressive states. The specimens consisted of 22 PTLD lesions, 18 acquired immunodeficiency syndrome-associated lymphomas, including 6 primary central nervous system lymphomas, and 2 cases of Hodgkin disease. The presence of latent EBV infection was determined by EBER in situ hybridization, and expression of EBV-LMP1 was detected by immunohistochemistry. Latent EBV infection, as determined by a positive EBER signal, was detected in 36 of 42 tumors. Of the EBER-positive specimens, 30 of 36 also expressed LMP1. Twenty-four of 30 LMP1-positive tumors, including both Hodgkin disease specimens, expressed TRAF1, compared with only 3 of 12 LMP1-negative tumors. This difference was statistically significant (P <.005). These results show frequent expression of TRAF1 at the protein level in LMP1-positive PTLD and related disorders and suggest an important role for LMP1-mediated TRAF1 signaling in the pathogenesis of EBV-positive tumors arising in immunosuppressive states.