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.     

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.     

MAGUK-Controlled Ubiquitination of CARMA1 Modulates Lymphocyte NF-κB Activity

The adaptor protein CARMA1 is required for antigen receptor-triggered activation of IKK and JNK in lymphocytes. Once activated, the events that subsequently turn off the CARMA1 signalosome are unknown. In this study, we found that antigen receptor-activated CARMA1 underwent lysine 48 (K48) polyubiquitination and proteasome-dependent degradation. The MAGUK region of CARMA1 was an essential player in this event; the SH3 and GUK domains contained the main ubiquitin acceptor sites, and deletion of a Hook domain (an important structure for maintaining inactive MAGUK proteins) between SH3 and GUK was sufficient to induce constitutive ubiquitination of CARMA1. A similar deletion promoted the ubiquitination of PSD-95 and Dlgh1, suggesting that a conserved mechanism may control the turnover of other MAGUK family protein complexes. Functionally, we demonstrated that elimination of MAGUK ubiquitination sites in CARMA1 resulted in elevated basal and inducible NF-κB and JNK activation as a result of defective K48 ubiquitination and increased persistence of this ubiquitination-deficient CARMA1 protein in activated lymphocytes. The coordination of degradation with the full activation of the CARMA1 molecule likely provides an intrinsic feedback control mechanism to balance lymphocyte activation upon antigenic stimulation.The CARD-containing MAGUK protein 1 (CARMA1, or CARD11) is regarded as an orchestrator of both T-cell-dependent and T-cell-independent immune responses due to its requirement in the activation of IKK and JNK signaling pathways downstream of antigen receptor (AR) ligation in B and T cells (3, 6, 8, 17). CARMA1 overexpression and/or mutations have also been associated with lymphomagenesis, as it promotes sustained activation of NF-κB-dependent cell survival (10, 16, 18). Structurally, CARMA1 is a multidomain adaptor protein containing a caspase recruitment (CARD) and a coiled-coil (CC) domain linked upstream of a region that is related to the MAGUK family of proteins. This MAGUK region contains a postsynaptic density 95/disc large/zona occludens 1 (PDZ), a SRC homology 3 (SH3), and a guanylate kinase-like (GUK) domain (2, 20). In addition, CARMA1 contains a flexible serine/threonine-rich linker that bridges the CC and MAGUK domains. Phosphorylation of this linker by protein kinase Cβ (PKCβ) or PKCθ controls the activation status of CARMA1 (13, 29, 30); thus, this region has been designated as the PKC-regulated domain (PRD) (22). It is likely that PRD phosphorylation destabilizes an inhibitory conformation in CARMA1 that exposes the various interaction domains required to assemble its downstream signaling components. Consistent with this model, deletion of the PRD results in a constitutively active CARMA1, resulting in high basal NF-κB activation (14, 30).Proximal downstream adaptors of CARMA1 include BCL10 and MALT1 (22). Genetic deletion of any of these proteins in cellular and animal models has revealed the importance of this pathway to immune cell function. While AR-induced activation of early signaling pathways, such as protein tyrosine phosphorylation, intracellular Ca²⁺ flux, and activation of extracellular signal-regulated kinase (ERK) and Akt are intact in CARMA1-, BCL10-, or MALT1-deficient lymphocytes, activation of NF-κB and JNK signaling pathways is markedly impaired (6, 24-26). This is manifested in defective lymphocyte proliferation and survival and in reduced immune responses.While the events leading to the activation of the IKK signaling complex downstream of CARMA1 have been well characterized, the signals that down-modulate this pathway are less well understood. Studies of BCL10 turnover have yielded some possible mechanisms (34). After cell activation, BCL10 is posttranslationally modified by both phosphorylation (possibly via IKKβ or CaMKII) and polyubiquitination (polyUb) and undergoes degradation that results in the down-modulation of NF-κB activity. Several ubiquitin protein ligases (E3s), including Itch, NEDD4, cIAP2, and βTrCP, have been reported to drive BCL10 ubiquitination in lymphocytes and to promote its degradation through either lysosomal or proteasomal pathways (7, 12, 27, 37). Overexpression of such E3s downregulates BCL10-dependent pathways, including NF-κB and the production of interleukin-2. Interestingly, antigen receptor-induced phosphorylation and degradation of BCL10 is not observed in the absence of CARMA1 (12), indicating a role for CARMA1 in BCL10 turnover.In this report, we demonstrate that endogenous CARMA1 is directly ubiquitinated and degraded by the proteasome in AR-activated lymphocytes. Structure-function analyses showed that the primary targets for ubiquitination within CARMA1 were localized within the MAGUK region. Mutation of all lysine residues (potential ubiquitin modification targets) to arginines within the MAGUK of CARMA1 produced a hyperactive molecule that promoted high NF-κB and JNK activation levels. Unlike the wild-type (WT) CARMA1 molecule, a lysine-to-arginine mutant CARMA1 was not modified by polyUb chains upon cell activation and had a resulting increase in protein stability. We identified a region between the SH3 and GUK domains that is highly similar to a region (termed the Hook region) that regulates the conformation of the MAGUK protein PSD-95. Notably, deletion of this Hook region was sufficient to trigger polyUb of CARMA1 as well as PSD-95 and Dlgh1, other MAGUK family proteins. These data suggest that activation of CARMA1 initiates a feedback mechanism controlled by the MAGUK domain that triggers ubiquitination and degradation of the CARMA1 signalosome, thereby limiting NF-κB and JNK signaling.     

