Vitamin D Receptor Inhibits Nuclear Factor κB Activation by Interacting with IκB Kinase β Protein

1,25-Dihydroxyvitamin D (1,25(OH)₂D₃) is known to suppress NF-κB activity, but the underlying mechanism remains poorly understood. Here we show that the vitamin D receptor (VDR) physically interacts with IκB kinase β (IKKβ) to block NF-κB activation. 1,25(OH)₂D₃ rapidly attenuates TNFα-induced p65 nuclear translocation and NF-κB activity in a VDR-dependent manner. VDR overexpression inhibits IKKβ-induced NF-κB activity. GST pull-down assays and coimmunoprecipitation experiments demonstrated that VDR physically interacts with IKKβ and that this interaction is enhanced by 1,25(OH)₂D₃. Protein mapping reveals that VDR-IKKβ interaction occurs between the C-terminal portions of the VDR and IKKβ proteins. Reconstitution of VDR^−/− cells with the VDR C terminus restores the ability to block TNFα-induced NF-κB activation and IL-6 up-regulation. VDR-IKKβ interaction disrupts the formation of the IKK complex and, thus, abrogates IKKβ phosphorylation at Ser-177 and abolishes IKK activity to phosphorylate IκBα. Consequently, stabilization of IκBα arrests p65/p50 nuclear translocation. Together, these data define a novel mechanism whereby 1,25(OH)₂D₃-VDR inhibits NF-κB activation.     

Hyperinvasive Meningococci Induce Intra-nuclear Cleavage of the NF-κB Protein p65/RelA by Meningococcal IgA Protease

Differential modulation of NF-κB during meningococcal infection is critical in innate immune response to meningococcal disease. Non-invasive isolates of Neisseria meningitidis provoke a sustained NF-κB activation in epithelial cells. However, the hyperinvasive isolates of the ST-11 clonal complex (ST-11) only induce an early NF-κB activation followed by a sustained activation of JNK and apoptosis. We show that this temporal activation of NF-κB was caused by specific cleavage at the C-terminal region of NF-κB p65/RelA component within the nucleus of infected cells. This cleavage was mediated by the secreted 150 kDa meningococcal ST-11 IgA protease carrying nuclear localisation signals (NLS) in its α-peptide moiety that allowed efficient intra-nuclear transport. In a collection of non-ST-11 healthy carriage isolates lacking NLS in the α-peptide, secreted IgA protease was devoid of intra-nuclear transport. This part of iga polymorphism allows non-invasive isolates lacking NLS, unlike hyperinvasive ST-11 isolates of N. meningitides habouring NLS in their α-peptide, to be carried asymptomatically in the human nasopharynx through selective eradication of their ability to induce apoptosis in infected epithelial cells.     

Ubiquitin-specific Peptidase 21 Inhibits Tumor Necrosis Factor ��-induced Nuclear Factor ��B Activation via Binding to and Deubiquitinating Receptor-interacting Protein 1

Ubiquitination and deubiquitination of receptor-interacting protein 1 (RIP1) play an important role in the positive and negative regulation of the tumor necrosis factor α (TNFα)-induced nuclear factor κB (NF-κB) activation. Using a combination of functional genomic and proteomic approaches, we have identified ubiquitin-specific peptidase 21 (USP21) as a deubiquitinase for RIP1. USP21 is constitutively associated with RIP1 and deubiquitinates RIP1 in vitro and in vivo. Notably, knockdown of USP21 in HeLa cells enhances TNFα-induced RIP1 ubiquitination, IκB kinase β (IKKβ), and NF-κB phosphorylation, inhibitor of NF-κB α (IκBα) phosphorylation and ubiquitination, as well as NF-κB-dependent gene expression. Therefore, our results demonstrate that USP21 plays an important role in the down-regulation of TNFα-induced NF-κB activation through deubiquitinating RIP1.     

Cyclophilin A (CypA) Interacts with NF-κB Subunit,p65/RelA,and Contributes to NF-κB Activation Signaling

Background

Peptidyl-prolyl isomerase cyclophilin A (CypA) plays important roles in signaling, protein translocation, inflammation, and cancer formation. However, little is known about the mechanisms by which CypA exerts its effects. C57BL/6 Ppia (encoding CypA)-deficient embryonic fibroblasts show reduced activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), the p65/RelA subunit, suggesting that CypA may mediate modulation of NF-κB activity to exert its biological effects.

Methodology

Western blotting and qRT-PCR analyses were used to evaluate the association of CypA deficiency with reduced activation of NF-κB/p65 at the protein level. GST pull-down and co-immunoprecipitation were used to examine interactions between CypA and p65/RelA. Truncation mutants and site-directed mutagenesis were used to determine the sequences of p65/RelA required for interactions with CypA. Enhancement of p65/RelA nuclear translocation by CypA was assessed by co-transfection and immunofluorescent imaging. Treatment of cells with cycloheximide that were harvested at various time points for Western blot analyses was carried out to evaluate p65/RelA protein stability. The functional activity of NF-κB was assessed by electrophoretic mobility-shift assays (EMSA), luciferase assays, and changes in expression levels of target genes.

