NF-κB-inducing Kinase Research Articles

Non‐canonical NF‐κB signaling activation and regulation: principles and perspectives

Nuclear-factor κB (NF-κB) transcription factors are activated by a wide variety of stimuli in diverse cell types and control key aspects of immune function and development. Receptor-mediated activation of NF-κB appears to occur through two distinct signaling pathways termed as the canonical and non-canonical NF-κB pathways. Although much work has demonstrated the physiological importance of non-canonical NF-κB signaling to immunity and its involvement in diverse pathologies, such as cancers and autoimmune disease, the architecture and regulation of the pathway is only beginning to be understood. The non-canonical pathway appears to be activated by a select set of receptors within the tumor necrosis factor superfamily, and we discuss the molecular mechanisms that connect ligation of these receptors to pathway activation. It has become increasingly clear that the key regulatory step of the pathway involves modulation of the post-translational degradation of NF-κB-inducing kinase (NIK), the central activating kinase of non-canonical NF-κB signaling. How NIK post-translational stability is controlled before and after receptor ligation is an important aspect of understanding non-canonical NF-κB signaling. Furthermore, how release of NF-κB dimers downstream of the pathway's activation is actually connected to its identified physiological and pathological roles is a key remaining question in the field.

The TNF receptor super family in immune regulation

The TNF receptor super family in immune regulation

Nuclear factor-κB2 represses Sp1-mediated transcription at the CD99 promoter.

Downregulation of the CD99 antigen on the surface of Hodgkin's lymphoma (HL) cells via EBV LMP1-mediated NF-κB suppression of Sp1 transcriptional activity is known to be associated with the appearance of pathogenic Reed-Sternberg cells. Here, we show that in addition, EBV LMP1 heterologous NF-κB activators such as CD30 and CD40 repress the CD99 promoter, which contains multiple Sp1-binding sites but no NF-κB binding sites. In addition, NF-κB-inducing kinase (NIK) repressed the CD99 promoter while NIK kinase mutants and JNK inhibitory protein failed to do so. Of the NF-κB subunits, NF-κB2 (p52) alone or in combination with other Rel subunits consistently inhibited the CD99, while NF-κB1 (p50) showed a marginal repressive effect. Furthermore, while transfection of LMP1 repressed the CD99 promoter in wild-type or NF-κB1 deficient MEFs, the same repression was not observed in NF-κB2 (p52)-deficient MEFs, indicating that NF-κB2 (p52) is required for LMP1-mediated repression of the CD99 promoter. Consistently, basal activity of the CD99 promoter was significantly higher in IKKα(-/-) and IKKβ(-/-) MEFs, but not in IKKΓ(-/-) MEFs compared to the wild-type control MEFs. Sp1-binding sites were directly used in the repression, because a synthetic Sp1 reporter with 10 Sp1-binding sites from the CD99 promoter was repressed by LMP1 or p52 transfection. These data indicate that LMP1-mediated NF-κB2 exhibits the major inhibitory role in the transcription at the CD99 promoter.

Splenic extramedullary hemopoiesis caused by a dysfunctional mutation in the NF-κB-inducing kinase gene

NF-κB-inducing kinase (NIK) plays critical roles in the development of lymph nodes and Peyer’s patches, and microarchitecture of the thymus and spleen via NF-κB activation. Alymphoplasia (aly/aly) mice have a point mutation in the NIK gene that causes a defect in the activation of an NF-κB member RelB. Here, we developed a novel method to determine the aly mutation by genetic typing using PCR. This method facilitated the easy establishment of a congeneic aly/aly mouse line. Indeed, we generated a mouse line with aly mutation on a BALB/cA background (BALB/cA-aly/aly). BALB/cA-aly/aly mice showed significant splenomegaly with extramedullary hemopoiesis, which was not significant in aly/aly mice on a C57BL/6 background. Interestingly, the splenomegaly and extramedullary hemopoiesis caused by the aly mutation was gender-dependent. These data together with previous reports on extramedullary hemopoiesis in RelB-deficient mice suggest that NIK–RelB signaling may be involved in the suppression of extramedullary hemopoiesis in adult mice.