RIP1 Cleavage in the Kinase Domain Regulates TRAIL-Induced NF-κB Activation and Lymphoma Survival

Although TRAIL is considered a potential anticancer agent, it enhances tumor progression by activating NF-κB in apoptosis-resistant cells. Cellular FLICE-like inhibitory protein (cFLIP) overexpression and caspase-8 activation have been implicated in TRAIL-induced NF-κB activation; however, the underlying mechanisms are unknown. Here, we report that caspase-8-dependent cleavage of RIP1 in the kinase domain (KD) and intermediate domain (ID) determines the activation state of the NF-κB pathway in response to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment. In apoptosis-sensitive cells, caspase-8 cleaves RIP1 in the KD and ID immediately after the recruitment of RIP1 to the receptor complex, impairing IκB kinase (IKK) recruitment and NF-κB activation. In apoptosis-resistant cells, cFLIP restricts caspase-8 activity, resulting in limited RIP1 cleavage and generation of a KD-cleaved fragment capable of activating NF-κB but not apoptosis. Notably, depletion of the cytoplasmic pool of TRAF2 and cIAP1 in lymphomas by CD40 ligation inhibits basal RIP1 ubiquitination but does not prompt cell death, due to CD40L-induced cFLIP expression and limited RIP1 cleavage. Inhibition of RIP1 cleavage at the KD suppresses NF-κB activation and cell survival even in cFLIP-overexpressing lymphomas. Importantly, RIP1 is constitutively cleaved in human and mouse lymphomas, suggesting that cFLIP-mediated and caspase-8-dependent limited cleavage of RIP1 is a new layer of mechanism that promotes NF-κB activation and lymphoma survival.     

Differential Effects of β-catenin and NF-κB Interplay in the Regulation of Cell Proliferation,Inflammation and Tumorigenesis in Response to Bacterial Infection

