Activation Of NF-kappaB Pathway Research Articles

Rosanortriterpenes A-B, Two Promising Agents from Rosa laevigata var. leiocapus, Alleviate Inflammatory Responses and Liver Fibrosis in In Vitro Cell Models.

Rosanortriterpenes A–B (RTA and RTB), two nortriterpenoids, are characteristic constituents in the fruits of Rosa laevigata var. leiocapus. However, pharmacological studies on these compounds are still scarce. In the present study, we aim to investigate the anti-inflammatory mechanisms associated with the effects of RTA–B in RAW264.7 macrophages and LO2 cells by detecting cell viabilities, nitric oxide (NO) production, tumour necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) production. Simultaneously, the anti-inflammatory action mechanisms of these two compounds were illustrated through western blot assay. Besides, the antihepatic fibrosis activities of these compounds have also been explored. The results demonstrated that RTA and RTB inhibited the production of NO, TNF-α, and IL-6 and suppressed liver fibrosis. RTA and RTB treatment also greatly inhibited the activation of NF-kappaB (NF-κB) pathway. Our study confirmed the promising anti-inflammatory and anti-liver fibrosis actions of RTA–B, suggesting that they might be developed as alternative and promising drugs for the treatment of hepatic inflammatory and fibrotic diseases.

Protective Effect of PBCA Nanoparticles Loaded with Thymulin Against the Relapsing-Remitting Form of Experimental Autoimmune Encephalomyelitis in Mice.

Relapsing–remitting experimental autoimmune encephalomyelitis (rEAE) in mice is a model that closely resembles relapsing–remitting multiple sclerosis in humans. This study aims to investigate a new approach to modulation of the inflammatory response in rEAE mice using a thymic peptide thymulin bound to polybutylcyanoacrylate (PBCA) nanoparticles. PBCA nanoparticles were used to prolong the presence of thymulin in the blood. Cytokine levels in blood were measured by ELISA; NF-κB and SAPK/JNK cascade activation, as well as Hsp72 and p53 protein expression, were measured by Western blotting. Animal health statuses were estimated using severity scores. Results showed that the cytokine response in rEAE was multi-staged: an early phase was accompanied by an increase in plasma interferon-γ, while the interleukin (IL)-17 response was markedly increased at a later stage. The stages were attributed to rEAE induction and maintenance phases. Thymulin significantly alleviated symptoms of rEAE and lowered plasma cytokine levels both in early and later stages of rEAE, and decreased NF-κB and SAPK/JNK cascade activation. Thymulin modulated NF-kappaB pathway activity via site-specific phosphorylation of RelA/p65 protein (at Ser276 and Ser536). The effect of nanoparticle-bound thymulin was more pronounced than the effect of free thymulin. Therefore, PBCA–thymulin can be considered a prospective treatment for this pathology.

Endothelial cells secreted endothelin-1 augments diabetic nephropathy via inducing extracellular matrix accumulation of mesangial cells in ETBR-/- mice.

Endothelin B receptor (ETBR) deficiency may contribute to the progression of diabetic nephropathy (DN) in a streptozotocin (STZ) model, but the underlying mechanism is not fully revealed. In this study, STZ-diabetic ETBR-/- mice was characterized by increased serum creatinine and urinary albumin, enhanced glomerulosclerosis, and upregulated ET-1 expression compared with STZ-diabetic WT mice. In vitro, HG conditioned media (CM) of ETBR-/- GENs promoted mesangial cell proliferation and upregulated ECM-related proteins, and ET-1 knockout in GENs or inhibition of ET-1/ETAR in mesangial cell suppressed mesangial cell proliferation and collagen IV formation. In addition, ET-1 was over-expressed in ETBR-/- GENs and was regulated by NF-kapapB pathway. ET-1/ETBR suppressed NF-kappaB to modulate ET-1 in GENs. Furthermore, ET-1/ETAR promoted RhoA/ROCK pathway in mesangial cells, and accelerated mesangial cell proliferation and ECM accumulation. Finally, in vivo experiments proved inhibition of NF-kappaB pathway ameliorated DN in ETBR-/- mice. These results suggest that in HG-exposed ETBR-/- GENs, suppression of ET-1 binding to ETBR activated NF-kappaB pathway, thus to secrete large amount of ET-1. Due to the communication between GENs and mesangial cells in diabetes, ET-1 binding to ETAR in mesangial cell promoted RhoA/ROCK pathway, thus to accelerate mesangial cell proliferation and ECM accumulation.

