Hepatitis C virus core protein activates nuclear factor kappa B-dependent signaling through tumor necrosis factor receptor-associated factor

Hepatitis C virus (HCV) core protein, a viral nucleocapsid, has been shown to affect various intracellular events including the nuclear factor kappaB (NF-kappaB) signaling supposedly associated with inflammatory response, cell proliferation, and apoptosis. In order to elucidate the effect of HCV core protein on the NF-kappaB signaling in HeLa and HepG2 cells, a reporter assay was utilized. HCV core protein significantly activated NF-kappaB signaling in a dose-dependent manner not only in HeLa and HepG2 cells transiently transfected with core protein expression plasmid, but also in HeLa cells induced to express core protein under the control of doxycycline. HCV core protein increased the DNA binding affinity of NF-kappaB in the electrophoretic mobility shift assay. Acetyl salicylic acid, an IKKbeta-specific inhibitor, and dominant negative form of IKKbeta significantly blocked NF-kappaB activation by HCV core protein, suggesting HCV core protein activates the NF-kappaB pathway mainly through IKKbeta. Moreover, the dominant negative forms of TRAF2/6 significantly blocked activation of the pathway by HCV core protein, suggesting HCV core protein mimics proinflammatory cytokine activation of the NF-kappaB pathway through TRAF2/6. In fact, HCV core protein activated interleukin-1beta promoter mainly through NF-kappaB pathway. Therefore, this function of HCV core protein may play an important role in the inflammatory reaction induced by this hepatotropic virus.     

Hepatitis C virus nonstructural 5B protein regulates tumor necrosis factor alpha signaling through effects on cellular IkappaB kinase

Hepatitis C virus (HCV) NS5B protein is a membrane-associated phosphoprotein that possesses an RNA-dependent RNA polymerase activity. We recently reported that NS5A protein interacts with TRAF2 and modulates tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB and Jun N-terminal protein kinase (JNK). Since NS5A and NS5B are the essential components of the HCV replication complex, we examined whether NS5B could modulate TNF-alpha-induced NF-kappaB and JNK activation. In this study, we have demonstrated that TNF-alpha-induced NF-kappaB activation is inhibited by NS5B protein in HEK293 and hepatic cells. Furthermore, NS5B protein inhibited both TRAF2- and IKK-induced NF-kappaB activation. Using coimmunoprecipitation assays, we show that NS5B interacts with IKKalpha. Most importantly, NS5B protein in HCV subgenomic replicon cells interacted with endogenous IKKalpha, and then TNF-alpha-mediated IKKalpha kinase activation was significantly decreased by NS5B. Using in vitro kinase assay, we have further found that NS5B protein synergistically activated TNF-alpha-mediated JNK activity in HEK293 and hepatic cells. These data suggest that NS5B protein modulates TNF-alpha signaling pathways and may contribute to HCV pathogenesis.     

TIRP,a novel Toll/interleukin-1 receptor (TIR) domain-containing adapter protein involved in TIR signaling

The Toll/interleukin-1 receptor (TIR) family members play important roles in host defense. These receptors signal through TIR domain-containing adapter proteins. In this report, we identified a novel TIR domain-containing adapter protein designated as TIRP. Co-immunoprecipitation experiments suggest that TIRP is associated with IL-1 receptors. TIRP also interacts with kinase-inactive mutants of IRAK and IRAK-4, IRAK-2, IRAK-M, and TRAF6. Overexpression of TIRP activates NF-kappaB and potentiates IL-1 receptor-mediated NF-kappaB activation. A dominant negative mutant of TIRP inhibits IL-1- but not tumor necrosis factor-triggered NF-kappaB activation. Moreover, TIRP-mediated NF-kappaB activation is inhibited by dominant negative mutants of IRAK, IRAK-2, TRAF6, and IKKbeta. Our findings suggest that TIRP is involved in IL-1-triggered NF-kappaB activation and functions upstream of IRAK, IRAK-2, TRAF6, and IKKbeta     

1Hepatitis C virus NS5A protein modulates c-Jun N-terminal kinase through interaction with tumor necrosis factor receptor-associated factor 2