Both β-catenin and NF-κB have been implicated in our laboratory as candidate factors in driving proliferation in an in vivo model of Citrobacter rodentium (CR)-induced colonic crypt hyper-proliferation and hyperplasia. Herein, we test the hypothesis that β-catenin and not necessarily NF-κB regulates colonic crypt hyperplasia or tumorigenesis in response to CR infection. When C57Bl/6 wild type (WT) mice were infected with CR, sequential increases in proliferation at days 9 and 12 plateaued off at day 19 and paralleled increases in NF-κB signaling. In Tlr4^−/− (KO) mice, a sequential but sustained proliferation which tapered off only marginally at day 19, was associated with TLR4-dependent and independent increases in NF-κB signaling. Similarly, increases in either activated or total β-catenin in the colonic crypts of WT mice as early as day 3 post-infection coincided with cyclinD1 and c-myc expression and associated crypt hyperplasia. In KO mice, a delayed kinetics associated predominantly with increases in non-phosphorylated (active) β-catenin coincided with increases in cyclinD1, c-myc and crypt hyperplasia. Interestingly, PKCζ-catalyzed Ser-9 phosphorylation and inactivation of GSK-3β and not loss of wild type APC protein accounted for β-catenin accumulation and nuclear translocation in either strain. In vitro studies with Wnt2b and Wnt5a further validated the interplay between the Wnt/β-catenin and NF-κB pathways, respectively. When WT or KO mice were treated with nanoparticle-encapsulated siRNA to β-catenin (si- β-Cat), almost complete loss of nuclear β-catenin coincided with concomitant decreases in CD44 and crypt hyperplasia without defects in NF-κB signaling. si-β-Cat treatment to Apc ^Min/+ mice attenuated CR-induced increases in β-catenin and CD44 that halted the growth of mutated crypts without affecting NF-κB signaling. The predominant β-catenin-induced crypt proliferation was further validated in a Castaneus strain (B6.CAST.11M) that exhibited significant crypt hyperplasia despite an attenuated NF-κB signaling. Thus, β-catenin and not necessarily NF-κB regulates crypt hyperplasia in response to bacterial infection.     

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.     

Ubiquitin-associated Domain-containing Ubiquitin Regulatory X (UBX) Protein UBXN1 Is a Negative Regulator of Nuclear Factor κB (NF-κB) Signaling

Excessive nuclear factor κB (NF-κB) activation should be precisely controlled as it contributes to multiple immune and inflammatory diseases. However, the negative regulatory mechanisms of NF-κB activation still need to be elucidated. Various types of polyubiquitin chains have proved to be involved in the process of NF-κB activation. Many negative regulators linked to ubiquitination, such as A20 and CYLD, inhibit IκB kinase activation in the NF-κB signaling pathway. To find new NF-κB signaling regulators linked to ubiquitination, we used a small scale siRNA library against 51 ubiquitin-associated domain-containing proteins and screened out UBXN1, which contained both ubiquitin-associated and ubiquitin regulatory X (UBX) domains as a negative regulator of TNFα-triggered NF-κB activation. Overexpression of UBXN1 inhibited TNFα-triggered NF-κB activation, although knockdown of UBXN1 had the opposite effect. UBX domain-containing proteins usually act as valosin-containing protein (VCP)/p97 cofactors. However, knockdown of VCP/p97 barely affected UBXN1-mediated NF-κB inhibition. At the same time, we found that UBXN1 interacted with cellular inhibitors of apoptosis proteins (cIAPs), E3 ubiquitin ligases of RIP1 in the TNFα receptor complex. UBXN1 competitively bound to cIAP1, blocked cIAP1 recruitment to TNFR1, and sequentially inhibited RIP1 polyubiquitination in response to TNFα. Therefore, our findings demonstrate that UBXN1 is an important negative regulator of the TNFα-triggered NF-κB signaling pathway by mediating cIAP recruitment independent of VCP/p97.     

