Interleukin-1-induced NF-kappaB activation is NEMO-dependent but does not require IKKbeta

Activation of NF-kappaB by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the IkappaB kinase (IKK) complex, which contains two kinases named IKKalpha and IKKbeta and a critical regulatory subunit named NEMO. Although we have previously demonstrated that NEMO associates with both IKKs, genetic studies reveal that only its interaction with IKKbeta is required for TNF-induced NF-kappaB activation. To determine whether NEMO and IKKalpha can form a functional IKK complex capable of activating the classical NF-kappaB pathway in the absence of IKKbeta, we utilized a panel of mouse embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. This confirmed that TNF-induced IkappaBalpha degradation absolutely requires NEMO and IKKbeta. In contrast, we consistently observed intact IkappaBalpha degradation and NF-kappaB activation in response to IL-1 in two separate cell lines lacking IKKbeta. Furthermore, exogenously expressed, catalytically inactive IKKbeta blocked TNF- but not IL-1-induced IkappaBalpha degradation in wild-type MEFs, and reconstitution of IKKalpha/beta double knockout cells with IKKalpha rescued IL-1- but not TNF-induced NF-kappaB activation. Finally, we have shown that incubation of IKKbeta-deficient MEFs with a cell-permeable peptide that blocks the interaction of NEMO with the IKKs inhibits IL-1-induced NF-kappaB activation. Our results therefore demonstrate that NEMO and IKKalpha can form a functional IKK complex that activates the classical NF-kappaB pathway in response to IL-1 but not TNF. These findings further suggest NEMO differentially regulates the fidelity of the IKK subunits activated by distinct upstream signaling pathways.     

NF-kappaB-inducing kinase restores defective IkappaB kinase activity and NF-kappaB signaling in intestinal epithelial cells

Cytokine-stimulated IkappaBalpha degradation is impaired in HT-29 and primary intestinal epithelial cells. To gain more insight into the mechanism of this defect, we dissected cytokine-induced NF-kappaB signaling pathway in HT-29 cells. IL-1beta and TNF, alone or in combination with IFNgamma, failed to induce IkappaBalpha or IkappaBbeta degradation in HT-29 cells. Despite similar 125I-IL-1beta binding, HT-29 cells displayed no IRAK degradation, a 75% reduction of IKK activity, and decreased IkappaBalpha phosphorylation, NF-kappaB DNA binding activity and IL-8 mRNA accumulation in response to IL-1beta compared to Caco-2 cells. Selective activation of NF-kappaB pathway by adenoviral delivery of NF-kappaB-inducing kinase (Ad5NIK) or IKKbeta (Ad5IKKbeta) strongly activated IKK activity (>20 fold) in HT-29 cells with concomitant endogenous IkappaBalpha serine 32 phosphorylation and total IkappaBalpha degradation. In addition, NF-kappaB DNA binding activity and IL-8 secretion is higher in Ad5NIK-infected than in IL-1beta-stimulated HT-29 cells. These data show that altered NF-kappaB signaling is associated with impaired stimulation of an upstream IKK activator.     

IkappaB kinase beta phosphorylates the K63 deubiquitinase A20 to cause feedback inhibition of the NF-kappaB pathway

Misregulation of NF-kappaB signaling leads to infectious, inflammatory, or autoimmune disorders. IkappaB kinase beta (IKKbeta) is an essential activator of NF-kappaB and is known to phosphorylate the NF-kappaB inhibitor, IkappaBalpha, allowing it to undergo ubiquitin-mediated proteasomal degradation. However, beyond IkappaBalpha, few additional IKKbeta substrates have been identified. Here we utilize a peptide library and bioinformatic approach to predict likely substrates of IKKbeta. This approach predicted Ser381 of the K63 deubiquitinase A20 as a likely site of IKKbeta phosphorylation. While A20 is a known negative regulator of innate immune signaling pathways, the mechanisms regulating the activity of A20 are poorly understood. We show that IKKbeta phosphorylates A20 in vitro and in vivo at serine 381, and we further show that this phosphorylation event increases the ability of A20 to inhibit the NF-kappaB signaling pathway. Phosphorylation of A20 by IKKbeta thus represents part of a novel feedback loop that regulates the duration of NF-kappaB signaling following activation of innate immune signaling pathways.     

