Autophagy is required for the activation of NFκB

It is well-established that the activation of the inhibitor of NFκB (IκBα) kinase (IKK) complex is required for autophagy induction by multiple stimuli. Here, we show that in autophagy-competent mouse embryonic fibroblasts (MEFs), distinct autophagic triggers, including starvation, mTOR inhibition with rapamycin and p53 inhibition with cyclic pifithrin α lead to the activation of IKK, followed by the phosphorylation-dependent degradation of IκBα and nuclear translocation of NFκB. Remarkably, the NFκB signaling pathway was blocked in MEFs lacking either the essential autophagy genes Atg5 or Atg7. In addition, we found that tumor necrosis factor α (TNFα)-induced NFκB nuclear translocation is abolished in both Atg5- and Atg7-deficient MEFs. Similarly, the depletion of essential autophagy modulators, including ATG5, ATG7, Beclin 1 and VPS34, by RNA interference inhibited TNFα-driven NFκB activation in two human cancer cell lines. In conclusion, it appears that, at least in some instances, autophagy is required for NFκB activation, highlighting an intimate crosstalk between these two stress response signaling pathways.     

The JNK/NFκB pathway is required to activate murine lymphocytes induced by a sulfated polysaccharide from Ecklonia cava

Background

The proven immunomodulatory and immune system activating properties of Ecklonia cava (E. cava) have been attributed to its plentiful polysaccharide content. Therefore, we investigated whether the sulfated polysaccharide (SP) of E. cava specifically activates the protein kinases (MAPKs) and nuclear factor-κB (NFκB) to incite immune responses.

Methods

To assess immune responsiveness, lymphocytes were isolated from spleens of ICR mice and cultured with SP and its inhibitors. Assays included ³H-thymidine incorporation, flow cytometry, real time polymerase chain reaction (rtPCR), enzyme linked immunosorbent assay (ELISA), intracellular cytokine assay, Western blot, and electrophoretic mobility shift assay (EMSA).

Results

SP dose-dependently increased the proliferation of lymphocytes without cytotoxicity. In particular, SP markedly enhanced the proliferation and differentiation of CD3⁺ mature T cells and CD45R/B220⁺ pan B cells. Additionally, SP increased the expression and/or production of IL-2, IgG_1a, and IgG_2b compared to that in untreated cells. The subsequent application of JNK (SP600125), NFκB (PDTC), and serine protease (TPCK) inhibitors significantly inhibited the proliferation and IL-2 production of SP-treated lymphocytes as well as the phosphorylation of JNK and IκB, the activation of nuclear NFκB p65, and binding of NFκB p65 DNA. Moreover, co-application of both JNK and NFκB inhibitors completely blocked the proliferation of lymphocytes even in the presence of SP.

Conclusion

These results suggest that SP induced T and B cell responses via both JNK and NFκB pathways.

General significance

The effect of SP on splenic lymphocyte activation was assayed here for the first time and indicated the underlying functional mechanism.     

TNFR1-induced activation of the classical NF-κB pathway

The molecular mechanisms underlying activation of the IκB kinase (IKK) complex are presumably best understood in the context of tumor necrosis factor (TNF) receptor-1 (TNFR1) signaling. In fact, it seems that most, if not all, proteins relevant for this process have been identified and extensive biochemical and genetic data are available for the role of these factors in TNF-induced IKK activation. There is evidence that protein modification-independent assembly of a core TNFR1 signaling complex containing TNFR1-associated death domain, receptor interacting kinase?1, TNF receptor-associated factor?2 and cellular inhibitor of apoptosis protein?1 and 2 starts a chain of nondegrading ubiquitination events that culminate in the recruitment and activation of IKK complex-stimulating kinases and the IKK complex itself. Here, we sum up the known details of TNFR1-induced IKK activation, address arising contradictions and discuss possible explanations resolving the apparent discrepancies.     