NF-κB inducing kinase (NIK) modulates melanoma tumorigenesis by regulating expression of pro-survival factors through the β-catenin pathway

Nuclear factor-κB (NF-κB) inducing kinase (NIK) is a MAP3K that regulates the activation of NF-κB. NIK is often highly expressed in tumor cells, including melanoma, but the significance of this in melanoma progression has been unclear. Tissue microarray analysis of NIK expression reveals that dysplastic nevi (n=22), primary (n=15) and metastatic melanoma (n=13) lesions showed a statistically significant elevation in NIK expression when compared to benign nevi (n=30). Moreover, when shRNA techniques were used to knock-down NIK, the resultant NIK-depleted melanoma cell lines exhibited decreased proliferation, increased apoptosis, and reduced tumor growth in a mouse xenograft model. As expected, when NIK was depleted there was decreased activation of the non-canonical NF-κB pathway, while canonical NF-κB activation remained intact. NIK depletion also resulted in reduced expression of genes that contribute to tumor growth, including CXCR4, c-MYC and c-MET, and pro-survival factors such as BCL2 and survivin. These changes in gene expression are not fully explained by the attenuation of the non-canonical NF-κB pathway. Shown here for the first time is the demonstration that NIK modulates β-catenin mediated transcription to promote expression of survivin. NIK-depleted melanoma cells exhibited down-regulation of survivin as well as other β-catenin regulated genes including c-MYC, c-MET and CCND2. These data indicate that NIK mediates both β-catenin and NF-κB regulated transcription to modulate melanoma survival and growth. Thus, NIK may be a promising therapeutic target for melanoma.

NF-κB Signaling Participates in Both RANKL- and IL-4–Induced Macrophage Fusion: Receptor Cross-Talk Leads to Alterations in NF-κB Pathways

NF-κB activation is essential for receptor activator for NF-κB ligand (RANKL)-induced osteoclast formation. IL-4 is known to inhibit the RANKL-induced osteoclast differentiation while at the same time promoting macrophage fusion to form multinucleated giant cells (MNG). Several groups have proposed that IL-4 inhibition of osteoclastogenesis is mediated by suppressing the RANKL-induced activation of NF-κB. However, we found that IL-4 did not block proximal, canonical NF-κB signaling. Instead, we found that IL-4 inhibited alternative NF-κB signaling and induced p105/50 expression. Interestingly, in nfκb1(-/-) bone marrow-derived macrophages (BMM), the formation of both multinucleated osteoclast and MNG induced by RANKL or IL-4, respectively, was impaired. This suggests that NF-κB signaling also plays an important role in IL-4-induced macrophage fusion. Indeed, we found that the RANKL-induced and IL-4-induced macrophage fusion were both inhibited by the NF-κB inhibitors IκB kinase 2 inhibitor and NF-κB essential modulator inhibitory peptide. Furthermore, overexpression of p50, p65, p52, and RelB individually in nfκb1(-/-) or nfκb1(+/+) BMM enhanced both giant osteoclast and MNG formation. Interestingly, knockdown of nfκb2 in wild-type BMM dramatically enhanced both osteoclast and MNG formation. In addition, both RANKL- and IL-4-induced macrophage fusion were impaired in NF-κB-inducing kinase(-/-) BMM. These results suggest IL-4 influences NF-κB pathways by increasing p105/p50 and suppressing RANKL-induced p52 translocation and that NF-κB pathways participate in both RANKL- and IL-4-induced giant cell formation.

NIK signaling in dendritic cells but not in T cells is required for the development of effector T cells and cell-mediated immune responses

The canonical NF-κB pathway is a driving force for virtually all aspects of inflammation. Conversely, the role of the noncanonical NF-κB pathway and its central mediator NF-κB-inducing kinase (NIK) remains poorly defined. NIK has been proposed to be involved in the formation of T(H)17 cells, and its absence in T(H) cells renders them incapable of inducing autoimmune responses, suggesting a T cell-intrinsic role for NIK. Upon systematic analysis of NIK function in cell-mediated immunity, we found that NIK signaling is dispensable within CD4(+) T cells but played a pivotal role in dendritic cells (DCs). We discovered that NIK signaling is required in DCs to deliver co-stimulatory signals to CD4(+) T cells and that DC-restricted expression of NIK is sufficient to restore T(H)1 and T(H)17 responses as well as cell-mediated immunity in NIK(-/-) mice. When CD4(+) T cells developed in the absence of NIK-sufficient DCs, they were rendered anergic. Reintroduction of NIK into DCs allowed developing NIK(-/-) CD4(+) T cells to become functional effector populations and restored the development of autoimmune disease. Therefore, our data suggest that a population of thymic DCs requires NIK to shape the formation of most αβ CD4(+) T effector lineages during early development.