Results

GST pull-down assays in vitro and co-immunoprecipitation analyses in vivo provided evidence for protein-protein interactions. These interactions were further supported by identification of a CypA-binding consensus-like sequence within NF-κB subunit p65 at the N-terminal 170–176 amino acid residues. Significantly, CypA provided stability for NF-κB p65 and promoted NF-κB p65 nuclear translocation, resulting in increased nuclear accumulation and enhanced NF-κB activity.

Conclusions

Our findings revealed important mechanisms that regulate NF-κB activation, and offer new insights into the role of CypA in aberrant activation of NF-κB-mediated signaling for altered expression of its target genes, resulting in pathological effects in various diseases.     

Indacaterol Inhibits Tumor Cell Invasiveness and MMP-9 Expression by Suppressing IKK/NF-κB Activation

The β₂ adrenergic receptor (ADRB2) is a G protein-coupled transmembrane receptor expressed in the human respiratory tract and widely recognized as a pharmacological target for treatments of asthma and chronic obstructive pulmonary disorder (COPD). Although a number of ADRB2 agonists have been developed for use in asthma therapy, indacaterol is the only ultra-long-acting inhaled β₂-agonist (LABA) approved by the FDA for relieving the symptoms in COPD patients.The precise molecular mechanism underlying the pharmacological effect of indacaterol, however, remains unclear. Here, we show that β-arrestin-2 mediates the internalization of ADRB2 following indacaterol treatment. Moreover, we demonstrate that indacaterol significantly inhibits tumor necrosis factor-α (TNF-α)-induced NF-κB activity by reducing levels of both phosphorylated-IKK and -IκBα, thereby decreasing NF-κB nuclear translocation and the expression of MMP-9, an NF-κB target gene. Subsequently, we show that indacaterol significantly inhibits TNF-α/NF-κB-induced cell invasiveness and migration in a human cancer cell line. In conclusion, we propose that indacaterol may inhibit NF-κB activity in a β-arrestin2-dependent manner, preventing further lung damage and improving lung function in COPD patients.     

Type I Interferon and NF-κB Activation Elicited by Herpes Simplex Virus gH/gL via αvβ3 Integrin in Epithelial and Neuronal Cell Lines

αvβ3 integrin represents a novel sensing system which detects herpes simplex virus (HSV) and bacterial constituents. In cooperation with Toll-like receptor 2 (TLR2), it elicits an innate response that leads to activation of type I interferon (IFN), NF-κB, and a specific set of cytokines. We report that this defensive branch is functional in cells which represent experimental models of epithelial, including keratinocytic, and neuronal cells. These are the major targets of HSV in vivo. HSV entered the three cell lines via distinct routes. Hence, the defensive response was independent of the route of virus entry. Soluble gH/gL sufficed to elicit type I IFN and NF-κB activation and represents the viral pathogen-associated molecular pattern (PAMP) of this defense system.     

NleC, a type III secretion protease, compromises NF-κB activation by targeting p65/RelA

The NF-κB signaling pathway is central to the innate and adaptive immune responses. Upon their detection of pathogen-associated molecular patterns, Toll-like receptors on the cell surface initiate signal transduction and activate the NF-κB pathway, leading to the production of a wide array of inflammatory cytokines, in attempt to eradicate the invaders. As a countermeasure, pathogens have evolved ways to subvert and manipulate this system to their advantage. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely related bacteria responsible for major food-borne diseases worldwide. Via a needle-like protein complex called the type three secretion system (T3SS), these pathogens deliver virulence factors directly to host cells and modify cellular functions, including by suppressing the inflammatory response. Using gain- and loss-of-function screenings, we identified two bacterial effectors, NleC and NleE, that down-regulate the NF-κB signal upon being injected into a host cell via the T3SS. A recent report showed that NleE inhibits NF-κB activation, although an NleE-deficient pathogen was still immune-suppressive, indicating that other anti-inflammatory effectors are involved. In agreement, our present results showed that NleC was also required to inhibit inflammation. We found that NleC is a zinc protease that disrupts NF-κB activation by the direct cleavage of NF-κB's p65 subunit in the cytoplasm, thereby decreasing the available p65 and reducing the total nuclear entry of active p65. More importantly, we showed that a mutant EPEC/EHEC lacking both NleC and NleE (ΔnleC ΔnleE) caused greater inflammatory response than bacteria carrying ΔnleC or ΔnleE alone. This effect was similar to that of a T3SS-defective mutant. In conclusion, we found that NleC is an anti-inflammatory bacterial zinc protease, and that the cooperative function of NleE and NleC disrupts the NF-κB pathway and accounts for most of the immune suppression caused by EHEC/EPEC.