Hypothesis of Opposite Interplay Between the Canonical WNT/beta-catenin Pathway and PPAR Gamma in Primary Central Nervous System Lymphomas.

Primary central nervous system lymphomas (PCNSLs) are angiocentric neoplasia which present dense monoclonal lymphocyte proliferation, and occur in brain parenchyma in 90% of the cases. Activated B-cell like Diffuse Large B-cell Lymphoma (ABC-DLBCL) subtype represents more than 90% of PCNSLs and is the most aggressive subtype with a cure rate of only 40%. One of the characteristics of ABC-DLBCL subtype is neuroinflammation through the activation of NF-kappaB pathway. c-Myc alterations and protein expression have been shown in aggressive DLBCL. c-Myc is considered as a key prognostic and predictive biomarker for survival in DLBCL, its expression is associated with worst survival rates. Although mRNA of c-Myc is increased by low levels gains of c-Myc, several studies have shown that c-Myc protein expression is overexpressed without c-Myc abnormalities. These high levels of c-Myc protein in DLBCL without genetic abnormalities suggest that c-Myc protein expression may be also increased by other mechanisms or signaling pathways which regulate its expression. In PCNSLs, the canonical WNT/beta- catenin pathway is upregulated while PPAR gamma is downregulated. The opposite interplay between WNT/beta-catenin pathway and PPAR gamma is reviewed here. Activation of WNT/beta-catenin pathway leads to the transcription of genes involved in cell proliferation, mitochondrial metabolism, protein synthesis, and tumor growth, such as c-Myc. PPAR gamma agonists induce the inhibition of several signaling pathways such as NF-kappaB, STAT, PI3K/Akt and WNT/beta-catenin pathway. Activation of PPAR gamma agonists may have a major negative key role in the regulation of PCNSLs progression.

Blastic Plasmacytoid Dendritic Cell Neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare malignancy derived from plasmacyoid dendritic cells whose biology, clinical features, and treatment options are increasingly better understood. TCF4 is a master regulator that drives donwstream transcriptional programs in BPDCN. In turn, TCF4 activity is dependent on the bromodomain and extra-terminal domain (BET) protein BRD4 whose inhibition provides a promising therapeutic vulnerability. Notably, TCF4 expression is a highly sensitive marker for BPDCN and augments diagnostic specificity alongside CD4, CD56, CD123, and TCL1. The gene expression profile of BPDCN is characterized by aberrant NF-kappaB pathway activation, while its genomic landscape is dominated by structural chromosomal alterations involving ETV6, MYC, and NR3C1, as well as mutations in epigenetic regulators particularly TET2. Advances in elucidating the biological characteristics of BPDCN are resulting in a more refined diagnostic approach and are opening novel therapeutic avenues for patients with this disease.

Nitrosporeusine analogue ameliorates Chandipura virus induced inflammatory response in CNS via NFκb inactivation in microglia.

Chandipura Virus (CHPV), a negative-stranded RNA virus belonging to the Rhabdoviridae family, has been previously reported to bring neuronal apoptosis by activating several factors leading to neurodegeneration. Following virus infection of the central nervous system, microglia, the ontogenetic and functional equivalents of macrophages in somatic tissues gets activated and starts secreting chemokines, thereby recruiting peripheral leukocytes into the brain parenchyma. In the present study, we have systemically examined the effect of CHPV on microglia and the activation of cellular signalling pathways leading to chemokine expression upon CHPV infection. Protein and mRNA expression profiles of chemokine genes revealed that CHPV infection strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. CHPV infection triggered the activation of signalling pathways mediated by mitogen-activated protein kinases, including p38, JNK 1 and 2, and nuclear factor κB (NF-kappaB). CHPV-induced expression of CXCL10 and CCL5 was achieved by the activation of p38 and NF-kappaB pathways. Considering the important role of inflammation in neurodegeneration, we have targeted NF-kappaB using a newly synthesised natural product nitrosporeusine analogue and showed incapability of microglial supernatant of inducing apoptosis in neurons after treatment.