Noncanonical NF-kappaB regulates inducible costimulator (ICOS) ligand expression and T follicular helper cell development.

Follicular helper T (Tfh) cells have a central role in mediating humoral immune responses. Generation of Tfh cells depends on both T-cell intrinsic factors and the supporting function of B cells, but the underlying molecular mechanisms are incompletely understood. Here we show that NF-κB-inducing kinase (NIK), a central component of the noncanonical NF-κB signaling pathway, is required for Tfh cell development. Unlike other known Tfh regulators, NIK acts by controlling the supporting function of B cells. NIK and its upstream BAFF receptor regulate B-cell expression of inducible costimulator ligand (ICOSL), a molecule required for Tfh cell generation. Consistently, injection of a recombinant ICOSL protein into NIK-deficient mice largely rescues their defect in Tfh cell development. We provide biochemical and genetic evidence indicating that the ICOSL gene is a specific target of the noncanonical NF-κB. Our findings suggest that the noncanonical NF-κB pathway regulates the development of Tfh cells by mediating ICOSL gene expression in B cells.

Specific effects of BCL10 Serine mutations on phosphorylations in canonical and noncanonical pathways of NF-κB activation following carrageenan

To determine the impact of B cell leukemia/lymphoma (BCL) 10 on the phosphorylation of crucial mediators in NF-κB-mediated inflammatory pathways, human colonic epithelial cells were exposed to carrageenan (CGN), a sulfated polysaccharide commonly used as a food additive and known to induce NF-κB nuclear translocation by both canonical and noncanonical pathways. Phosphorylations of intermediates in inflammatory cascades, including NF-κB-inducing kinase (NIK) at Thr(559), transforming growth factor-β-activating kinase (TAK) 1 at Thr(184), Thr(187), and Ser(192), and inhibitory factor κBα (IκBα) at Ser(32), were examined following mutation of BCL10 at Ser(138) and at Ser(218). Specific phosphoantibodies were used for detection by enzyme-linked immunosorbent assay, immunoblot, and confocal microscopy of differences in phosphorylation following transfection by mutated BCL10. Both mutations demonstrated dominant-negative effects, with inhibition of phospho(Ser(32))-IκBα to less than control levels. Both of the BCL10 mutations reduced the CGN-induced increases in nuclear RelA and p50, but only the Ser(138) mutation inhibited the CGN-induced increases in nuclear RelB and p52 and in NIK Thr(559) phosphorylation. Hence, the phosphorylation of BCL10 Ser(138), but not Ser(218), emerged as a critical event in activation of the noncanonical pathway of NF-κB activation. Either BCL10 Ser(138) or Ser(218) mutation inhibited the phosphorylation of TAK1 at Thr(184) and at Thr(187), but not at Ser(192). These findings indicate that BCL10 phosphorylations act upstream of phosphorylations of NIK, TAK1, and IκBα and differentially affect the canonical and noncanonical pathways of NF-κB activation.

Molecular mechanism of inflammatory response in mouse liver caused by exposure to CeCl3

