The CARMA3-Bcl10-MALT1 Signalosome Promotes Angiotensin II-dependent Vascular Inflammation and Atherogenesis

The CARMA1, Bcl10, and MALT1 proteins together constitute a signaling complex (CBM signalosome) that mediates antigen-dependent activation of NF-κB in lymphocytes, thereby representing a cornerstone of the adaptive immune response. Although CARMA1 is restricted to cells of the immune system, the analogous CARMA3 protein has a much wider expression pattern. Emerging evidence suggests that CARMA3 can substitute for CARMA1 in non-immune cells to assemble a CARMA3-Bcl10-MALT1 signalosome and mediate G protein-coupled receptor activation of NF-κB. Here we show that one G protein-coupled receptor, the type 1 receptor for angiotensin II, utilizes this mechanism for activation of NF-κB in endothelial and vascular smooth muscle cells, thereby inducing pro-inflammatory signals within the vasculature, a key factor in atherogenesis. Further, we demonstrate that Bcl10-deficient mice are protected from developing angiotensin-dependent atherosclerosis and aortic aneurysms. By uncovering a novel vascular role for the CBM signalosome, these findings illustrate that CBM-dependent signaling has functions outside the realm of adaptive immunity and impacts pathobiology more broadly than previously known.     

Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation

Antigen receptors activate pathways that control cell survival, proliferation, and differentiation. Two important targets of antigen receptors, NF-κB and Jun N-terminal kinase (JNK), are activated downstream of CARMA1, a scaffolding protein that nucleates a complex including BCL10, MALT1, and other IκB kinase (IKK)-signalosome components. Somatic mutations that constitutively activate CARMA1 occur frequently in diffuse large B cell lymphoma (DLBCL) and mediate essential survival signals. Mechanisms that downregulate this pathway might thus yield important therapeutic targets. Stimulation of antigen receptors induces not only BCL10 activation but also its degradation downstream of CARMA1, thereby ultimately limiting signals to its downstream targets. Here, using lymphocyte cell models, we identify a kinase-independent requirement for TAK1 and its adaptor, TAB1, in antigen receptor-induced BCL10 degradation. We show that TAK1 acts as an adaptor for E3 ubiquitin ligases that target BCL10 for degradation. Functionally, TAK1 overexpression restrains CARMA1-dependent activation of NF-κB by reducing BCL10 levels. TAK1 also promotes counterselection of NF-κB-addicted DLBCL lines by a dual mechanism involving kinase-independent degradation of BCL10 and kinase-dependent activation of JNK. Thus, by directly promoting BCL10 degradation, TAK1 counterbalances NF-κB and JNK signals essential for the activation and survival of lymphocytes and CARMA1-addicted lymphoma types.     

From MALT lymphoma to the CBM signalosome: Three decades of discovery

The advent of molecular cytogenetics has led to the elucidation of genetic abnormalities that cause various congenital and oncological disorders. In B-cell lymphoma, for example, a number of chromosomal translocations have been identified in and associated with the etiology of specific subtypes of lymphoma. Several recurrent chromosomal translocations have been identified in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). Cloning and characterization of the products of three mutually exclusive translocation breakpoints found in MALT lymphoma led to the discovery of a novel NFκB-activating complex comprising the CARMA, Bcl10 and MALT1 proteins. This “CBM signalosome” acts downstream of the antigen receptors in lymphocytes as well as a number of non-lymphoid cell-surface receptors involved in a variety of biological processes. CBM signalosome activity is important for normal cellular functions and is perturbed in neoplastic and inflammatory disorders, making it a viable target for novel therapeutic design.Key words: chromosomal translocation, cytogenetics, lymphoma, MALT1, CARMA1, CARMA3, Bcl10, NFκB, inflammation, atherosclerosis     

MALT1-Ubiquitination Triggers Non-Genomic NF-κB/IKK Signaling upon Platelet Activation

We have recently shown that IKK complex plays an important non-genomic role in platelet function, i.e., regulates SNARE machinery-dependent membrane fusion. In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex. Therefore, the present studies investigated the mechanism by which IKK signaling is regulated in the context of the platelet proteasome. It was found that platelets express a functional proteasome, and form CARMA/MALT1/Bcl10 (CBM) complex when activated. Using a pharmacological inhibitor, the proteasome was found to regulate platelet function (aggregation, integrin activation, secretion, phosphatidylserine exposure and changes in intracellular calcium). It was also found to regulate thrombogenesis and physiologic hemostasis. We also observed, upon platelet activation, that MALT1 is ubiquitinated, and this coincides with the activation of the IKK/NF-κB-signaling pathway. Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex. Thus, our data demonstrate that MALT1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing platelet signals to the NF-κB pathway.     