The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) is a phosphoprotein possessing various functions. We have previously reported that the HCV NS5A protein interacts with tumor necrosis factor (TNF) receptor-associated factor (TRAF) domain of TRAF2 (Park, K.-J., Choi, S.-H., Lee, S. Y., Hwang, S. B., and Lai, M. M. C. (2002) J. Biol. Chem. 277, 13122-13128). Both TNF-alpha- and TRAF2-mediated nuclear factor-kappaB (NF-kappaB) activations were inhibited by NS5A-TRAF2 interaction. Because TRAF2 is required for the activation of both NF-kappaB and c-Jun N-terminal kinase (JNK), we investigated HCV NS5A protein for its potential capacity to modulate TRAF2-mediated JNK activity. Using in vitro kinase assay, we have found that NS5A protein synergistically activated both TNF-alpha- and TRAF2-mediated JNK in human embryonic kidney 293T cells. Furthermore, synergism of NS5A-mediated JNK activation was inhibited by dominant-negative form of MEK kinase 1. Our in vivo binding data show that NS5A does not inhibit interaction between TNF receptor-associated death domain and TRAF2 protein, indicating that NS5A and TRAF2 may form a ternary complex with TNF receptor-associated death domain. These results indicate that HCV NS5A protein modulates TNF signaling of the host cells and may play a role in HCV pathogenesis.     

Hepatitis C virus core protein inhibits Fas- and tumor necrosis factor alpha-mediated apoptosis via NF-kappaB activation

The effects of hepatitis C virus (HCV) proteins on anti-Fas (CD95/APO-1) antibody- and tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis in different human cell lines were investigated by magnetic concentration of cells which transiently produced the exogenous protein. HepG2 cells, which produced whole HCV proteins, became resistant to anti-Fas-induced apoptotic cell death. Furthermore, the core protein among HCV proteins had a key role in protecting the various cells from apoptosis mediated by not only anti-Fas but also TNF-alpha. We also found that the core functioned in the activation of nuclear factor kappaB (NF-kappaB) in all cells examined. Deletion analysis of the core revealed that the region required for NF-kappaB activation was closely correlated with that for its antiapoptotic function. In addition, we revealed in some cases that the antiapoptotic effect of the core was restrained by coproduction of the inhibitor of NF-kappaB, IkappaB-alpha protein. These results demonstrated that the core inhibits Fas- and TNF-alpha-mediated apoptotic cell death via a mechanism dependent on the activation of NF-kappaB in particular cell lines.     

TNAP, a novel repressor of NF-kappaB-inducing kinase, suppresses NF-kappaB activation

NF-kappaB-inducing kinase (NIK) has been implicated as an essential component of NF-kappaB activation. However, the regulatory mechanism of NIK signaling remains elusive. We have identified a novel NIK interacting protein, TNAP (for TRAFs and NIK-associated protein). In mammalian cells, TNAP physically interacts with NIK, TRAF2, and TRAF3 but not IKK1 or IKK2. TNAP specifically inhibits NF-kappaB activation induced by tumor necrosis factor (TNF)-alpha, TNF receptor 1, TRADD, RIP, TRAF2, and NIK but does not affect IKK1- and IKK2-mediated NF-kappaB activation. Knockdown of TNAP by lentiviral-mediated small interference RNA potentiates TNF-alpha-induced NF-kappaB activation. TNAP suppresses NIK kinase activity and subsequently reduces p100 processing, p65 phosphorylation, and IkappaBalpha degradation. These data suggest that TNAP is a repressor of NIK activity and regulates both the classical and alternative NF-kappaB signaling pathways.     

Sequestration of TRAF2 into stress granules interrupts tumor necrosis factor signaling under stress conditions

The cellular stress response (SR) is a phylogenetically conserved protection mechanism that involves inhibition of protein synthesis through recruitment of translation factors such as eIF4G into insoluble stress granules (SGs) and blockade of proinflammatory responses by interruption of the signaling pathway from tumor necrosis factor alpha (TNF-alpha) to nuclear factor-kappaB (NF-kappaB) activation. However, the link between these two physiological phenomena has not been clearly elucidated. Here we report that eIF4GI, which is a scaffold protein interacting with many translation factors, interacts with TRAF2, a signaling molecule that plays a key role in activation of NF-kappaB through TNF-alpha. These two proteins colocalize in SGs during cellular exposure to stress conditions. Moreover, TRAF2 is absent from TNFR1 complexes under stress conditions even after TNF-alpha treatment. This suggests that stressed cells lower their biological activities by sequestration of translation factors and TRAF2 into SGs through a protein-protein interaction.     