To investigate the molecular mechanism of inflammatory response in the mouse liver caused by exposure to CeCl₃, we measured the liver indices, and cerium content, evaluated the liver histopathological section, detected serum biochemical parameters of liver function, and the immunoglobulin M (IgM) content, analyzed the liver mRNA and protein expression levels of Toll-like receptor 2, 4 (TLR2, TLR4), and inflammatory cytokines in liver using real-time quantitative reverse transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. The results showed that exposure to CeCl₃ decreased body weight and caused cerium accumulation in the mouse liver and histopathological changes of liver (such as inflammatory cell infiltration). Furthermore, biochemical assays suggested that CeCl3 could promote the activities of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, pseudocholinesterase, and leucine aminopeptidase, decrease serum IgM, upregulate the levels of TLR2, TLR4, nuclear factor-κB (NF-κB), NF-κBp52, NF-κBp65, NF-κB-inducing kinase (NIK), IκB kinase α (IKK-α), IκB kinase β (IKK-β), and tumor necrosis factor-α (TNF-α) expression, and suppress NF-κB-inhibiting factor (IκB) and interleukin-2 (IL-2) expression in liver. Taken together, the inflammation of mice liver caused by exposure to CeCl₃ might be closely associated with the alteration of inflammatory cytokine expressions in the mouse liver, the signal-transducing events happening in CeCl₃-induced macrophages of liver sequentially might occur via activation of TLRs→TNF-α→NIK→IκB kinase (including IKK1, IKK2)→NF-κB (including NF-κBP52, NF-κBP65)→ inflammation.

Genetic and epigenetic loss of miR-31 activates NIK-dependent NF-κB pathway in Adult T-cell Leukemia

Although crucial roles of microRNA have begun to emerge, detailed studies with ATL patients have not been achieved. Using 40 primary ATL samples and 22 samples of normal CD4+ T-cells, we determined the microRNA signatures of ATL and revealed loss of miR-31, which has recently been reported as a metastasis-associated miRNA. All ATL cases invariably showed undetectable or very low levels of miR-31, clearly implying that miR-31 loss is involved in ATL development. As a novel miR-31 target gene, we identified NF-κB inducing kinase (NIK) that plays central roles in noncanonical signaling and constitutive activation of NF-κB in various cancers, including ATL. Restoration of miR-31 downregulated the levels of NIK and NF-κB activity, resulting in reduction of malignant phenotypes, containing proliferative index, anti-apoptosis, and chemotaxis in ATL cells. Furthermore, lentivirus-introduced miR-31 could induce strong apoptosis in primary tumor cells freshly isolated from ATL patients, indicating pivotal functions of miR-31 as a tumor suppressor. Global copy number profiling demonstrated that 21 cases out of 168 (12.5%) have genomic loss of 9p21 containing miR-31 region. Furthermore, expression profiling and ChIP assay showed requirement of overexpression of histone methyltransferase in epigenetic suppression of miR-31 and aberrant NF-κB activation in primary ATL cells. Knockdown of methyltransferase complex restored the miR-31 expression and consequently inhibited NIK-dependent NF-κB cascade. These findings illustrated that genetic and epigenetic abnormalities link to NF-κB activation through the loss of miR-31. Considering aberrant epigenomics associated with cancers, the emerging relationship provides us a conceptual advance in understanding the broad-acting oncogenic signaling.

Activation and induction of cytosolic phospholipase A2 by TNF‐α mediated through Nox2, MAPKs, NF‐κB, and p300 in human tracheal smooth muscle cells

Cytosolic phospholipase A(2) (cPLA(2)) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA(2) expression and PGE(2) synthesis in human tracheal smooth muscle cells (HTSMCs) remain unknown. Here, we report that TNF-α-induced cPLA(2) protein and mRNA expression, PGE(2) production, and phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, which were attenuated by pretreatment with a ROS scavenger [N-acetyl-L-cysteine, (NAC)] and the inhibitors of NADPH oxidase [apocynin (APO) and diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNA of Nox2, p47(phox) , MEK1, p42, p38, or JNK2. TNF-α-induced cPLA(2) expression was also inhibited by pretreatment with a selective NF-κB inhibitor [helenalin (HLN)] or transfection with dominant negative mutants of NF-κB inducing kinase (NIK) or IκB kinase (IKK)α/β. TNF-α-induced NF-κB translocation was blocked by pretreatment with NAC, DPI, APO, or HLN, but not by U0126, SB202190, or SP600125. In addition, pretreatment with curcumin (a p300 inhibitor) or transfection with p300 siRNA blocked cPLA(2) expression and PGE(2) synthesis induced by TNF-α. We further confirmed that p300 was associated with the cPLA(2) promoter which was dynamically linked to histone H4 acetylation stimulated by TNF-α, determined by chromatin immunoprecipitation assay. Association of p300 and histone H4 to cPLA(2) promoter was inhibited by U0126, SB202190, and SP600125. These results suggested that in HTSMCs, activation of p47(phox) , MAPKs, NF-κB, and p300 is essential for TNF-α-induced cPLA(2) expression and PGE(2) release.