Oral Administration of Transforming Growth Factor-β1 (TGF-β1) Protects the Immature Gut from Injury via Smad Protein-dependent Suppression of Epithelial Nuclear Factor κB (NF-κB) Signaling and Proinflammatory Cytokine Production

Inflammatory immune responses play an important role in mucosal homeostasis and gut diseases. Nuclear factor κB (NF-κB), central to the proinflammatory cascade, is activated in necrotizing enterocolitis (NEC), a devastating condition of intestinal injury with extensive inflammation in premature infants. TGF-β is a strong immune suppressor and a factor in breast milk, which has been shown to be protective against NEC. In an NEC animal model, oral administration of the isoform TGF-β1 activated the downstream effector Smad2 in intestine and significantly reduced NEC incidence. In addition, TGF-β1 suppressed NF-κB activation, maintained levels of the NF-κB inhibitor IκBα in the intestinal epithelium, and systemically decreased serum levels of IL-6 and IFN-γ. The immature human fetal intestinal epithelial cell line H4 was used as a reductionistic model of the immature enterocyte to investigate mechanism. TGF-β1 pretreatment inhibited the TNF-α-induced IκBα phosphorylation that targets the IκBα protein for degradation and inhibited NF-κB activation. Chromatin immunoprecipitation (ChIP) assays demonstrated decreased NF-κB binding to the promoters of IL-6, IL-8, and IκBα in response to TNF-α with TGF-β1 pretreatment. These TGF-β1 effects appear to be mediated through the canonical Smad pathway as silencing of the TGF-β central mediator Smad4 resulted in loss of the TGF-β1 effects. Thus, TGF-β1 is capable of eliciting anti-inflammatory effects by inhibiting NF-κB specifically in the intestinal epithelium as well as by decreasing systemic IL-6 and IFN-γ levels. Oral administration of TGF-β1 therefore can potentially be used to protect against gastrointestinal diseases.     

Functional Metagenomics: A High Throughput Screening Method to Decipher Microbiota-Driven NF-κB Modulation in the Human Gut

Background/Aim

The human intestinal microbiota plays an important role in modulation of mucosal immune responses. To study interactions between intestinal epithelial cells (IECs) and commensal bacteria, a functional metagenomic approach was developed. One interest of metagenomics is to provide access to genomes of uncultured microbes. We aimed at identifying bacterial genes involved in regulation of NF-κB signaling in IECs. A high throughput cell-based screening assay allowing rapid detection of NF-κB modulation in IECs was established using the reporter-gene strategy to screen metagenomic libraries issued from the human intestinal microbiota.

Methods

A plasmid containing the secreted alkaline phosphatase (SEAP) gene under the control of NF-κB binding elements was stably transfected in HT-29 cells. The reporter clone HT-29/kb-seap-25 was selected and characterized. Then, a first screening of a metagenomic library from Crohn''s disease patients was performed to identify NF-κB modulating clones. Furthermore, genes potentially involved in the effect of one stimulatory metagenomic clone were determined by sequence analysis associated to mutagenesis by transposition.

Results

The two proinflammatory cytokines, TNF-α and IL-1β, were able to activate the reporter system, translating the activation of the NF-κB signaling pathway and NF-κB inhibitors, BAY 11-7082, caffeic acid phenethyl ester and MG132 were efficient. A screening of 2640 metagenomic clones led to the identification of 171 modulating clones. Among them, one stimulatory metagenomic clone, 52B7, was further characterized. Sequence analysis revealed that its metagenomic DNA insert might belong to a new Bacteroides strain and we identified 2 loci encoding an ABC transport system and a putative lipoprotein potentially involved in 52B7 effect on NF-κB.

Conclusions

We have established a robust high throughput screening assay for metagenomic libraries derived from the human intestinal microbiota to study bacteria-driven NF-κB regulation. This opens a strategic path toward the identification of bacterial strains and molecular patterns presenting a potential therapeutic interest.     