Inhibition of nuclear factor-kappaB activation is essential for membrane-associated TNF-alpha-induced apoptosis in HL-60 cells

The killing of tumour cells that are resistant to soluble TNF-alpha (sTNF-alpha) by the membrane-bound form of TNF-alpha (mTNF-alpha) suggests that different intracellular signalling pathways are involved. We found that mTNF-alpha induced apoptosis in HL-60 cells and failed to cause degradation of inhibitor of kappa B alpha (IkappaB-alpha) and translocation and activation of nuclear factor kappa B (NF-kappaB), whereas sTNF-alpha failed to induce apoptosis, but lowered cytoplasmic inhibitor of kappa B alpha, induced translocation of NF-kappaB to the nucleus and experimentally increased activity of the regulated luciferase. Furthermore, mTNF-alpha upregulated the expression of TNF receptor associated factor (TRAF) 1 and failed to induce TRAF1 and TRAF2 membrane translocation, but led to cytoplasmic colocalization. In contrast, sTNF-alpha stimulated the expression of TRAF1 and TRAF2, recruiting both molecules onto the cell membrane poststimulation. These results suggest that the increased susceptibility of HL-60 cells to mTNF-alpha may be due to the failure of TRAF2 membrane translocation caused by the upregulation of TRAF1 expression and formation of a TRAF1/TRAF2 complex in the cytoplasm, thereby inhibiting NF-kappaB activation and inducing apoptosis.     

TRAF2 phosphorylation modulates tumor necrosis factor alpha-induced gene expression and cell resistance to apoptosis

TRAF2 is an adaptor protein that regulates the activation of the c-Jun N-terminal kinase (JNK) and IkappaB kinase (IKK) signaling cascades in response to tumor necrosis factor alpha (TNF-alpha) stimulation. Although the downstream events in TNF-alpha signaling are better understood, the membrane-proximal events are still elusive. Here, we demonstrate that TNF-alpha and cellular stresses induce TRAF2 phosphorylation at serine 11 and that this phosphorylation is required for the expression of a subset of NF-kappaB target genes. Although TRAF2 phosphorylation had a minimal effect on the TNF-alpha-induced rapid and transient IKK activation, it was essential for secondary and prolonged IKK activation. Consistent with this, TRAF2 phosphorylation is not required for its recruitment to the TNFR1 complex in response to TNF-alpha stimulation but is required for its association with a cytoplasmic complex containing RIP1 and IKK. In addition, TRAF2 phosphorylation was essential for the full TNF-alpha-induced activation of JNK. Notably, TRAF2 phosphorylation increased both basal and inducible c-Jun and NF-kappaB activities and rendered cells resistant to stress-induced apoptosis. Moreover, TRAF2 was found to be constitutively phosphorylated in some lymphomas. These results unveil a new, finely tuned mechanism for TNF-alpha-induced IKK activation modulated by TRAF2 phosphorylation and suggest that TRAF2 phosphorylation contributes to elevated levels of basal NF-kappaB activity in certain human cancers.     

Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression

NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.     

Hepatitis C virus core protein inhibits tumor necrosis factor alpha-mediated apoptosis by a protective effect involving cellular FLICE inhibitory protein

We have previously shown that hepatitis C virus (HCV) core protein modulates multiple cellular processes, including those that inhibit tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis. In this study, we have investigated the signaling mechanism for inhibition of TNF-alpha-mediated apoptosis in human hepatoma (HepG2) cells expressing core protein alone or in context with other HCV proteins. Activation of caspase-3 and the cleavage of DNA repair enzyme poly(ADP-ribose) polymerase were inhibited upon TNF-alpha exposure in HCV core protein-expressing HepG2 cells. In vivo protein-protein interaction studies displayed an association between TNF receptor 1 (TNFR1) and TNFR1-associated death domain protein (TRADD), suggesting that the core protein does not perturb this interaction. A coimmunoprecipitation assay also suggested that HCV core protein does not interfere with the TRADD-Fas-associated death domain protein (FADD)-procaspase-8 interaction. Further studies indicated that HCV core protein expression inhibits caspase-8 activation by sustaining the expression of cellular FLICE (FADD-like interleukin-1beta-converting enzyme)-like inhibitory protein (c-FLIP). Similar observations were also noted upon expression of core protein in context to other HCV proteins expressed from HCV full-length plasmid DNA or a replicon. A decrease in endogenous c-FLIP by specific small interfering RNA induced TNF-alpha-mediated apoptotic cell death and caspase-8 activation. Taken together, our results suggested that the TNF-alpha-induced apoptotic pathway is inhibited by a sustained c-FLIP expression associated with the expression of HCV core protein, which may play a role in HCV-mediated pathogenesis.     