Abstract 2889: NF-κB inducing kinase modulates melanoma tumorigenesis by regulating pro-survival factors through β-catenin

Abstract Nuclear factor-κB (NF-κB) contributes to the progression of many cancers due to its role in regulating the transcription of genes important for inflammation, proliferation, apoptosis, and survival. In melanoma, high NF-κB activity results from constitutive elevation of IKKβ activity, a major kinase of this pathway. Targeted deletion of IKKβ prevents Ras induced melanoma tumor formation in INK4a/ARF-/- mice, underscoring the importance of NF-κB in melanoma. We have demonstrated that the NF-κB inducing kinase (NIK) is elevated in many melanoma cell lines. To determine the role of NIK overexpression in melanoma, we knocked down NIK using shRNA in WM115 and Hs294T human melanoma cell lines. NIK knock-down melanoma cells exhibited increased apoptosis and decreased proliferation. Similarly, NIK depletion substantially decreased tumor burden of WM115 cells in a xenograft model. Analysis of gene expression microarray data suggests that NIK modulates expression of c-Myc, c-Met, CCND2 and CXCR4, genes which are crucial for melanoma progression. Consistent with the apoptotic phenotype of NIK knock-down melanoma cells, expression of survival factors, such as Bcl2, cIAP1 and survivin, were attenuated in NIK knock-down melanoma cells. Intriguingly, Bcl2 and survivin expression levels (mRNA and protein) were also diminished in NIK knock-out mouse embryonic fibroblasts (MEFs), suggesting that these pro-survival genes are under the regulation of NIK. However, NF-κB activity is not reduced in NIK knock down cells, possibly due to the compensatory activation of IKKβ. Thus, we explored the possibility that NIK affects expression of survival factors in a NF-κB independent manner. Our data suggest that NIK regulates subcellular localization of β-catenin and modulates expression of survival genes such as survivin. Our findings reveal a novel mechanism by which NIK modulates pro-survival genes and suggest that NIK may be a promising therapeutic target for melanoma malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2889. doi:10.1158/1538-7445.AM2011-2889

Abstract 1022: TNFα-induced apoptosis is reduced, but inflammation is increased by combined exposure with common food additive carrageenan

Abstract Tumor necrosis factor (TNF)-α, a homotrimeric, pleiotropic cytokine, mediates both apoptosis and inflammation. Following exposure to TNF-α, activation of caspases leads to apoptotic cell death and stimulation of an inflammatory cascade through the non-canonical pathway of NF-κB activation leads to increased nuclear RelB and p52. In contrast, the common food additive carrageenan (CGN), which contains sulfated galactose residues, stimulates inflammation through both the canonical and non-canonical pathways of NF-κB activation. CGN-induced inflammation utilizes B-cell leukemia/lymphoma (BCL)-10 to activate a pathway of innate immunity that is initiated by interaction with toll-like receptor (TLR)-4. CGN also stimulates inflammation through a reactive oxygen species-mediated pathway. In a series of cell-based experiments, colonic epithelial cells and mouse embryonic fibroblasts were treated with TNF-α and CGN, in order to simulate the possible effects of exposure to dietary CGN in the setting of TNF-α-mediated effects. Marked increase in secretion of Interleukin (IL)-8 occurred, attributable to synergistic effects on phospho-NIK in the non-canonical pathway. TNF-α induced the ubiquitination of TNF receptor-associated factor (TRAF)-2 which interacts with NFκB-inducing kinase (NIK), and CGN induced phosphorylation of BCL10, which is required for NIK-phosphorylation. These results suggest that TNF-α and CGN in combination act to increase NIK phosphorylation, thereby increasing activation of the non-canonical pathway of NF-κB activation. In contrast, the apoptotic effects of TNF-α, including activation of caspase-8 and poly [ADP-ribose] polymerase (PARP)-1 fragmentation, were markedly reduced in the presence of CGN. CGN exposure caused reduction in Fas by cDNA microarray and by ELISA. The study results demonstrate that exposure to CGN drives TNF-α stimulated cells toward inflammation, rather than toward apoptotic cell death. The findings suggest that CGN exposure may compromise the effectiveness of anti-TNF-α therapy. Average consumption of CGN in the typical Western diet is reported to be 250 mg/day, in contrast to therapeutic glycosides, such as digoxin that are prescribed in doses of ∼0.1 mg/day. Ingestion of CGN-containing foods has the potential to both inhibit the pro-apoptotic effect and to increase the pro-inflammatory effect of TNF-α and may contribute to malignant transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1022. doi:10.1158/1538-7445.AM2011-1022