P006 A NEW MODEL OF SPONTANEOUS INNATE IMMUNE MEDIATED COLITIS

IBD is driven by excessive inflammation involving reciprocal interactions between innate and adaptive immune cells. To better understand how innate immunity contributes to IBD, colitis models that occur in the absence of adaptive immunity are needed. TNFAIP3 is a cytosolic protein that blocks innate immune receptor activation of NF-kappaB and MAPK pathways. We generated villin-TNFAIP3 mice to investigate its role in intestinal epithelial cells. We crossed these mice to mice lacking adaptive immune cells (RAG1-/-) and discovered that these mice develop colitis. All mice are C57Bl/6. v-TNFAIP3 mice and RAG1-/- mice were interbred (TRAG mice) and colitis was assessed. Antibiotics were given to investigate the role of microbes. Anti-TNF and anti-Thy1 treatments were used to test the role of TNF and innate lymphoid cell (ILCs). Rorc-/- mice were used to test the role of ILC3s. LPLs and cytokines were measured in mucosal tissues. Villin-TNFAIP3 x RAG1-/- mice (TRAG mice) developed 100% penetrant early onset colitis which was prevented by antibiotics but not by anti-TNF treatment. Inflammation was restricted to the colon and not transmissible to RAG1-/- or TNFAIP3 littermates. TRAG colitis involved increased mucosal infiltration of ILCs and was prevented by anti-Thy1 antibody treatment. TRAG colitis was not prevented on a ILC3-deficient (Rorc-/-) background. The colitis in TRAG mice was characterized by increased expression of IFNg and IFNg-target genes. We are therefore investigating whether innate immune colitis in TRAG mice is driven by IFNg and potentially ILC1 cells. This is a new robust model of colitis that occurs spontaneously in the absence of adaptive immune cells, is driven by microbes but is not transmissible, and is also driven by ILC but occurs in the absence of ILC3s. Further investigation of this model will provide new insight into innate immune mechanisms that drive colitis.

Abstract 397: Aldehyde-Modified Hdl Has CD36-dependent Pro-Atherogenic Effects in Macrophages

The role of high density lipoproteins (HDL) in protecting against cardiovascular disease is compromised when HDL undergoes modification during conditions of oxidative stress; however, the mechanisms underlying these changes in HDL function are not well defined. Reactive aldehydes such as acrolein (a major component in cigarette smoke) or major products of lipid peroxidation such as 4-hydroxynonenal (HNE) or malondialdehyde (MDA) are known to oxidize HDL in cardiovascular disease. To test the hypothesis that modification of HDL with aldehydes impairs HDL’s athero-protective functions in macrophages, we first measured the ability of modified HDL to protect against foam cell formation. Cholesterol-loaded peritoneal macrophages isolated from wild-type C57Bl/J mice were incubated with native HDL, acrolein-modified HDL (acro-HDL), HNE-modified HDL (HNE-HDL) or MDA-modified HDL (MDA-HDL) for 24 h. Contrary to native HDL, oxidized forms of HDL were unable to prevent foam cell formation as shown by increased Oil red-O staining. Next, using a Boyden chamber assay, we demonstrated that acro- and MDA-HDL had impaired abilities to promote macrophage migration (64% and 67% of native HDL cell migration, respectively). Finally, using a secreted alkaline phosphatase reporter THP-1 cell-based assay, we determined that acro-HDL promotes activation of the pro-inflammatory NFkappaB pathway. Interestingly, immunoblot and quantitative RT-PCR analyses revealed that incubation of macrophages with acro- and MDA-HDL leads to increased expression of the pro-atherogenic receptor, cluster of differentiation 36 (CD36). Therefore, we repeated the foam cell formation and migration experiments using similar ligands, but this time, in CD36-null peritoneal macrophages. We found that both of these functions were dependent on CD36; however, the extent of the functional changes varied based on the type of oxidative modification present on HDL. In conclusion, modification of HDL with reactive aldehydes generates a particle that has pro-atherogenic effects in macrophages, many of which are dependent on CD36.

Hypoxia/ischemia promotes CXCL10 expression in cardiac microvascular endothelial cells by NFkB activation