The protein kinase C-responsive inhibitory domain of CARD11 functions in NF-kappaB activation to regulate the association of multiple signaling cofactors that differentially depend on Bcl10 and MALT1 for association

The activation of NF-κB by T-cell receptor (TCR) signaling is critical for T-cell activation during the adaptive immune response. CARD11 is a multidomain adapter that is required for TCR signaling to the IκB kinase (IKK) complex. During TCR signaling, the region in CARD11 between the coiled-coil and PDZ domains is phosphorylated by protein kinase Cθ (PKCθ) in a required step in NF-κB activation. In this report, we demonstrate that this region functions as an inhibitory domain (ID) that controls the association of CARD11 with multiple signaling cofactors, including Bcl10, TRAF6, TAK1, IKKγ, and caspase-8, through an interaction that requires both the caspase recruitment domain (CARD) and the coiled-coil domain. Consistent with the ID-mediated control of their association, we demonstrate that TRAF6 and caspase-8 associate with CARD11 in T cells in a signal-inducible manner. Using an RNA interference rescue assay, we demonstrate that the CARD, linker 1, coiled-coil, linker 3, SH3, linker 4, and GUK domains are each required for TCR signaling to NF-κB downstream of ID neutralization. Requirements for the CARD, linker 1, and coiled-coil domains in signaling are consistent with their roles in the association of CARD11 with Bcl10, TRAF6, TAK1, caspase-8, and IKKγ. Using Bcl10- and MALT1-deficient cells, we show that CARD11 can recruit signaling cofactors independently of one another in a signal-inducible manner.     

Neurotropin Suppresses Inflammatory Cytokine Expression and Cell Death through Suppression of NF-κB and JNK in Hepatocytes

Inflammatory response and cell death in hepatocytes are hallmarks of chronic liver disease, and, therefore, can be effective therapeutic targets. Neurotropin® (NTP) is a drug widely used in Japan and China to treat chronic pain. Although NTP has been demonstrated to suppress chronic pain through the descending pain inhibitory system, the action mechanism of NTP remains elusive. We hypothesize that NTP functions to suppress inflammatory pathways, thereby attenuating disease progression. In the present study, we investigated whether NTP suppresses inflammatory signaling and cell death pathways induced by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) in hepatocytes. NTP suppressed nuclear factor-κB (NF-κB) activation induced by IL-1β and TNFα assessed by using hepatocytes isolated from NF-κB-green fluorescent protein (GFP) reporter mice and an NF-κB-luciferase reporter system. The expression of NF-κB target genes, Il6, Nos2, Cxcl1, ccl5 and Cxcl2 induced by IL-1β and TNFα was suppressed after NTP treatment. We also found that NTP suppressed the JNK phosphorylation induced by IL-1β and TNFα. Because JNK activation contributes to hepatocyte death, we determined that NTP treatment suppressed hepatocyte death induced by IL-1β and TNFα in combination with actinomycin D. Taken together, our data demonstrate that NTP attenuates IL-1β and TNFα-mediated inflammatory cytokine expression and cell death in hepatocytes through the suppression of NF-κB and JNK. The results from the present study suggest that NTP may become a preventive or therapeutic strategy for alcoholic and non-alcoholic fatty liver disease in which NF-κB and JNK are thought to take part.     

Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181

NF-κB activation downstream of antigen receptor engagement is a highly regulated event required for lymphocyte activation during the adaptive immune response. The pathway is often dysregulated in lymphoma, leading to constitutive NF-κB activity that supports the aberrant proliferation of transformed lymphocytes. To identify novel regulators of antigen receptor signaling to NF-κB, we developed bioluminescence resonance energy transfer-based interaction cloning (BRIC), a screening strategy that can detect protein-protein interactions in live mammalian cells in a high-throughput manner. Using this strategy, we identified the RING finger protein RNF181 as an interactor of CARD11, a key signaling scaffold in the antigen receptor pathway. We present evidence that RNF181 functions as an E3 ubiquitin ligase to inhibit antigen receptor signaling to NF-κB downstream of CARD11. The levels of the obligate signaling protein Bcl10 are reduced by RNF181 even prior to signaling, and Bcl10 can serve as a substrate for RNF181 E3 ligase activity in vitro. Furthermore, RNF181 limits the proliferation of human diffuse large B cell lymphoma cells that depend upon aberrant CARD11 signaling to NF-κB for growth and survival in culture. Our results define a new regulatory checkpoint that can modulate the output of CARD11 signaling to NF-κB in both normal and transformed lymphocytes.     

