p97/VCP promotes Cullin‐RING‐ubiquitin‐ligase/proteasome‐dependent degradation of IκBα and the preceding liberation of RelA from ubiquitinated IκBα

Cullin‐RING‐ubiquitin‐ligase (CRL)‐dependent ubiquitination of the nuclear factor kappa B (NF‐κB) inhibitor IκBα and its subsequent degradation by the proteasome usually precede NF‐κB/RelA nuclear activity. Through removal of the CRL‐activating modification of their cullin subunit with the ubiquitin (Ub)‐like modifier NEDD8, the COP9 signalosome (CSN) opposes CRL Ub‐ligase activity. While RelA phosphorylation was observed to mediate NF‐κB activation independent of Ub‐proteasome‐pathway (UPP)‐dependent turnover of IκBα in some studies, a strict requirement of the p97/VCP ATPase for both, IκBα degradation and NF‐κB activation, was reported in others. In this study, we thus aimed to reconcile the mechanism for tumour necrosis factor (TNF)‐induced NF‐κB activation. We found that inducible phosphorylation of RelA is accomplished in an IKK‐complex‐dependent manner within the NF‐κB/RelA‐IκBα‐complex contemporaneous with the phosphorylation of IκBα, and that RelA phosphorylation is not sufficient to dissociate NF‐κB/RelA from IκBα. Subsequent to CRL‐dependent IκBα ubiquitination functional p97/VCP is essentially required for efficient liberation of (phosphorylated) RelA from IκBα, preceding p97/VCP‐promoted timely and efficient degradation of IκBα as well as simultaneous NF‐κB/RelA nuclear translocation. Collectively, our data add new facets to the knowledge about maintenance of IκBα and RelA expression, likely depending on p97/VCP‐supported scheduled basal NF‐κB activity, and the mechanism of TNF‐induced NF‐κB activation.     

Protein–protein interactions involving IKKγ (NEMO) that promote the activation of NF‐κB

Inhibitor of κB kinase (IKK) gamma (IKKγ), also referred to as nuclear factor κB (NF‐κB) essential modulator (NEMO), is an important regulatory component of the IKK complex. The IKK complex is a signalosome that catalyzes the inducible phosphorylation of IκB proteins, which is a key step that leads to the activation of NF‐κB. The exact functions of IKKγ (NEMO) as part of the IKK complex have not yet been fully elucidated. This mini‐review covers 16 proteins that have been reported to bind to IKKγ and lead to the enhancement of the activities of the IKK complex, thus resulting in NF‐κB activation. The major mechanisms by which these interactions are mediated involve the recognition of ubiquitinated upstream signaling components by IKKγ or the modification of IKKγ itself by ubiquitination. Additional mechanisms include the sumoylation or phosphorylation of IKKγ and the modification of the tertiary or quaternary structure of IKKγ. J. Cell. Physiol. 223:558–561, 2010. © 2010 Wiley‐Liss, Inc.     

Influenza A virus‐encoded NS1 virulence factor protein inhibits innate immune response by targeting IKK

The IKK/NF‐κB pathway is an essential signalling process initiated by the cell as a defence against viral infection like influenza virus. This pathway is therefore a prime target for viruses attempting to counteract the host response to infection. Here, we report that the influenza A virus NS1 protein specifically inhibits IKK‐mediated NF‐κB activation and production of the NF‐κB induced antiviral genes by physically interacting with IKK through the C‐terminal effector domain. The interaction between NS1 and IKKα/IKKβ affects their phosphorylation function in both the cytoplasm and nucleus. In the cytoplasm, NS1 not only blocks IKKβ‐mediated phosphorylation and degradation of IκBα in the classical pathway but also suppresses IKKα‐mediated processing of p100 to p52 in the alternative pathway, which leads to the inhibition of nuclear translocation of NF‐κB and the subsequent expression of downstream NF‐κB target genes. In the nucleus, NS1 impairs IKK‐mediated phosphorylation of histone H3 Ser 10 that is critical to induce rapid expression of NF‐κB target genes. These results reveal a new mechanism by which influenza A virus NS1 protein counteracts host NF‐κB‐mediated antiviral response through the disruption of IKK function. In this way, NS1 diminishes antiviral responses to infection and, in turn, enhances viral pathogenesis.     