CXCL10, the chemokine with potent chemotactic activity on immune cells and other non-immune cells expressing its receptor CXCR3, has been demonstrated to involve in myocardial infarction, which was resulted from hypoxia/ischemia. The cardiac microvascular endothelial cells (CMECs) are the first cell type which is implicated by hypoxia/ischemia. However, the potential molecular mechanism by which hypoxia/ischemia regulates the expression of CXCL10 in CMECs remains unclear. In the present study, the expression of CXCL10 was firstly examined by real-time PCR and ELISA analysis. Several potential binding sites (BS) for transcription factors including NF-kappaB (NFkB), HIF1 alpha (HIF1α) and FoxO3a were identified in the promoter region of CXCL10 gene from −2000bp to −1bp using bioinformatics software. Luciferase reporter gene vectors for CXCL10 promoter and for activation of above transcription factors were constructed. The activation of NFkB, hypoxia-inducible transcription factor-1 alpha (HIF-1α) and FoxO3a was also analyzed by Western blotting. It was shown that the production of CXCL10 in CMECs was significantly increased by hypoxia/ischemia treatment, in parallel with the activation of CXCL10 promoter examined by reporter gene vector system. Furthermore, transcription factors including NFkB, HIF1α and FoxO3a were activated by hypoxia/ischemia in CMECs. However, over-expression of NFkB, but not that of HIF1α or FoxO3a, significantly promoted the activation of CXCL10 promoter reporter gene. These findings indicated that CXCL10 production in CMECs was significantly increased by hypoxia/ischemia, at least in part, through activation of NFkB pathway and subsequently binding to CXCL10 promoter, finally promoted the transcription of CXCL10 gene.

Anticancer cardamonin analogs suppress the activation of NF-kappaB pathway in lung cancer cells

NF-kappaB pathway has been proven to be critical to survival of lung cancer cells, and many natural products from plants were shown to inhibit the activation of this pathway. In this study, we investigated the effects of two cardamonin analogs, 4,4'-dihydroxylchalcone (DHC) and 4,4'-dihydroxy-2'-methoxychalcone (DHMC), on survival of lung cancer cells and the involved mechanisms. MTT assay revealed that the two compounds potently decreased the survival of immortalized and primary lung cancer cells. DHC and DHMC were able to induce apoptosis in A549 and NCI-H460 cells. Immunoblotting, immunofluorescent staining, and luciferase reporter further demonstrated that the two compounds suppressed the activation of NF-kappaB pathway in lung cancer cells. PMA-mediated NF-kappaB reactivation abrogated the effect of DHC and DHMC on lung cancer cells. DHC and DHMC were also shown to suppress the growth of A549 xenograft in mice. Collectively, we verified two cardamonin analogs as novel compounds suppressing NF-kappaB signaling for lung cancer therapy.

TNF Receptor-Associated Factor 1 is a Major Target of Soluble TWEAK

Soluble tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), in contrast to membrane TWEAK and TNF, is only a weak activator of the classical NFκB pathway. We observed that soluble TWEAK was regularly more potent than TNF with respect to the induction of TNF receptor-associated factor 1 (TRAF1), a NFκB-controlled signaling protein involved in the regulation of inflammatory signaling pathways. TNF-induced TRAF1 expression was efficiently blocked by inhibition of the classical NFκB pathway using the IKK2 inhibitor, TPCA1. In contrast, in some cell lines, TWEAK-induced TRAF1 production was only partly inhibited by TPCA1. The NEDD8-activating enzyme inhibitor MLN4924, however, which inhibits classical and alternative NFκB signaling, blocked TNF- and TWEAK-induced TRAF1 expression. This suggests that TRAF1 induction by soluble TWEAK is based on the cooperative activity of the two NFκB signaling pathways. We have previously shown that oligomerization of soluble TWEAK results in ligand complexes with membrane TWEAK-like activity. Oligomerization of soluble TWEAK showed no effect on the dose response of TRAF1 induction, but potentiated the ability of soluble TWEAK to trigger production of the classical NFκB-regulated cytokine IL8. Transfectants expressing soluble TWEAK and membrane TWEAK showed similar induction of TRAF1 while only the membrane TWEAK expressing cells robustly stimulated IL8 production. These data indicate that soluble TWEAK may efficiently induce a distinct subset of the membrane TWEAK-targeted genes and argue again for a crucial role of classical NFκB pathway-independent signaling in TWEAK-induced TRAF1 expression. Other TWEAK targets, which can be equally well induced by soluble and membrane TWEAK, remain to be identified and the relevance of the ability of soluble TWEAK to induce such a distinct subset of membrane TWEAK-targeted genes for TWEAK biology will have to be clarified in future studies.