IL-10 Inhibits the NF-κB and ERK/MAPK-Mediated Production of Pro-Inflammatory Mediators by Up-Regulation of SOCS-3 in Trypanosoma cruzi-Infected Cardiomyocytes

Trypanosoma cruzi (T. cruzi) infection produces an intense inflammatory response which is critical for the control of the evolution of Chagas’ disease. Interleukin (IL)-10 is one of the most important anti-inflammatory cytokines identified as modulator of the inflammatory reaction. This work shows that exogenous addition of IL-10 inhibited ERK1/2 and NF-κB activation and reduced inducible nitric oxide synthase (NOS2), metalloprotease (MMP) -9 and MMP-2 expression and activities, as well as tumour necrosis factor (TNF)-α and interleukin (IL)-6 expression, in T. cruzi-infected cardiomyocytes. We found that T. cruzi and IL-10 promote STAT3 phosphorylation and up-regulate the expression of suppressor of cytokine signalling (SOCS)-3 thereby preventing NF-κB nuclear translocation and ERK1/2 phosphorylation. Specific knockdown of SOCS-3 by small interfering RNA (siRNA) impeded the IL-10-mediated inhibition of NF-κB and ERK1/2 activation. As a result, the levels of studied pro-inflammatory mediators were restored in infected cardiomyocytes. Our study reports the first evidence that T. cruzi up- regulates SOCS-3 expression and highlights the relevance of IL-10 in the modulation of pro-inflammatory response of cardiomyocytes in Chagas’ disease.     

TRAF Family Member-associated NF-κB Activator (TANK) Inhibits Genotoxic Nuclear Factor κB Activation by Facilitating Deubiquitinase USP10-dependent Deubiquitination of TRAF6 Ligase

DNA damage-induced NF-κB activation plays a critical role in regulating cellular response to genotoxic stress. However, the molecular mechanisms controlling the magnitude and duration of this genotoxic NF-κB signaling cascade are poorly understood. We recently demonstrated that genotoxic NF-κB activation is regulated by reversible ubiquitination of several essential mediators involved in this signaling pathway. Here we show that TRAF family member-associated NF-κB activator (TANK) negatively regulates NF-κB activation by DNA damage via inhibiting ubiquitination of TRAF6. Despite the lack of a deubiquitination enzyme domain, TANK has been shown to negatively regulate the ubiquitination of TRAF proteins. We found TANK formed a complex with MCPIP1 (also known as ZC3H12A) and a deubiquitinase, USP10, which was essential for the USP10-dependent deubiquitination of TRAF6 and the resolution of genotoxic NF-κB activation upon DNA damage. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of TANK in human cells significantly enhanced NF-κB activation by genotoxic treatment, resulting in enhanced cell survival and increased inflammatory cytokine production. Furthermore, we found that the TANK-MCPIP1-USP10 complex also decreased TRAF6 ubiquitination in cells treated with IL-1β or LPS. In accordance, depletion of USP10 enhanced NF-κB activation induced by IL-1β or LPS. Collectively, our data demonstrate that TANK serves as an important negative regulator of NF-κB signaling cascades induced by genotoxic stress and IL-1R/Toll-like receptor stimulation in a manner dependent on MCPIP1/USP10-mediated TRAF6 deubiquitination.     

CARD9 Mediates Dectin-2-induced IκBα Kinase Ubiquitination Leading to Activation of NF-κB in Response to Stimulation by the Hyphal Form of Candida albicans

The scaffold protein CARD9 plays an essential role in anti-fungus immunity and is implicated in mediating Dectin-1/Syk-induced NF-κB activation in response to Candida albicans infection. However, the molecular mechanism by which CARD9 mediates C. albicans-induced NF-κB activation is not fully characterized. Here we demonstrate that CARD9 is involved in mediating NF-κB activation induced by the hyphal form of C. albicans hyphae (Hyphae) but not by its heat-inactivated unicellular form. Our data show that inhibiting Dectin-2 expression selectively blocked Hyphae-induced NF-κB, whereas inhibiting Dectin-1 mainly suppressed zymosan-induced NF-κB, indicating that Hyphae-induced NF-κB activation is mainly through Dectin-2 and not Dectin-1. Consistently, we find that the hyphae stimulation induces CARD9 association with Bcl10, an adaptor protein that functions downstream of CARD9 and is also involved in C. albicans-induced NF-κB activation. This association is dependent on Dectin-2 but not Dectin-1 following the hyphae stimulation. Finally, we find that although both CARD9 and Syk are required for Hyphae-induced NF-κB activation, they regulate different signaling events in which CARD9 mediates IκBα kinase ubiquitination, whereas Syk regulates IκBα kinase phosphorylation. Together, our data demonstrated that CARD9 is selectively involved in Dectin-2-induced NF-κB activation in response to C. albicans hyphae challenging.