Cyanidin-3-O-β-glucoside inhibits lipopolysaccharide-induced inflammatory response in mouse mastitis model

Cyanidin-3-O-β-glucoside (C3G) (CAS number 7084-24-4), a typical anthocyanin pigment that exists in the human diet, has been reported to have anti-inflammatory properties. However, the effect of C3G on lipopolysaccharide (LPS)-induced mastitis and the molecular mechanisms have not been investigated. In this study, we detected the protective effects of C3G on a LPS-induced mouse mastitis model and investigated the molecular mechanisms in LPS-stimulated mouse mammary epithelial cells (MMECs). Our results showed that C3G could attenuate mammary histopathologic changes and myeloperoxidase activity, and inhibit TNF-α, interleukin (IL)-1β, and IL-6 production caused by LPS. Meanwhile, C3G dose-dependently inhibited TNF-α and IL-6 in LPS-stimulated MMECs. C3G suppressed LPS-induced nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) activation. Furthermore, C3G disrupted the formation of lipid rafts by depleting cholesterol. Moreover, C3G activated liver X receptor (LXR)-ABCG1-dependent cholesterol efflux. Knockdown of LXRα abrogated the anti-inflammatory effects of C3G. In conclusion, C3G has a protective effect on LPS-induced mastitis. The promising anti-inflammatory mechanisms of C3G are associated with upregulation of the LXRα-ABCG1 pathway which result in disrupting lipid rafts by depleting cholesterol, thereby suppressing toll-like receptor 4-mediated NF-κB and IRF3 signaling pathways induced by LPS.     

NRP/Optineurin Cooperates with TAX1BP1 to Potentiate the Activation of NF-κB by Human T-Lymphotropic Virus Type 1 Tax Protein

Nuclear factor (NF)-κB is a major survival pathway engaged by the Human T-Lymphotropic Virus type 1 (HTLV-1) Tax protein. Tax1 activation of NF-κB occurs predominantly in the cytoplasm, where Tax1 binds NF-κB Essential Modulator (NEMO/IKKγ) and triggers the activation of IκB kinases. Several independent studies have shown that Tax1-mediated NF-κB activation is dependent on Tax1 ubiquitination. Here, we identify by co-immunoprecipitation assays NEMO-Related Protein (NRP/Optineurin) as a binding partner for Tax1 in HTLV-1 infected and Tax1/NRP co-expressing cells. Immunofluorescence studies reveal that Tax1, NRP and NEMO colocalize in Golgi-associated structures. The interaction between Tax1 and NRP requires the ubiquitin-binding activity of NRP and the ubiquitination sites of Tax1. In addition, we observe that NRP increases the ubiquitination of Tax1 along with Tax1-dependent NF-κB signaling. Surprisingly, we find that in addition to Tax1, NRP interacts cooperatively with the Tax1 binding protein TAX1BP1, and that NRP and TAX1BP1 cooperate to modulate Tax1 ubiquitination and NF-κB activation. Our data strongly suggest for the first time that NRP is a critical adaptor that regulates the assembly of TAX1BP1 and post-translationally modified forms of Tax1, leading to sustained NF-κB activation.     

Cleavage of Signal Regulatory Protein α (SIRPα) Enhances Inflammatory Signaling

Signal regulatory protein α (SIRPα) is a membrane glycoprotein immunoreceptor abundant in cells of monocyte lineage. SIRPα ligation by a broadly expressed transmembrane protein, CD47, results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, resulting in the inhibition of NF-κB signaling in macrophages. Here we observed that proteolysis of SIRPα during inflammation is regulated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), resulting in the generation of a membrane-associated cleavage fragment in both THP-1 monocytes and human lung epithelia. We mapped a charge-dependent putative cleavage site near the membrane-proximal domain necessary for ADAM10-mediated cleavage. In addition, a secondary proteolytic cleavage within the membrane-associated SIRPα fragment by γ-secretase was identified. Ectopic expression of a SIRPα mutant plasmid encoding a proteolytically resistant form in HeLa cells inhibited activation of the NF-κB pathway and suppressed STAT1 phosphorylation in response to TNFα to a greater extent than expression of wild-type SIRPα. Conversely, overexpression of plasmids encoding the proteolytically cleaved SIRPα fragments in cells resulted in enhanced STAT-1 and NF-κB pathway activation. Thus, the data suggest that combinatorial actions of ADAM10 and γ-secretase on SIRPα cleavage promote inflammatory signaling.