NF-κB hyper-activation by HTLV-1 tax induces cellular senescence, but can be alleviated by the viral anti-sense protein HBZ

Activation of I-κB kinases (IKKs) and NF-κB by the human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, is thought to promote cell proliferation and transformation. Paradoxically, expression of Tax in most cells leads to drastic up-regulation of cyclin-dependent kinase inhibitors, p21(CIP1/WAF1) and p27(KIP1), which cause p53-/pRb-independent cellular senescence. Here we demonstrate that p21(CIP1/WAF1)-/p27(KIP1)-mediated senescence constitutes a checkpoint against IKK/NF-κB hyper-activation. Senescence induced by Tax in HeLa cells is attenuated by mutations in Tax that reduce IKK/NF-κB activation and prevented by blocking NF-κB using a degradation-resistant mutant of I-κBα despite constitutive IKK activation. Small hairpin RNA-mediated knockdown indicates that RelA induces this senescence program by acting upstream of the anaphase promoting complex and RelB to stabilize p27(KIP1) protein and p21(CIP1/WAF1) mRNA respectively. Finally, we show that down-regulation of NF-κB by the HTLV-1 anti-sense protein, HBZ, delay or prevent the onset of Tax-induced senescence. We propose that the balance between Tax and HBZ expression determines the outcome of HTLV-1 infection. Robust HTLV-1 replication and elevated Tax expression drive IKK/NF-κB hyper-activation and trigger senescence. HBZ, however, modulates Tax-mediated viral replication and NF-κB activation, thus allowing HTLV-1-infected cells to proliferate, persist, and evolve. Finally, inactivation of the senescence checkpoint can facilitate persistent NF-κB activation and leukemogenesis.     

Activation of the IκB kinase complex by HTLV-1 Tax requires cytosolic factors involved in Tax-induced polyubiquitination

Activation of NF-κB by human T cell leukaemia virus type 1 Tax is thought to be crucial in T-cell transformation and the onset of adult T cell leukaemia. Tax activates NF-κB through activation of the IκB kinase (IKK) complex, similar to cytokine-induced NF-κB activation, which involves active signalling complex formation using polyubiquitin chains as a platform. Although polyubiquitination of Tax was reported to be required for IKK activation, most studies have been performed using intact cells, in which secondary NF-κB activation can be induced by various cytokines that are secreted due to Tax-mediated primary NF-κB activation. Therefore, a cell-free assay system, in which IKK can be activated by adding highly purified recombinant Tax to cytosolic extract, was used to analyse Tax-induced IKK activation. In contrast to the cytosolic extract, the purified IKK complex was not activated by Tax, whereas, it was efficiently activated by MEKK1, that does not require polyubiquitination to activate IKK. Moreover, Tax-induced IKK activation was blocked when the cytosolic extract was mixed with either lysine-free, methylated or K63R ubiquitin. These results obtained through our cell-free assay suggest that K63-linked polyubiquitination is critical, but linear polyubiquitination is dispensable or insufficient for Tax-induced IKK activation.     

Role of K63-linked polyubiquitination in NF-κB signalling: which ligase catalyzes and what molecule is targeted?

Nuclear factor-κB (NF-κB) is a master regulator of immunity and also involved in malignant transformation. It has been widely accepted that Lys-48 (K48)-linked polyubiquitination plays a critical role in NF-κB signalling by targeting inhibitor of NF-κB (IκB), thereby leading to its degradation by the proteasome. Alternatively, studies on IL-1 and TNF signalling have revealed that proteins modified with K63-linked polyubiquitin chains do not undergo the proteasomal degradation, instead, function as the signalling platforms required for the activation of the IκB kinase (IKK) complex. From the studies on lymphoid malignancies, human T cell leukaemia virus 1-derived protein, Tax, has been shown to activate the IKK complex, although the mechanism is largely unknown. Recently, Shibata et al. (Activation of the IκB kinase complex by HTLV-1 Tax requires cytosolic factors involved in Tax-induced polyubiquitination. J. Biochem. 150: 679-686, 2011) has established a cell free IKK assay system and demonstrated that recombinant Tax protein can activate the IKK complex in a K63-linked-polyubiquitination-dependent manner. This cell free assay system will be useful for the identification of various key players responsible for Tax-induced IKK activation.     