NIK overexpression intrinsically inhibits regulatory T cell function (168.9)

Abstract Activation of the non-canonical, or alternative, NF-κB pathway depends on NF-κB-inducing kinase (NIK). We have shown that NIK is intrinsically essential for T cells to cause lethal graft-versus-host disease and to respond to OX40 costimulation. We went on to investigate the effects of NIK overexpression in T cells by crossing inducible NIK transgenic mice to CD4-Cre inducer mice and found that modest NIK overexpression limited to T cells (CD4-NIKtg) caused fatal autoimmunity by 4-weeks of age, resembling the phenotype described in FoxP3-/- and scurfy mice. We hypothesized that NIK overexpression in regulatory T cells (Treg) affects their suppressive phenotype. Here we show that NIKtg Treg cells obtained from healthy bone marrow chimeras fail to suppress WT conventional T cell (Tconv) proliferation in vitro. In addition, NIKtg Tconv obtained from these mice are resistant to suppression by WT Treg cells. Transient NIK overexpression in vitro also represses Treg cell function, and preliminary experiments suggest that NIK overexpression destabilizes the Treg phenotype. Lastly, we investigated the effect of more extreme NIK overexpression using a mutant NIK transgene that is not targeted for degradation (NIKΔTRAF3). Contrary to modest NIK upregulation, excessive NIK accumulation is lethal to T cells. Together, these results show that NIK plays a critical T-cell intrinsic role in both Tconv and Treg, and that its tight regulation is crucial for T cell regulation and survival.

SMG1 and NIK regulate apoptosis induced by Smac mimetic compounds.

Smac mimetic compounds (SMCs) are experimental small molecules that induce tumour necrosis factor alpha (TNFα)-dependent cancer cell death by targeting the inhibitor of apoptosis proteins. However, many cancer cell lines are resistant to SMC-mediated apoptosis despite the presence of TNFα. To add insight into the mechanism of SMC-resistance, we used functional siRNA-based kinomic and focused chemical screens and identified suppressor of morphogenesis in genitalia-1 (SMG1) and NF-κB-inducing kinase (NIK) as novel protective factors. Both SMG1 and NIK prevent SMC-mediated apoptosis likely by maintaining FLICE inhibitory protein (c-FLIP) levels to suppress caspase-8 activation. In SMC-resistant cells, the accumulation of NIK upon SMC treatment enhanced the activity of both the classical and alternative nuclear factor-κB pathways, and increased c-FLIP mRNA levels. In parallel, persistent SMG1 expression in SMC-resistant cells repressed SMC-mediated TNFα-induced JNK activation and c-FLIP levels were sustained. Importantly, SMC-resistance is overcome by depleting NIK and SMG1, which appear to facilitate the downregulation of c-FLIP in response to SMC and TNFα treatment, leading to caspase-8-dependent apoptosis. Collectively, these data show that SMG1 and NIK function as critical repressors of SMC-mediated apoptosis by potentially converging on the regulation of c-FLIP metabolism.

In TNF-stimulated Cells, RIPK1 Promotes Cell Survival by Stabilizing TRAF2 and cIAP1, which Limits Induction of Non-canonical NF-κB and Activation of Caspase-8

RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-κB, but also regulates caspase-8 activation and cell death. Although overexpression of RIPK1 can cause caspase-8-dependent cell death, when RIPK1(-/-) cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities. To determine how RIPK1 promotes cell survival, we compared wild-type and RIPK1(-/-) cells treated with TNF. Although TRAF2 levels remained constant in TNF-treated wild-type cells, TNF stimulation of RIPK1(-/-) cells caused TRAF2 and cIAP1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-κB2 to p52, a decrease in levels of cFLIP(L), and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1.