COP9 signalosome controls the Carma1–Bcl10–Malt1 complex upon T‐cell stimulation

The Carma1–Bcl10–Malt1 (CBM) complex connects T‐cell receptor (TCR) signalling to the canonical IκB kinase (IKK)/NF (nuclear factor)‐κB pathway. Earlier studies have indicated that the COP9 signalosome (CSN), a pleiotropic regulator of the ubiquitin/26S proteasome system, controls antigen responses in T cells. The CSN is required for the degradation of the NF‐κB inhibitor IκBα, but other molecular targets involved in T‐cell signalling remained elusive. Here, we identify the CSN subunit 5 (CSN5) as a new interactor of Malt1 and Carma1. T‐cell activation triggers the recruitment of the CSN to the CBM complex, and CSN downregulation impairs TCR‐induced IKK activation. Furthermore, the CSN is required for maintaining the stability of Bcl10 in response to T‐cell activation. Taken together, our data provide evidence for a functional link between the evolutionarily conserved CSN and the adaptive immunoregulatory CBM complex in T cells.     

LIGHT/IFN‐γ triggers β cells apoptosis via NF‐κB/Bcl2‐dependent mitochondrial pathway

LIGHT recruits and activates naive T cells in the islets at the onset of diabetes. IFN‐γ secreted by activated T lymphocytes is involved in beta cell apoptosis. However, whether LIGHT sensitizes IFNγ‐induced beta cells destruction remains unclear. In this study, we used the murine beta cell line MIN6 and primary islet cells as models for investigating the underlying cellular mechanisms involved in LIGHT/IFNγ – induced pancreatic beta cell destruction. LIGHT and IFN‐γ synergistically reduced MIN6 and primary islet cells viability; decreased cell viability was due to apoptosis, as demonstrated by a significant increase in Annexin V⁺ cell percentage, detected by flow cytometry. In addition to marked increases in cytochrome c release and NF‐κB activation, the combination of LIGHT and IFN‐γ caused an obvious decrease in expression of the anti‐apoptotic proteins Bcl‐2 and Bcl‐xL, but an increase in expression of the pro‐apoptotic proteins Bak and Bax in MIN6 cells. Accordingly, LIGHT deficiency led to a decrease in NF‐κB activation and Bak expression, and peri‐insulitis in non‐obese diabetes mice. Inhibition of NF‐κB activation with the specific NF‐κB inhibitor, PDTC (pyrrolidine dithiocarbamate), reversed Bcl‐xL down‐regulation and Bax up‐regulation, and led to a significant increase in LIGHT‐ and IFN‐γ‐treated cell viability. Moreover, cleaved caspase‐9, ‐3, and PARP (poly (ADP‐ribose) polymerase) were observed after LIGHT and IFN‐γ treatment. Pretreatment with caspase inhibitors remarkably attenuated LIGHT‐ and IFNγ‐induced cell apoptosis. Taken together, our results indicate that LIGHT signalling pathway combined with IFN‐γ induces beta cells apoptosis via an NF‐κB/Bcl2‐dependent mitochondrial pathway.     

Integrity of IKK/NF‐κB Shields Thymic Stroma That Suppresses Susceptibility to Autoimmunity,Fungal Infection,and Carcinogenesis

A pathogenic connection between autoreactive T cells, fungal infection, and carcinogenesis has been demonstrated in studies of human autoimmune polyendocrinopathy‐candidiasis‐ectodermal dystrophy (APECED) as well as in a mouse model in which kinase‐dead Ikkα knock‐in mice develop impaired central tolerance, autoreactive T cell–mediated autoimmunity, chronic fungal infection, and esophageal squamous cell carcinoma, which recapitulates APECED. IκB kinase α (IKKα) is one subunit of the IKK complex required for NF‐κB activation. IKK/NF‐κB is essential for central tolerance establishment by regulating the development of medullary thymic epithelial cells (mTECs) that facilitate the deletion of autoreactive T cells in the thymus. In this review, we extensively discuss the pathogenic roles of inborn errors in the IKK/NF‐κB loci in the phenotypically related diseases APECED, immune deficiency syndrome, and severe combined immunodeficiency; differentiate how IKK/NF‐κB components, through mTEC (stroma), T cells/leukocytes, or epithelial cells, contribute to the pathogenesis of infectious diseases, autoimmunity, and cancer; and highlight the medical significance of IKK/NF‐κB in these diseases.     