Proteins that bind to IKKγ (NEMO) and down-regulate the activation of NF-κB

Inhibitor of κB kinase (IKK) gamma (IKKγ), also referred to as nuclear factor κB (NF-κB) essential modulator (NEMO), is an important component of the IKK complex. Following the exposure of cells to NF-κB-inducing stimuli, the IKK complex catalyzes the phosphorylation of inhibitor of κB (IκB) proteins, which is a critical step that leads to the activation of NF-κB via the canonical pathway. The exact functions of IKKγ as part of the IKK complex have not been fully elucidated. A number of proteins have been identified as directly interacting with IKKγ and modulating the activity of the IKK complex. This mini review covers eight proteins that have been reported to bind to IKKγ and lead to the suppression of the activities of the IKK complex and hence result in the down-regulation of the activation of NF-κB. The reported mechanisms by which these interactions suppress the activation of the IKK complex include the deubiquitination of IKKγ and competition with upstream activators for binding to IKKγ.     

Peptidylarginine deiminase 2 suppresses inhibitory {kappa}B kinase activity in lipopolysaccharide-stimulated RAW 264.7 macrophages

Peptidylarginine deiminases (PADs) are enzymes that convert arginine to citrulline in proteins. In this study, we examined PAD-mediated citrullination and its effect on pro-inflammatory activity in the macrophage cell line RAW 264.7. Citrullination of 45-65-kDa proteins was induced when cells were treated with lipopolysaccharide (LPS; 1 μg/ml). Protein citrullination was suppressed by the intracellular calcium chelator BAPTA/AM (30 μM). LPS treatment up-regulated COX-2 levels in cells. Interestingly, overexpressing PAD2 reduced LPS-mediated COX-2 up-regulation by 50%. PAD2 overexpression also reduced NF-κB activity, determined by NF-κB-driven luciferase activity. The effect of PAD2 on NF-κB activity was further examined by using HEK 293 cells transfected with NF-κB luciferase, IκB β/γ kinase (IKKβ/γ) subunits, and PAD2. IKKβ increased NF-κB activity, but this increase was markedly suppressed when PAD2 was present in cells. IKKβ-mediated NF-κB activation was further enhanced by IKKγ in the presence of calcium ionophore A23187. However, this stimulatory effect of IKKβ/γ was abolished by PAD2. Coimmunoprecipitation of cell lysates showed that IKKγ and PAD2 can coimmunoprecipitate in the presence of the Ca(2+) ionophore. IKKγ coimmunoprecipitated truncation mutants, PAD2(1-385) and PAD2(355-672). The substitution of Gln-358 (a putative ligand for Ca(2+) binding) with an Ala abolished coimmunoprecipitation. Conversely, PAD2 coimmunoprecipitated truncation mutants IKKγ(1-196) and IKKγ(197-419). In other experiments, treating RAW 264.7 cells with LPS induced citrullination in the immunoprecipitates of IKKγ. In vitro citrullination assay showed that incubation of purified PAD2 and IKKγ proteins in the presence of Ca(2+) citrullinated IKKγ. These results demonstrate that PAD2 interacts with IKKγ and suppresses NF-κB activity.     

Differential usage of NF-κB activating signals by IL-1β and TNF-α in pancreatic beta cells

The cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α induce β-cell death in type 1 diabetes via NF-κB activation. IL-1β induces a more marked NF-κB activation than TNF-α, with higher expression of genes involved in β-cell dysfunction and death. We show here a differential usage of the IKK complex by IL-1β and TNF-α in β-cells. While TNF-α uses IKK complexes containing both IKKα and IKKβ, IL-1β induces complexes with IKKα only; this effect is achieved by induction of IKKβ degradation via the proteasome. Both IKKγ and activation of the TRAF6-TAK1-JNK pathway are involved in IL-1β-induced IKKβ degradation.     