Optineurin is required for CYLD-dependent inhibition of TNFα-induced NF-κB activation

The nuclear factor kappa B (NF-κB) regulates genes that function in diverse cellular processes like inflammation, immunity and cell survival. The activation of NF-κB is tightly controlled and the deubiquitinase CYLD has emerged as a key negative regulator of NF-κB signalling. Optineurin, mutated in certain glaucomas and amyotrophic lateral sclerosis, is also a negative regulator of NF-κB activation. It competes with NEMO (NF-κB essential modulator) for binding to ubiquitinated RIP (receptor interacting protein) to prevent NF-κB activation. Recently we identified CYLD as optineurin-interacting protein. Here we have analysed the functional significance of interaction of optineurin with CYLD. Our results show that a glaucoma-associated mutant of optineurin, H486R, is altered in its interaction with CYLD. Unlike wild-type optineurin, the H486R mutant did not inhibit tumour necrosis factor α (TNFα)-induced NF-κB activation. CYLD mediated inhibition of TNFα-induced NF-κB activation was abrogated by expression of the H486R mutant. Upon knockdown of optineurin, CYLD was unable to inhibit TNFα-induced NF-κB activation and showed drastically reduced interaction with ubiquitinated RIP. The level of ubiquitinated RIP was increased in optineurin knockdown cells. Deubiquitination of RIP by over-expressed CYLD was abrogated in optineurin knockdown cells. These results suggest that optineurin regulates NF-κB activation by mediating interaction of CYLD with ubiquitinated RIP thus facilitating deubiquitination of RIP.     

Ligand-induced internalization of TNF receptor 2 mediated by a di-leucin motif is dispensable for activation of the NFκB pathway

Endocytosis is an important mechanism to regulate tumor necrosis factor (TNF) signaling. In contrast to TNF receptor 1 (TNFR1; CD120a), the relevance of receptor internalization for signaling as well as the fate and route of internalized TNF receptor 2 (TNFR2; CD120b) is poorly understood. To analyze the dynamics of TNFR2 signaling and turnover at the plasma membrane we established a human TNFR2 expressing mouse embryonic fibroblast cell line in a TNFR1^−/−/TNFR2^−/− background. TNF stimulation resulted in a decrease of constitutive TNFR2 ectodomain shedding. We hypothesized that reduced ectodomain release is a result of TNF/TNFR2 complex internalization. Indeed, we could demonstrate that TNFR2 was internalized together with its ligand and cytoplasmic binding partners. Upon endocytosis the TNFR2 signaling complex colocalized with late endosome/lysosome marker Rab7 and entered the lysosomal degradation pathway. Furthermore, we identified a di-leucin motif in the cytoplasmic part of TNFR2 suggesting clathrin-dependent internalization of TNFR2. Internalization defective TNFR2 mutants are capable to signal, i.e. activate NFκB, demonstrating that the di-leucin motif dependent internalization is dispensable for this response. We therefore propose that receptor internalization primarily serves as a negative feed-back to limit TNF responses via TNFR2.     

Fungal substances as modulators of NF-κB activation pathway

MCF7 breast cancer cell line, carrying a luciferase reporter gene under the control of nuclear factor kappa B (NF-κB)-responsive promoter, was established and used for the screening of fungal organic extracts for their ability to interfere with the NF-κB activation pathway. Twenty-eight crude fungal extracts, out of 242, were found to inhibit NF-κB reporter activity by more than 40%. Furthermore, positive extracts were used to evaluate their antiproliferative activity as well as their ability to influence the phosphorylation and degradation levels of IκBa. Fungal extracts prepared from Marasmius oreades and Cyathus striatus showed significant inhibitory effects on the NF-κB activation pathway. Taken together, our results support the notion of the presence of novel activities that might be utilized as cancer therapeutics.     

Direct molecular interactions between Beclin 1 and the canonical NFκB activation pathway

General (macro)autophagy and the activation of NFκB constitute prominent responses to a large array of intracellular and extracellular stress conditions. The depletion of any of the three subunits of the inhibitor of NFκB (IκB) kinase (IKKα, IKKβ, IKKγ/NEMO), each of which is essential for the canonical NFκB activation pathway, limits autophagy induction by physiological or pharmacological triggers, while constitutive active IKK subunits suffice to stimulate autophagy. The activation of IKK usually relies on TGFβ-activated kinase 1 (TAK1), which is also necessary for the optimal induction of autophagy in multiple settings. TAK1 interacts with two structurally similar co-activators, TAK1-binding proteins 2 and 3 (TAB2 and TAB3). Importantly, in resting conditions both TAB2 and TAB3 bind the essential autophagic factor Beclin 1, but not TAK1. In response to pro-autophagic stimuli, TAB2 and TAB3 dissociate from Beclin 1 and engage in stimulatory interactions with TAK1. The inhibitory interaction between TABs and Beclin 1 is mediated by their coiled-coil domains (CCDs). Accordingly, the overexpression of either TAB2 or TAB3 CCD stimulates Beclin 1- and TAK1-dependent autophagy. These results point to the existence of a direct molecular crosstalk between the canonical NFκB activation pathway and the autophagic core machinery that guarantees the coordinated induction of these processes in response to stress.     