LINC00667 promotes Wilms’ tumor metastasis and stemness by sponging miR‐200b/c/429 family to regulate IKK‐β

Wilms' tumor, also known as nephroblastoma, is a kind of pediatric renal cancer. Previous studies have indicated that microRNAs (miRNAs) regulate various cancers progression. However, whether miR‐200 family regulated Wilms' tumor progression remains to be elucidated. In our study, miR‐200b/c/429 expression was downregulated in Wilms' tumor tissue samples from 25 patients. And data from three independent analyses of quantitative real‐time polymerase chain reaction revealed that the expression of miR‐200b/c/429 was downregulated in Wilms' tumor cell lines. Functionally, Cell counting kit‐8 assay revealed that cell viability was reduced by overexpressing miR‐200b/c/429. Transwell assay manifested that cell migration and invasion was hindered by miR‐200b/c/429 overexpression. Sphere‐forming and western blot assays demonstrated that miR‐200b/c/429 overexpression suppressed the sphere formation ability. Mechanically, nuclear factor‐κB (NF‐κB) pathway was confirmed to be associated with Wilms' tumor progression; miR‐200b/c/429 overexpression inactivated NF‐κB pathway as miR‐200b/c/429 was identified to target IκB kinase β (IKK‐β), an NF‐κB pathway‐related gene. Moreover, miR‐200b/c/429 was sponged by LINC00667 in Wilms' tumor cells. LINC00667 competitively bound with miR‐200b/c/429 to regulate IKK‐β expression and then activated NF‐κB pathway in Wilms' tumor. Subsequently, rescue assays illustrated that silencing of IKK‐β could reverse the effect of miR‐200b/c/429 inhibition on the progression of sh‐LINC00667‐transfected Wilms' tumor cells. In summary, LINC00667 promoted Wilms' tumor progression by sponging miR‐200b/c/429 family to regulate IKK‐β.     

Silencing of ATP2B1‐AS1 contributes to protection against myocardial infarction in mouse via blocking NFKBIA‐mediated NF‐κB signalling pathway

Myocardial infarction (MI) is an acute coronary syndrome that refers to tissue infarction of the myocardium. This study aimed to investigate the effect of long intergenic non‐protein‐coding RNA (lincRNA) ATPase plasma membrane Ca²⁺ transporting 1 antisense RNA 1 (ATP2B1‐AS1) against MI by targeting nuclear factor‐kappa‐B inhibitor alpha (NFKBIA) and mediating the nuclear factor‐kappa‐B (NF‐κB) signalling pathway. An MI mouse model was established and idenepsied by cardiac function evaluation. It was determined that ATP2B1‐AS1 was highly expressed, while NFKBIA was poorly expressed and NF‐κB signalling pathway was activated in MI mice. Cardiomyocytes were extracted from mice and introduced with a series of mouse ATP2B1‐AS1 vector, NFKBIA vector, siRNA‐mouse ATP2B1‐AS1 and siRNA‐NFKBIA. The expression of NF‐κBp50, NF‐κBp65 and IKKβ was determined to idenepsy whether ATP2B1‐AS1 and NFKBIA affect the NF‐κB signalling pathway, the results of which suggested that ATP2B1‐AS1 down‐regulated the expression of NFKBIA and activated the NF‐κB signalling pathway in MI mice. Based on the data from assessment of cell viability, cell cycle, apoptosis and levels of inflammatory cytokines, either silencing of mouse ATP2B1‐AS1 or overexpression of NFKBIA was suggested to result in reduced cardiomyocyte apoptosis and expression of inflammatory cytokines, as well as enhanced cardiomyocyte viability. Our study provided evidence that mouse ATP2B1‐AS1 silencing may have the potency to protect against MI in mice through inhibiting cardiomyocyte apoptosis and inflammation, highlighting a great promise as a novel therapeutic target for MI.     

Defective immune responses in mice lacking LUBAC‐mediated linear ubiquitination in B cells

The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF‐κB pathway, which is important for B‐cell development and function. Here, we describe a mouse model (B‐HOIP^Δlinear) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF‐κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B‐HOIP^Δlinear mice due to defective activation of the IKK complex; however, B‐cell receptor (BCR)‐mediated activation of the NF‐κB and ERK pathways was unaffected. B‐HOIP^Δlinear mice show impaired B1‐cell development and defective antibody responses to thymus‐dependent and thymus‐independent II antigens. Taken together, these data suggest that LUBAC‐mediated linear polyubiquitination is essential for B‐cell development and activation, possibly via canonical NF‐κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR.     

Apoptosis‐inducing effect of garcinol is mediated by NF‐κB signaling in breast cancer cells