The tyrosine kinase Syk regulates TPL2 activation signals

Tpl2/Cot is a serine/threonine kinase that plays a key physiological role in the regulation of immune responses to pro-inflammatory stimuli, including tumor necrosis factor-alpha (TNF-alpha). TNF-alpha stimulates the JNK, ERK, and p38 mitogen-activated protein kinases and the NF-kappaB pathway by recruiting RIP1 and TRAF2 to the TNF receptor 1. Here we showed that Tpl2 activation by TNF-alpha signals depends on the integrity of the Tpl2-interacting proteins RIP1 and TRAF2, which are required for the engagement of the ERK mitogen-activated protein kinase pathway. However, neither RIP1 nor TRAF2 overexpression was sufficient to activate Tpl2 and ERK. We also showed that Tpl2 activation by TNF-alpha depends on a tyrosine kinase activity that is detected in TNF-alpha-stimulated cells. Based on both genetic and biochemical evidence, we concluded that in a variety of cell types, Syk is the tyrosine kinase that plays an important role in the activation of Tpl2 upstream of ERK. These data therefore dissect the TNF receptor 1 proximal events that regulate Tpl2 and ERK and highlight a role for RIP1, TRAF2, and Syk in this pathway.     

FLN29, a novel interferon- and LPS-inducible gene acting as a negative regulator of toll-like receptor signaling

Lipopolysaccharide (LPS) activates macrophages through toll-like receptor (TLR) 4. Although the mechanism of the TLR signaling pathway has been well documented, the mechanism of the negative regulation in response to LPS, particularly LPS tolerance, is still poorly understood. In this study we identified and characterized a novel interferon- and LPS-inducible gene, FLN29, which contains a TRAF6-related zinc finger motif and TRAF family member-associated NF-kappaB activator-related sequences. The induction of FLN29 was dependent on STAT1. The forced expression of FLN29 in macrophage-like RAW cells resulted in the suppression of TLR-mediated NF-kappaB and mitogen-activated protein kinase activation, while a reduced expression of FLN29 by small interfering RNA partly cancelled the down-regulation of LPS signaling. Furthermore, we demonstrated that NF-kappaB activation induced by TRAF6 and TAB2 was impaired by co-expression of FLN29, suggesting FLN29 may regulate the downstream of TRAF6. Taken together, FLN29 is a new negative feedback regulator of TLR signaling.     

TRAF6 is a critical signal transducer in IL-33 signaling pathway

IL-33 has been shown to induce Th2 responses by signaling through the IL-1 receptor-related protein, ST2L. However, the signal transduction pathways activated by the ST2L have not been characterized. Here, we found that IL-33-induced monocyte chemoattractant protein (MCP)-1, MCP-3 and IL-6 expression was significantly inhibited in TNF receptor-associated Factor 6 (TRAF6)-deficient MEFs. IL-33 rapidly induced the formation of ST2L complex containing IL-1 receptor-associated kinase (IRAK), however, lack of TRAF6 abolished the recruitment of IRAK to ST2L. Consequently, p38, JNK and Nuclear factor-kappaB (NF-kappaB) activation induced by IL-33 was completely inhibited in TRAF6-deficient MEFs. On the other hand, IL-33-induced ERK activation was observed regardless of the presence of TRAF6. The introduction of TRAF6 restored the efficient activation of p38, JNK and NF-kappaB in TRAF6 deficient MEFs, resulting in the induction of MCP-1, MCP-3 and IL-6 expression. Moreover, IL-33 augmented autoubiquitination of TRAF6 and the reconstitution of TRAF6 mutant (C70A) that is defective in its ubiquitin ligase activity failed to restore IL-33-induced p38, JNK and NF-kappaB activation. Thus, these data demonstrate that TRAF6 plays a pivotal role in IL-33 signaling pathway through its ubiquitin ligase activity.     

Both amino- and carboxyl-terminal domains of TRAF3 negatively regulate NF-kappaB activation induced by OX40 signaling

OX40 is a member of the tumor necrosis factor receptor (TNF-R) superfamily. We observed that overexpression of OX40 activated NF-kappaB, which was inhibited by dominant negative forms of TRAF2, NF-kappaB-inducing kinase (NIK), and IkappaB kinase (IKK) alpha. This indicates that OX40 signaling leads to NF-kappaB activation through the same cascade as TNF-R2. We then investigated the negative regulatory function of TRAF3 on OX40-induced NF-kappaB activation. TRAF3 blocked OX40-, TRAF2-induced NF-kappaB activation, but not NIK- and IKKalpha-induced NF-kappaB activation, indicating that TRAF3 blocks the pathway between TRAF2 and NIK. C-terminal deletion mutants as well as the N-terminal deletion mutant of TRAF3 inhibited NF-kappaB activation induced by OX40 or TRAF2. Since TRAF3 bound to OX40 through the C-terminal TRAF domain, the C-terminal domain is likely to work as a dominant negative mutant to compete the recruitment of TRAF2 to the receptor, which transmits the signal from OX40 to the downstream, NIK kinase. On the other hand, the N-terminal domain of TRAF3 seems to affect the downstream of TRAF2 binding. Thus, it is suggested that TRAF3 actively inhibits NF-kappaB activation induced by OX40.