TNFR2 increases the sensitivity of ligand-induced activation of the p38 MAPK and NF-κB pathways and signals TRAF2 protein degradation in macrophages

Tumour necrosis factor (p55 or p60) receptor (TNFR) 1 is the major receptor that activates pro-inflammatory signalling and induces gene expression in response to TNF. Consensus is lacking for the function of (p75 or p80) TNFR2 but experiments in mice have suggested neuro-, cardio- and osteo-protective and anti-inflammatory roles. It has been shown in various cell types to be specifically required for the induction of TNFR-associated factor-2 (TRAF2) degradation and activation of the alternative nuclear factor (NF)-kappaB pathway, and to contribute to the activation of mitogen-activated protein kinases (MAPK) and the classical NF-kappaB pathway. We have investigated the signalling functions of TNFR2 in primary human and murine macrophages. We find that in these cells TNF induces TRAF2 degradation, and this is blocked in TNFR2−/− macrophages. TRAF2 has been previously reported to be required for TNF-induced activation of p38 MAPK. However, TRAF2 degradation does not inhibit TNF-induced tolerance of p38 MAPK activation. Neither TNF, nor lipopolysaccharide treatment, induced activation of the alternative NF-kappaB pathway in macrophages. Activation by TNF of the p38 MAPK and NF-kappaB pathways was blocked in TNFR1−/− macrophages. In contrast, although TNFR2−/− macrophages displayed robust p38 MAPK activation and IkappaBα degradation at high concentrations of TNF, at lower doses the concentration dependence of signalling was weakened by an order of magnitude. Our results suggest that, in addition to inducing TRAF2 protein degradation, TNFR2 also plays a crucial auxiliary role to TNFR1 in sensitising macrophages for the ligand-induced activation of the p38 MAPK and classical NF-kappaB pro-inflammatory signalling pathways.

Autophagy facilitates ventilator-induced lung injury partly through activation of NF-kappaB pathway

Mechanical ventilation is an important supportive therapy in the intensive care unit (ICU) to assist the critically ill patients with respiratory failure. But longer ventilation time has been proven to contribute to the lung injury which has been recognized as ventilator-induced lung injury (VILI). Recently studies have suggested that NF-κB signaling pathways may play a critical role in the process of inflammation and autophagy, and autophagy can reduce the damage of VILI partly by activating the NF-κB pathways. Thus, we propose that autophagy may facilitate ventilator-induced lung injury partly through activation of NF-κB pathway, which might be a new potential therapeutic target for ventilator-induced lung injury. Although the exact mechanism of autophagy and its exact role in the VILI need to be further explored, at least it provides us a potential target in the future prevention of VILI.

The Crystal Structure of the Catalytic Domain of the NF-κB Inducing Kinase Reveals a Narrow but Flexible Active Site

The NF-κB inducing kinase (NIK) regulates the non-canonical NF-κB pathway downstream of important clinical targets including BAFF, RANKL, and LTβ. Despite numerous genetic studies associating dysregulation of this pathway with autoimmune diseases and hematological cancers, detailed molecular characterization of this central signaling node has been lacking. We undertook a systematic cloning and expression effort to generate soluble, well-behaved proteins encompassing the kinase domains of human and murine NIK. Structures of the apo NIK kinase domain from both species reveal an active-like conformation in the absence of phosphorylation. ATP consumption and peptide phosphorylation assays confirm that phosphorylation of NIK does not increase enzymatic activity. Structures of murine NIK bound to inhibitors possessing two different chemotypes reveal conformational flexibility in the gatekeeper residue controlling access to a hydrophobic pocket. Finally, a single amino acid difference affects the ability of some inhibitors to bind murine and human NIK with the same affinity.

Haemozoin Induces Early Cytokine-Mediated Lysozyme Release from Human Monocytes through p38 MAPK- and NF-kappaB- Dependent Mechanisms

Malarial pigment (natural haemozoin, HZ) is a ferriprotoporphyrin IX crystal produced by Plasmodium parasites after haemoglobin catabolism. HZ-fed human monocytes are functionally compromised, releasing increased amounts of pro-inflammatory molecules, including cytokines, chemokines and cytokine-related proteolytic enzyme Matrix Metalloproteinase-9 (MMP-9), whose role in complicated malaria has been recently suggested. In a previous work HZ was shown to induce through TNFalpha production the release of monocytic lysozyme, an enzyme stored in gelatinase granules with MMP-9. Here, the underlying mechanisms were investigated. Results showed that HZ lipid moiety promoted early but not late lysozyme release. HZ-dependent lysozyme induction was abrogated by anti-TNFalpha/IL-1beta/MIP-1alpha blocking antibodies and mimicked by recombinant cytokines. Moreover, HZ early activated either p38 MAPK or NF-kappaB pathways by inducing: p38 MAPK phosphorylation; cytosolic I-kappaBalpha phosphorylation and degradation; NF-kappaB nuclear translocation and DNA-binding. Inhibition of both routes through selected molecules (SB203580, quercetin, artemisinin, parthenolide) prevented HZ-dependent lysozyme release. These data suggest that HZ-triggered overproduction of TNFalpha, IL-1beta and MIP-1alpha mediates induction of lysozyme release from human monocytes through activation of p38 MAPK and NF-kappaB pathways, providing new evidence on mechanisms underlying the HZ-enhanced monocyte degranulation in falciparum malaria and the potential role for lysozyme as a new affordable marker in severe malaria.