Garcinol, obtained from Garcinia indica in tropical regions, is used for its numerous biological effects. Its anti‐cancer activity has been suggested but the mechanism of action has not been studied in‐detail, especially there is no report on its action against breast cancer cells. Here we tested our hypothesis that garcinol may act as an anti‐proliferative and apoptosis‐inducing agent against breast cancer cell lines. Using multiple techniques such as MTT, Histone‐DNA ELISA, Annexin V‐PI staining, Western blot for activated caspases and cleaved PARP, homogenous caspase‐3/7 fluorometric assay and EMSA, we investigated the mechanism of apoptosis‐inducing effect of garcinol in ER‐positive MCF‐7 and ER‐negative MDA‐MB‐231 cells. We found that garcinol exhibits dose‐dependent cancer cell‐specific growth inhibition in both the cell lines with a concomitant induction of apoptosis, and has no effect on non‐tumorigenic MCF‐10A cells. Our results suggested induction of caspase‐mediated apoptosis in highly metastatic MDA‐MB‐231 cells by garcinol. Down‐regulation of NF‐κB signaling pathway was observed to be the mechanism of apoptosis‐induction. Garcinol inhibited constitutive NF‐κB activity, which was consistent with down‐regulation of NF‐κB‐regulated genes. This is the first report on anti‐proliferative and apoptosis‐inducing action of garcinol against human breast cancer cells and the results suggest that this natural compound merits investigation as a potential chemo‐preventive/‐therapeutic agent, especially against breast cancer. J. Cell. Biochem. 109: 1134–1141, 2010. © 2010 Wiley‐Liss, Inc.     

Mechanistic study of saikosaponin‐d (Ssd) on suppression of murine T lymphocyte activation

Saikosaponin‐d (Ssd) is a triterpene saponin derived from the medicinal plant, Bupleurum falcatum L. (Umbelliferae). Previous findings showed that Ssd exhibits a variety of pharmacological and immunomodulatory activities including anti‐inflammatory, anti‐bacterial, anti‐viral and anti‐cancer effects. In the current study we have investigated the effects of Ssd on activated mouse T lymphocytes through the NF‐κB, NF‐AT and AP‐1 signaling pathways, cytokine secretion, and IL‐2 receptor expression. The results demonstrated that Ssd not only suppressed OKT3/CD28‐costimulated human T cell proliferation, it also inhibited PMA, PMA/Ionomycin and Con A‐induced mouse T cell activation in vitro. The inhibitory effect of Ssd on PMA‐induced T cell activation was associated with down‐regulation of NF‐κB signaling through suppression of IKK and Akt activities. In addition, Ssd suppressed both DNA binding activity and the nuclear translocation of NF‐AT and activator protein 1 (AP‐1) of the PMA/Ionomycin‐stimulated T cells. The cell surface markers like IL‐2 receptor (CD25) were also down‐regulated together with decreased production of pro‐inflammatory cytokines of IL‐6, TNF‐α and IFN‐γ. These results indicate that the NF‐κB, NF‐AT and AP‐1 (c‐Fos) signaling pathways are involved in the T cell inhibition evoked by Ssd, so it can be a potential candidate for further study in treating T cell‐mediated autoimmune conditions. J. Cell. Biochem. 107: 303–315, 2009. © 2009 Wiley‐Liss, Inc.     

Infection of epithelial cells with Chlamydia trachomatis inhibits TNF‐induced apoptosis at the level of receptor internalization while leaving non‐apoptotic TNF‐signalling intact

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of medical importance. C. trachomatis develops inside a membranous vacuole in the cytosol of epithelial cells but manipulates the host cell in numerous ways. One prominent effect of chlamydial infection is the inhibition of apoptosis in the host cell, but molecular aspects of this inhibition are unclear. Tumour necrosis factor (TNF) is a cytokine with important roles in immunity, which is produced by immune cells in chlamydial infection and which can have pro‐apoptotic and non‐apoptotic signalling activity. We here analysed the signalling through TNF in cells infected with C. trachomatis. The pro‐apoptotic signal of TNF involves the activation of caspase‐8 and is controlled by inhibitor of apoptosis proteins. We found that in C. trachomatis‐infected cells, TNF‐induced apoptosis was blocked upstream of caspase‐8 activation even when inhibitor of apoptosis proteins were inhibited or the inhibitor of caspase‐8 activation, cFLIP, was targeted by RNAi. However, when caspase‐8 was directly activated by experimental over‐expression of its upstream adapter Fas‐associated protein with death domain, C. trachomatis was unable to inhibit apoptosis. Non‐apoptotic TNF‐signalling, particularly the activation of NF‐κB, initiates at the plasma membrane, while the activation of caspase‐8 and pro‐apoptotic signalling occur subsequently to internalization of TNF receptor and the formation of a cytosolic signalling complex. In C. trachomatis‐infected cells, NF‐κB activation through TNF was unaffected, while the internalization of the TNF–TNF‐receptor complex was blocked, explaining the lack of caspase‐8 activation. These results identify a dichotomy of TNF signalling in C. trachomatis‐infected cells: Apoptosis is blocked at the internalization of the TNF receptor, but non‐apoptotic signalling through this receptor remains intact, permitting a response to this cytokine at sites of infection.