Sam68 is required for both NF-κB activation and apoptosis signaling by the TNF receptor

The RNA-binding protein Sam68 is implicated in various cellular processes including RNA metabolism, apoptosis, and signal transduction. Here we identify a role of Sam68 in TNF-induced NF-κB activation and apoptosis. We found that Sam68 is recruited to the TNF receptor, and its deficiency dramatically reduces RIP recruitment and ubiquitylation. It also impairs cIAP1 recruitment and maintenance of recruited TRAF2 at the TNF receptor. In its absence, activation of the TAK1-IKK kinase complex is defective, greatly reducing signal transduction. Sam68 is also found as a part of the TNF-induced cytoplasmic caspase-8-FADD complex. RIP is not recruited to this complex in Sam68 knockout cells, and caspase activation is virtually absent. These findings delineate previously unknown functions for Sam68 in the TNF signaling pathway, where it acts as a signaling adaptor both in the membrane-associated complex I and in the cytoplasmic complex II, regulating both NF-κB activation and apoptosis.     

Uev1A amino terminus stimulates poly-ubiquitin chain assembly and is required for NF-κB activation

The ubiquitin (Ub)-conjugating enzyme variants (Uev) Uev1A and Mms2 interact with Ubc13 to form heterodimeric complexes with different biological functions. Uev1A-Ubc13 is involved in NF-κB activation while Mms2-Ubc13 is required for the DNA-damage response. The structural comparison of the core domains of these two Uevs reveals no obvious difference, suggesting that the amino terminal extension of Uev1A plays a critical role in the functional determination. Indeed, truncated Uev1A lacking the N-terminal extension behaves like Mms2, while a chimeric protein containing the N-terminal Uev1A fused to Mms2 functionally resembles Uev1A. Interestingly, the N-terminal extension of Uev1A also dictates whether to assemble di- or poly-Ub chains in an in vitro reaction. Both thermodynamic measurements and enzymatic assays revealed that the Uev1A N-terminal extension weakens the Uev-Ubc13 interaction; however, other means capable of causing a reduced Uev1A-Ubc13 affinity and poly-Ub chain assembly do not necessarily promote NF-κB activation, indicating that the poly-Ub chain formation is not the only component contributed by the N-terminal extension of Uev1A. The physiological relevance of the Uev1A N-terminal truncation is presented and discussed.     

Drug inhibition profile prediction for NFκB pathway in multiple myeloma

Nuclear factor κB (NFκB) activation plays a crucial role in anti-apoptotic responses in response to the apoptotic signaling during tumor necrosis factor (TNFα) stimulation in Multiple Myeloma (MM). Although several drugs have been found effective for the treatment of MM by mainly inhibiting NFκB pathway, there are not any quantitative or qualitative results of comparison assessment on inhibition effect between different drugs either used alone or in combinations. Computational modeling is becoming increasingly indispensable for applied biological research mainly because it can provide strong quantitative predicting power. In this study, a novel computational pathway modeling approach is employed to comparably assess the inhibition effects of specific drugs used alone or in combinations on the NFκB pathway in MM and to predict the potential synergistic drug combinations.     

SIGNR1 ligation on murine peritoneal macrophages induces IL-12 production through NFκB activation

We have previously shown that murine resident peritoneal macrophages (PEMs) are activated in response to uptake of oligomannose-coated liposomes (OMLs), leading to production of interleukin (IL)-12. To understand the mechanism of activation of PEMs by OMLs, in the present study we investigated the role of a mannose-binding C-type lectin receptor, SIGNR1, in production of proinflammatory cytokines by PEMs, in which SIGNR1 acts as a physiological receptor for OMLs. Engagement of SIGNR1 on PEMs with an anti-SIGNR1-specific rat IgM antibody, ERTR9, induced production of IL-12 and tumor necrosis factor (TNF)-α from PEMs, while secretion of IL-6 and IL-1β was not detected with the same treatment. The level of phosphorylated IκB kinase in PEMs also increased in response to ERTR9 treatment of the cells. Treatment of PEMs with a specific nuclear factor kappa-B (NFκB) inhibitor, BAY11-7082, reduced ERTR9-dependent IL-12 production. Intraperitoneal treatment with BAY11-7082 also led to reduction of subsequent OML-induced IL-12 production from PEMs. These results indicate that SIGNR1-mediated intercellular signaling may induce production of cytokines such as IL-12 through NFκB activation.