Targeted Repression of Overexpressed CD30 Downregulates NF-κB and ERK1/2 Pathway in Hodgkin Lymphoma Cell Lines

We previously reported that CD30 is induced during lymphocyte transformation and that overexpressed CD30 can transduce ligand-independent signals in Hodgkin lymphoma (HL) cells. However, its biological consequence is not fully addressed. In this study, we examined the effects of targeted repression of overexpressed CD30 on cell signaling and proliferation using small-interfering RNAs (siRNAs) in HL cell lines. Repression of CD30 inhibited cellular proliferation through reduced activation of IkappaB kinase (IKK) and extracellular signal-regulated kinase (ERK) 1/2 in both B- and T-HL cell lines. These HL cell lines bear one or more defects in negative regulators of nuclear factor (NF)-kappaB signaling, including A20, cylindromatosis tumor suppressor protein (CYLD), and IkappaBalpha, and when CD30 is repressed, they show reduced activation of the canonical NF-kappaB pathway. This suggests that CD30 governs NF-kappaB and ERK1/2 signaling pathways, and is involved in the proliferation of HL cells. Defective mutations in negative regulators of NF-kappaB signaling appear to promote CD30-initiated basal NF-kappaB activation. These results indicate that CD30 is involved in the tumorigenic process of HL, and that it may be useful as a therapeutic target for the treatment of HL.

Genetic Disruption of KEAP1/CUL3 E3 Ubiquitin Ligase Complex Components is a Key Mechanism of NF-KappaB Pathway Activation in Lung Cancer

Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) (IKK-β/IKK-2), which activates NF-κB, is a substrate of the KEAP1-CUL3-RBX1 E3-ubiquitin ligase complex, implicating this complex in NF-κB pathway regulation. We investigated complex component gene disruption as a novel genetic mechanism of NF-κB activation in non-small cell lung cancer. A total of 644 tumor- and 90 cell-line genomes were analyzed for gene dosage status of the individual complex components and IKBKB. Gene expression of these genes and NF-κB target genes were analyzed in 48 tumors. IKBKB protein levels were assessed in tumors with and without complex or IKBKB genetic disruption. Complex component knockdown was performed to assess effects of the E3-ligase complex on IKBKB and NF-κB levels, and phenotypic importance of IKBKB expression was measured by pharmacological inhibition. We observed strikingly frequent genetic disruption (42%) and aberrant expression (63%) of the E3-ligase complex and IKBKB in the samples examined. Although both adenocarcinomas and squamous cell carcinomas showed complex disruption, the patterns of gene disruption differed. IKBKB levels were elevated with complex disruption, knockdown of complex components increased activated forms of IKBKB and NF-κB proteins, and IKBKB inhibition detriments cell viability, highlighting the biological significance of complex disruption. NF-κB target genes were overexpressed in samples with complex disruption, further demonstrating the effect of complex disruption on NF-κB activity. Gene dosage alteration is a prominent mechanism that disrupts each component of the KEAP1-CUL3-RBX1 complex and its NF-κB stimulating substrate, IKBKB. Herein, we show that, multiple component disruption of this complex represents a novel mechanism of NF-κB activation in non-small cell lung cancer.