TWEAK induces NF-kappaB2 p100 processing and long lasting NF-kappaB activation

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily that has been shown to induce angiogenesis, apoptosis in tumor cells, and NF-kappaB activation through binding to its receptor, fibroblast growth factor-inducible 14. We have identified TWEAK as an inducer of constitutive NF-kappaB activation by expression cloning, and we report here sequential regulation by TWEAK of two separate signaling cascades for NF-kappaB activation, the NF-kappaB essential modulator-dependent and -independent signaling pathways. Upon TWEAK stimulation, IkappaBalpha is rapidly phosphorylated, generating NF-kappaB DNA-binding complexes containing p50 and RelA in a manner dependent on the canonical IkappaB kinase complex. Unlike TNF-alpha, TWEAK stimulation results in prolonged NF-kappaB activation with a transition of the DNA-binding NF-kappaB components from RelA- to RelB-containing complexes by 8 h, and the latter remained active in binding at least until 24 h post-stimulation. This long lasting activation is accompanied by the proteasome-mediated processing of NF-kappaB2/p100, which does not depend on the NF-kappaB essential modulator but requires IkappaB kinase 1 and functional NF-kappaB-inducing kinase activity. Finally, we show that fibroblast growth factor-inducible 14 with a mutation at its TNF receptor-associated factor (TRAF)-binding site cannot activate NF-kappaB and that TWEAK fails to induce the p100 processing and IkappaBalpha phosphorylation in cells deficient for TRAF2 and TRAF5. Our results thus identify TWEAK as a novel physiological regulator of the non-canonical pathway for NF-kappaB activation.     

Triterpenoid CDDO-Me blocks the NF-kappaB pathway by direct inhibition of IKKbeta on Cys-179

The novel oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) and the C-28 methyl ester (CDDO-Me) induce apoptosis of human tumor cells by disruption of redox balance and are currently in clinical trials. The present studies show that CDDO and CDDO-Me block tumor necrosis factoralpha-induced targeting of NF-kappaB p65 to the nucleus. CDDO-Me also blocked tumor necrosis factor alpha-induced phosphorylation of IkappaBalpha. In concert with these results, we found that CDDO-Me inhibits IkappaBalpha kinasebeta (IKKbeta) activity in cells. In support of a direct mechanism, CDDO-Me inhibited recombinant IKKbeta activity in vitro. The results also demonstrate that (i) CDDO and CDDO-Me form adducts with IKKbeta, but not IKKbeta with mutation of Cys-179 to Ala, and (ii) CDDO-Me inhibits IKKbeta by a mechanism dependent on oxidation of Cys-179. These findings indicate that CDDO and CDDO-Me directly block IKKbeta activity and thereby the NF-kappaB pathway by interacting with Cys-179 in the IKKbeta activation loop.     

MUC1 oncoprotein activates the IkappaB kinase beta complex and constitutive NF-kappaB signalling

Nuclear factor-kappaB (NF-kappaB) is constitutively activated in diverse human malignancies by mechanisms that are not understood. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and, similarly to NF-kappaB, blocks apoptosis and induces transformation. This study demonstrates that overexpression of MUC1 in human carcinoma cells is associated with constitutive activation of NF-kappaB p65. We show that MUC1 interacts with the high-molecular-weight IkappaB kinase (IKK) complex in vivo and that the MUC1 cytoplasmic domain binds directly to IKKbeta and IKKgamma. Interaction of MUC1 with both IKKbeta and IKKgamma is necessary for IKKbeta activation, resulting in phosphorylation and degradation of IkappaBalpha. Studies in non-malignant epithelial cells show that MUC1 is recruited to the TNF-R1 complex and interacts with IKKbeta-IKKgamma in response to TNFalpha stimulation. TNFalpha-induced recruitment of MUC1 is dependent on TRADD and TRAF2, but not the death-domain kinase RIP1. In addition, MUC1-mediated activation of IKKbeta is dependent on TAK1 and TAB2. These findings indicate that MUC1 is important for physiological activation of IKKbeta and that overexpression of MUC1, as found in human cancers, confers sustained induction of the IKKbeta-NF-kappaB p65 pathway.     

Role of NF-kappaB signaling pathway in increased tumor necrosis factor-alpha-induced apoptosis of lymphocytes in aged humans

In human aging, lymphocytes display increased sensitivity to tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. TNF-alpha induces both survival and apoptotic signals. The survival signal is mediated by the activation of NF-kappaB. Although a role of certain proapoptotic molecules in aging has been reported, a role of altered NF-kappaB signaling pathway has not been explored in detail. In this study, we have compared TNF-alpha-induced activation of NF-kappaB, phosphorylation of IkappaBalpha, and the expression of IKKbeta between lymphocytes from young and aged humans. Furthermore, we have explored a role of IKKbeta in increased susceptibility of lymphocytes from aged humans to TNF-alpha-induced apoptosis. Lymphocytes from aged humans displayed decreased activation of NF-kappaB, reduced phosphorylation of IkappaBalpha, and decreased expression of IKKbeta. In addition, overexpression of IKKbeta in lymphocytes from aged humans normalized TNF-alpha-induced apoptosis to the level of young subjects. These data suggest a deficiency of NF-kappaB signaling pathway and a role of IKKbeta, at least in part, for increased sensitivity of lymphocytes from aged humans to TNF-alpha-induced apoptosis.     