The MC159 protein from the molluscum contagiosum poxvirus inhibits NF-κB activation by interacting with the IκB kinase complex

Molluscum contagiosum virus (MCV) causes persistent neoplasms in healthy and immunocompromised people. Its ability to persist likely is due to its arsenal of viral immunoevasion proteins. For example, the MCV MC159 protein inhibits TNF-R1-induced NF-κB activation and apoptosis. The MC159 protein is a viral FLIP and, as such, possesses two tandem death effector domains (DEDs). We show in this article that, in human embryonic kidney 293 T cells, the expression of wild-type MC159 or a mutant MC159 protein containing the first DED (MC159 A) inhibited TNF-induced NF-κB, or NF-κB activated by PMA or MyD88 overexpression, whereas a mutant protein lacking the first DED (MC159 B) did not. We hypothesized that the MC159 protein targeted the IκB kinase (IKK) complex to inhibit these diverse signaling events. Indeed, the MC159 protein, but not MC159 B, coimmunoprecipitated with IKKγ. MC159 coimmunoprecipitated with IKKγ when using mouse embryonic fibroblasts that lack either IKKα or IKKβ, suggesting that the MC159 protein interacted directly with IKKγ. MC159-IKKγ coimmunoprecipitations were detected during infection of cells with either MCV isolated from human lesions or with a recombinant MC159-expressing vaccinia virus. MC159 also interacts with TRAF2, a signaling molecule involved in NF-κB activation. However, mutational analysis of MC159 failed to reveal a correlation between MC159-TRAF2 interactions and MC159's inhibitory function. We propose that MC159-IKK interactions, but not MC159-TRAF2 interactions, are responsible for inhibiting NF-κB activation.     

Enterovirus 71 2C protein inhibits TNF-α-mediated activation of NF-κB by suppressing IκB kinase β phosphorylation

Enterovirus 71 (EV71), a single, positive-stranded RNA virus, has been regarded as the most important neurotropic enterovirus after the eradication of the poliovirus. EV71 infection can cause hand, foot, and mouth disease or herpangina. Cytokine storm with elevated levels of proinflammatory and inflammatory cytokines, including TNF-α, has been proposed to explain the pathogenesis of EV71-induced disease. TNF-α-mediated NF-κB signaling pathway plays a key role in inflammatory response. We hypothesized that EV71 might also moderate host inflammation by interfering with this pathway. In this study, we tested this hypothesis and identified EV71 2C protein as an antagonist of TNF-α-mediated activation of NF-κB signaling pathway. Expression of 2C protein significantly reduced TNF-α-mediated NF-κB activation in 293T cells as measured by gene reporter and gel mobility shift assays. Furthermore, overexpression of TNFR-associated factor 2-, MEK kinase 1-, IκB kinase (IKK)α-, or IKKβ-induced NF-κB activation, but not constitutively active mutant of IKKβ (IKKβ SS/EE)-induced NF-κB activation, was inhibited by 2C protein. These data together suggested that the activation of IKKβ is most likely targeted by 2C; this notion was further strengthened by immunoblot detection of IKKβ phosphorylation and IκBα phosphorylation and degradation. Coimmunoprecipitation and colocalization of 2C and IKKβ expressed in mammalian cells provided compelling evidence that 2C interacts with IKKβ. Collectively, our data indicate that EV71 2C protein inhibits IKKβ activation and thus blocks NF-κB activation.     

TRAF2 Suppresses Basal IKK Activity in Resting Cells and TNFα Can Activate IKK in TRAF2 and TRAF5 Double Knockout Cells

Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF5 are adapter proteins involved in TNFα-induced activation of the c-Jun N-terminal kinase and nuclear factor κB (NF-κB) pathways. Currently, TNFα-induced NF-κB activation is believed to be impaired in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells. Here, we report instead that T2/5 DKO cells exhibit high basal IκB kinase (IKK) activity and elevated expression of NF-κB-dependent genes in unstimulated conditions. Although TNFα-induced receptor-interacting protein 1 ubiquitination is indeed impaired in T2/5 DKO cells, TNFα stimulation further increases IKK activity in these cells, resulting in significantly elevated expression of NF-κB target genes to a level higher than that in wild-type cells. Inhibition of NIK in T2/5 DKO cells attenuates basal IKK activity and restores robust TNFα-induced IKK activation to a level comparable with that seen in wild-type cells. This suggests that TNFα can activate IKK in the absence of TRAF2 and TRAF5 expression and receptor-interacting protein 1 ubiquitination. In addition, both the basal and TNFα-induced expression of anti-apoptotic proteins are normal in T2/5 DKO cells, yet these DKO cells remain sensitive to TNFα-induced cell death, due to the impaired recruitment of anti-apoptotic proteins to the TNFR1 complex in the absence of TRAF2. Thus, our data demonstrate that TRAF2 negatively regulates basal IKK activity in resting cells and inhibits TNFα-induced cell death by recruiting anti-apoptotic proteins to the TNFR1 complex rather than by activating the NF-κB pathway.     

The E3 ubiquitin ligase mind bomb-2 (MIB2) protein controls B-cell CLL/lymphoma 10 (BCL10)-dependent NF-κB activation

B-cell CLL/lymphoma 10 (BCL10) is crucial for the activation of NF-κB in numerous immune receptor signaling pathways, including the T-cell receptor (TCR) and B-cell receptor signaling pathways. However, the molecular mechanisms that lead to signal transduction from BCL10 to downstream NF-κB effector kinases, such as TAK1 and components of the IKK complex, are not entirely understood. Here we used a proteomic approach and identified the E3 ligase MIB2 as a novel component of the activated BCL10 complex. In vitro translation and pulldown assays suggest direct interaction between BCL10 and MIB2. Overexpression experiments show that MIB2 controls BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ/NEMO, as well as recruitment and activation of TAK1. Knockdown of MIB2 inhibited BCL10-dependent NF-κB activation. Together, our results identify MIB2 as a novel component of the activated BCL10 signaling complex and a missing link in the BCL10-dependent NF-κB signaling pathway.     

Role of IKK/NF-κB signaling in extinction of conditioned place aversion memory in rats

The inhibitor κB protein kinase/nuclear factor κB (IKK/NF-κB) signaling pathway is critical for synaptic plasticity. However, the role of IKK/NF-κB in drug withdrawal-associated conditioned place aversion (CPA) memory is unknown. Here, we showed that inhibition of IKK/NF-κB by sulphasalazine (SSZ; 10 mM, i.c.v.) selectively blocked the extinction but not acquisition or expression of morphine-induced CPA in rats. The blockade of CPA extinction induced by SSZ was abolished by sodium butyrate, an inhibitor of histone deacetylase. Thus, the IKK/NF-κB signaling pathway might play a critical role in the extinction of morphine-induced CPA in rats and might be a potential pharmacotherapy target for opiate addiction.     

Activation of IKK/NF-κB provokes renal inflammatory responses in guanylyl cyclase/natriuretic peptide receptor-A gene-knockout mice

The present study was aimed at determining the consequences of the disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) on proinflammatory responses of nuclear factor kappa B, inhibitory kappa B kinase, and inhibitory kappa B alpha (NF-κB, IKK, IκBα) in the kidneys of mutant mice. The results showed that the disruption of Npr1 enhanced the renal NF-κB binding activity by 3.8-fold in 0-copy (-/-) mice compared with 2-copy (+/+) mice. In parallel, IKK activity and IκBα protein phosphorylation were increased by 8- and 11-fold, respectively, in the kidneys of 0-copy mice compared with wild-type mice. Interestingly, IκBα was reduced by 80% and the expression of proinflammatory cytokines and renal fibrosis were significantly enhanced in 0-copy mice than 2-copy mice. Treatment of 0-copy mice with NF-κB inhibitors andrographolide, pyrrolidine dithiocarbamate, and etanercept showed a substantial reduction in renal fibrosis, attenuation of proinflammatory cytokines gene expression, and significantly reduced IKK activity and IkBα phosphorylation. These findings indicate that the systemic disruption of Npr1 activates the renal NF-κB pathways in 0-copy mice, which transactivates the expression of various proinflammatory cytokines to initiate renal remodeling; however, inhibition of NF-κB pathway repairs the abnormal renal pathology in mutant mice.