Preferential expression of NF-κB-inducing kinase in blood vessels of rheumatoid arthritis synovial tissue containing ectopic lymphoid neogenesis

BackgroundApproximately 30% of rheumatoid arthritis (RA) synovial tissues (ST) is characterised by ectopic lymphoid neogenesis (ELN). This results in structures that may resemble germinal centres. Nuclear factor-κB (NF-κB) transcription factors...

A Single Nucleotide Polymorphism in NF-κB Inducing Kinase Is Associated with Mortality in Septic Shock

We tested the hypothesis that single nucleotide polymorphisms (SNPs) within genes of the NF-κB pathway are associated with altered clinical outcome of septic shock patients. We genotyped 59 SNPs in the NF-κB pathway in a discovery cohort of septic shock patients (St. Paul's Hospital [SPH], N = 589), which identified the C allele of rs7222094 T/C within MAP3K14 (NF-κB inducing kinase; NIK) associated with increased 28-d mortality (uncorrected p = 0.00024, Bonferroni corrected p = 0.014). This result was replicated in a second cohort of septic shock patients (Vasopressin and Septic Shock Trial [VASST; N = 616]) in which the CC genotype of rs7222094 was associated with increased 28-d mortality (Cox regression: SPH cohort hazard ratio [HR], 1.35; 95% confidence interval [CI], 1.12-1.64; p = 0.002 Caucasian only; and VASST cohort HR, 1.24; 95% CI, 1.00-1.52; p = 0.048 Caucasian only). Patients having the CC genotype of rs7222094 in SPH experienced more renal and hematological dysfunction (p = 0.003 and p = 0.011), while patients of the VASST cohort with the rs7222094 CC genotype showed the same trend toward more renal dysfunction. In lymphoblastoid cell lines, we found the rs7222094 genotype most strongly associated with mRNA expression of CXCL10, a chemokine regulated by NF-κB. Accordingly, we measured CXCL10 protein levels and found that the CC genotype of rs7222094 was associated with significantly lower levels than those of the TT genotype in lymphoblastoid cell lines (p < 0.05) and in septic shock patients (p = 0.017). This suggests that the CC genotype of NIK rs7222094 is associated with increased mortality and organ dysfunction in septic shock patients, perhaps due to altered regulation of NF-κB pathway genes, including CXCL10.

Lymphotoxin‐β receptor activation by lymphotoxin‐α1β2 and LIGHT promotes tumor growth in an NFκB‐dependent manner

Lymphotoxin beta receptor (LTβR) activation on mouse fibrosarcoma cells (BFS-1) results in enhanced solid tumor growth paralleled by increased angiogenesis induced by the expression of pro-angiogenic CXCL2. In our study, we demonstrate that both functional ligands of the LTβR, namely LTα(1) β(2) and LIGHT, are involved in the activation of LTβR in solid fibrosarcomas. To identify whether the lymphocyte population is involved in the activation of LTβR in these fibrosarcoma tumors, we used conditional LTβ-deficient mice that specifically lack LTβ expression either on T cells (T-LTβ(-/-)) or on B cells (B-LTβ(-/-)). Solid tumor growth was reduced in both mouse strains when compared to tumor growth in wild-type mice, indicating the participation of both T and B host lymphocytes in the activation of LTβR in these tumors. Tumor growth was also reduced in LIGHT-deficient mice, suggesting a contribution of this ligand to the activation of LTβR in BFS-1 fibrosarcomas. LTβR signaling can involve IκBα and/or NFκB-inducing kinase (NIK) for subsequent NFκB activation in different types of cells. Expression of a dominant negative form of IκBα or of a dominant negative mutant of NIK resulted in decreased activation of NFκB signaling and reduced expression of pro-angiogenic CXCL2 in vitro. Moreover, expression of dominant negative form of NIK or an IκBα repressor in these fibrosarcoma cells resulted in reduced solid tumor growth in vivo, suggesting that both IκBα and NIK are involved in pro-angiogenic signaling after LTβR activation. Our data support the idea that the ablation of LTβR signaling should be considered for cancer treatment.