2‐Methoxystypandrone represses RANKL‐mediated osteoclastogenesis by down‐regulating formation of TRAF6–TAK1 signalling complexes

2-Methoxystypandrone (2-MS) is a naphthoquinone isolated from Polygonum cuspidatum, a Chinese herb used to treat bone diseases. Here we have determined whether 2-MS antagonised osteoclast development and bone resorption. RAW264.7 cells were treated with receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) to induce differentiation into osteoclasts. RT-PCR and Western blot were used to analyse osteoclast-associated gene expression and signalling pathways. The number of multinuclear osteoclasts, actin rings and resorption pit formation were markedly inhibited by 2-MS, targeting osteoclast differentiation at an early stage and without significant cytotoxicity. The anti-resorption effect of 2-MS was accompanied by decreasing dendritic cell-specific transmembrane protein and matrix metalloproteinase-9 (MMP-9) mRNA expression. RANKL-increased MMP-9 gelatinolytic activity was also attenuated by concurrent, but not by subsequent addition of 2-MS. 2-MS markedly inhibited not only the RANKL-triggered nuclear translocations of NF-kappaB, c-Fos and nuclear factor of activated T cells c1 (NFATc1), but also the subsequent NFATc1 induction. Degradation of IkappaB and phosphorylation of mitogen-activated protein kinases were also suppressed. RANKL facilitated the formation of signaling complexes of tumour necrosis factor receptor-associated factor 6 and transforming growth factor beta-activated kinase 1 (TRAF6-TAK1), important for osteoclastogenesis and formation of such signalling complexes was prevented by 2-MS. The anti-osteoclastogenic effects of 2-MS could reflect the block of RANKL-induced association of TRAF6-TAK1 complexes with consequent decrease of IkappaB-mediated NF-kappaB and mitogen-activated protein kinases-mediated c-Fos activation pathways and suppression of NFATc1 and other gene expression, essential for bone resorption.

Qualitative and quantitative differences in the intensity of Fas-mediated intracellular signals determine life and death in T cells

Fas stimulation has been reported to promote the activation and proliferation of T lymphocytes, but the intracellular signalling pathways that mediate non-apoptotic responses to Fas are poorly defined. To distinguish between the activation signalling and the death-inducing pathway downstream of Fas, we generated a novel T cell line expressing a chimeric hCD8-FasC protein and found that stimulation with the anti-CD8 antibodies induced tyrosine phosphorylation of TCR-proximal proteins, activation of Raf-1/ERK, p38 and JNK, and increased expression of CD69, Fas, and Fas ligand. Stimulation of hCD8-FasC-induced activation of an atypical NF-kappaB pathway, partial cleavage of caspases, and increased expression of TRAF1, FLIP(L) and FLIP(S), thereby protecting T cells from FasL-mediated apoptosis. The proliferative response transmitted through hCD8-FasC chimeric receptors was converted into death signals when cells were stimulated, resulting in increased expression of IL-2 and Nur77 and increased caspase cleavage. Surprisingly, both the enhanced expression of FLIP(L) and FLIP(S) and the complete inhibition of FLIP(S) expression were functionally associated with cell death induction. These findings imply that Fas is able to trigger intracellular signalling events driving both apoptosis and activation of T cells but that cell fate is determined by quantitative and qualitative differences in intracellular signalling following Fas stimulation.

Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2

TNF receptor-associated factor 2 (TRAF2) is a key component in NF-κB signaling triggered by TNF–α 1,2. Genetic evidence indicates that TRAF2 is necessary for polyubiquitination of receptor interacting protein 1 (RIP1) 3 that then serves as a platform for recruitment and stimulation of IκB kinase (IKK) leading to activation of the transcription factor NF-κB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyzes the ubiquitination of RIP1 is lacking. TRAF2 binds to sphingosine kinase 1 (SphK1) 4, one of the isoenzymes that generates the pro-survival lipid mediator sphingosine-1-phosphate (S1P) inside cells. Here we show that SphK1 and production of S1P is necessary for Lys 63-linked polyubiquitination of RIP1, phosphorylation of IKK and IκBα, and IκBα degradation, leading to NF-κB activation. Surprisingly, these responses were mediated by intracellular S1P independently of its cell surface G protein-coupled receptors. S1P specifically binds to TRAF2 at the N-terminal RING domain and stimulates its E3 ligase activity. S1P, but not dihydro-S1P, dramatically increased recombinant TRAF2-catalyzed Lys 63- but not Lys 48-linked polyubiquitination of RIP1 in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH13 or UbcH5a. Our data reveal that TRAF2 is a novel intracellular target of S1P, and that S1P is the missing co-factor for TRAF2 E3 ubiquitin ligase activity, suggesting a new paradigm for regulation of Lys 63-linked polyubiquitination. These results also highlight the key role of SphK1 and its product S1P in TNF-α signaling and the canonical NF-κB activation pathway important in inflammatory, anti-apoptotic, and immune processes.