IKKgamma inhibits activation of NF-kappaB by NIK

IKKgamma is a component of the IKK complex, which regulates NF-kappaB activity. To investigate the role of IKKgamma, we expressed wild type IKKgamma containing 412 amino acids, and deletion mutants containing residues 1-312 and 101-412, using murine IKKgamma cDNA. In a co-transfection assay with a CAT reporter plasmid, NIK activated NF-kappaB-dependent gene expression approximately two fold and this expression was inhibited by co-transfection of a wild type IKKgamma expression plasmid. In binding assays IKKgamma inhibited the association of IkappaBalpha with IKKbeta and the subsequent phosphorylation of IkappaBalpha that is activated by NIK. Inhibition by IKKgamma also occurred in an assay with a dominant negative mutant of NIK but not with a C-terminal deletion mutant of IKKgamma, indicating that the C-terminal 100 amino acids of IKKgamma are important for negative regulation of NF-kappaB activation. In addition, the interaction of IKKbeta with IKKgamma was inhibited by co-transfection with a NIK expression plasmid. Our results suggest that overexpression of IKKgamma inhibits activation of NF-kappaB by NIK by competing with NIK for interaction with IKKbeta.     

TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.     

Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB

Apaf-1 and Nod1 are members of a protein family, each of which contains a caspase recruitment domain (CARD) linked to a nucleotide-binding domain, which regulate apoptosis and/or NF-kappaB activation. Nod2, a third member of the family, was identified. Nod2 is composed of two N-terminal CARDs, a nucleotide-binding domain, and multiple C-terminal leucine-rich repeats. Although Nod1 and Apaf-1 were broadly expressed in tissues, the expression of Nod2 was highly restricted to monocytes. Nod2 induced nuclear factor kappaB (NF-kappaB) activation, which required IKKgamma and was inhibited by dominant negative mutants of IkappaBalpha, IKKalpha, IKKbeta, and IKKgamma. Nod2 interacted with the serine-threonine kinase RICK via a homophilic CARD-CARD interaction. Furthermore, NF-kappaB activity induced by Nod2 correlated with its ability to interact with RICK and was specifically inhibited by a truncated mutant form of RICK containing its CARD. The identification of Nod2 defines a subfamily of Apaf-1-like proteins that function through RICK to activate a NF-kappaB signaling pathway.     

Inhibition of interleukin-1beta -induced NF-kappa B activation by calcium/calmodulin-dependent protein kinase kinase occurs through Akt activation associated with interleukin-1 receptor-associated kinase phosphorylation and uncoupling of MyD88

Calcium/calmodulin-dependent protein kinase kinase (CaMKK) and Akt are two multifunctional kinases involved in many cellular responses. Although Akt and Ca(2+) signals have been implicated in NF-kappaB activation in response to certain stimuli, these results are still controversial, and the mechanism(s) involved remains unknown. In this study, we show the roles that CaMKK and Akt play in regulating interleukin-1beta (IL-1beta)-induced NF-kappaB signaling. In human embryonic kidney 293 cells, IL-1beta induces IkappaB kinase beta (IKKbeta) activation, IkappaBalpha degradation, NF-kappaB transactivation, and weak Akt activation. A CaMKK inhibitor (KN-93) and phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002) do not inhibit IL-1beta-induced NF-kappaB activation. However, IL-1beta-induced NF-kappaB activity is attenuated by increased intracellular calcium in response to ionomycin, UTP, or thapsigargin or by overexpression of CaMKKc and/or Akt. Ionomycin and CaMKKc overexpression increases Akt phosphorylation on Thr(308) and enzyme activity. Under these conditions or upon overexpression of wild type Akt, IL-1beta-induced IKKbeta activity is diminished. Furthermore, a dominant negative mutant of Akt abolishes IKKbeta inhibition by CaMKKc and ionomycin, suggesting that Akt acts as a mediator of CaMKK signaling to inhibit IL-1beta-induced IKK activity at an upstream target site. We have also identified a novel interaction between CaMKK-stimulated Akt and interleukin-1 receptor-associated kinase 1 (IRAK1), which plays a key role in IL-1beta-induced NF-kappaB activation. CaMKKc and Akt overexpression decreases IRAK1-mediated NF-kappaB activity and its association with MyD88 in response to IL-1beta stimulation. Furthermore, CaMKKc and Akt overexpression increases IRAK1 phosphorylation at Thr(100), and point mutation of this site abrogates the inhibitory effect of Akt on IRAK1-mediated NF-kappaB activation. Taken together, these results indicate a novel regulatory mechanism for IL-1beta signaling and suggest that CaMKK-dependent Akt activation inhibits IL-1beta-induced NF-kappaB activation through interference with the coupling of IRAK1 to MyD88.