Inhibition of I��B Kinase by Vaccinia Virus Virulence Factor B14

The IκB kinase (IKK) complex is a key regulator of signal transduction pathways leading to the induction of NF-κB-dependent gene expression and production of pro-inflammatory cytokines. It therefore represents a major target for the development of anti-inflammatory therapeutic drugs and may be targeted by pathogens seeking to diminish the host response to infection. Previously, the vaccinia virus (VACV) strain Western Reserve B14 protein was characterised as an intracellular virulence factor that alters the inflammatory response to infection by an unknown mechanism. Here we demonstrate that ectopic expression of B14 inhibited NF-κB activation in response to TNFα, IL-1β, poly(I:C), and PMA. In cells infected with VACV lacking gene B14R (vΔB14) there was a higher level of phosphorylated IκBα but a similar level of IκBα compared to cells infected with control viruses expressing B14, suggesting B14 affects IKK activity. Direct evidence for this was obtained by showing that B14 co-purified and co-precipitated with the endogenous IKK complex from human and mouse cells and inhibited IKK complex enzymatic activity. Notably, the interaction between B14 and the IKK complex required IKKβ but not IKKα, suggesting the interaction occurs via IKKβ. B14 inhibited NF-κB activation induced by overexpression of IKKα, IKKβ, and a constitutively active mutant of IKKα, S176/180E, but did not inhibit a comparable mutant of IKKβ, S177/181E. This suggested that phosphorylation of these serine residues in the activation loop of IKKβ is targeted by B14, and this was confirmed using Ab specific for phospho-IKKβ.     

Microbial-induced immunomodulation by targeting the NF-κB system

Virtually all eukaryotes have developed defense mechanisms to efficiently counter potential threats from prokaryotic microorganisms; an example is the conserved nuclear factor-kappaB (NF-κB) signaling system. However, bacterial pathogens and commensals have in turn evolved highly effective counter mechanisms to modulate this immune regulatory circuit. Modifications in ubiquitin, ubiquitin-like (Ubl) proteins such as neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) and other post-translational modifications (PTMs) in the NF-κB system represent attractive targets for microbial manipulation. In this review, we describe recent advances in understanding the different strategies that bacteria have evolved to interfere with PTMs in NF-κB signal transmission.     

Decoy receptor 3 suppresses TLR2-mediated B cell activation by targeting NF-κB

Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam(3)CSK(4)-, and LPS-mediated B cell proliferation but did not affect cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-βR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-κB p65 activation and NF-κB-driven luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-κB.     

Regulation of ΔNp63α by NFκB

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be downregulated in response to several DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents. We show that ΔNp63α interacts with NFκB in presence of cisplatin. We find that NFκB promotes ubiquitin-mediated proteasomal degradation of ΔNp63α. Chemotherapy-induced stimulation of NFκB leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of NFκB with siRNA-mediated silencing NFκB expression attenuates chemotherapy induced degradation of ΔNp63α. These data demonstrate that NFκB plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of NFκB may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage.Key words: ΔNp63α, NFκB, ubiquitination, cisplatin, head and neck cancer     

Palmitate-induced PTP1B expression is mediated by ceramide-JNK and nuclear factor κB (NF-κB) activation

Palmitate induces PTP1B expression in skeletal muscle cells. The purpose of this study was to investigate the mechanisms responsible for palmitate-induced PTP1B expression in mouse skeletal muscle cells. Three truncated fragments of PTP1B promoter were cloned into PGL3-basic vector and the promoter activity of PTP1B was assessed in C2C12 cells exposed to palmitate either in the presence or in the absence of several inhibitors to study the biochemical pathways involved. EMSA was performed to examine binding of NF-κB to NF-κB consensus sequence and PTP1B oligonucelotides in the cells treated with palmitate. Lentiviral PTP1B-shRNA was used to knockdown PTP1B in myotubes. The phosphorylation and protein levels of IRS-1 and Akt were detected by western blot. 0.5mM palmitate induced PTP1B promoter activity in fragment -1715/+59 by 50% (p<0.01). Palmitate increased NF-κB binding to both NF-κB consensus sequence and one NF-κB sequence (-920 to -935) in PTP1B promoter. Incubation of C2C12 cells with different concentrations of C2-ceramide enhanced PTP1B promoter activity dose-dependently. Inhibitors of de novo ceramide synthesis prevented palmitate-induced PTP1B promoter activity in myotubes. In addition, inhibitor of JNK pathway prevented ceramide-induced PTP1B promoter activity in myotubes. Knockdown of PTP1B also prevented ceramide-reduced IRS-1 and Akt phosphorylations in the myotubes. Exposure of the cells to PMA and calphostin C, an inhibitor of PKC, did not affect the activity of PTP1B promoter. Our data provide the evidence that the mechanism by which palmitate increased the expression of PTP1B seems to be through a mechanism involving the activation of ceramide-JNK and NF-κB pathways.     

Bmi-1 Promotes Glioma Angiogenesis by Activating NF-κB Signaling

Angiogenesis in glioma is associated with the poor prognosis of the disease and closely correlates with the highly invasive phenotype of glioma cells, which represents the most challenging impediment against the currently glioma treatments. Bmi-1, an onco-protein, has been implicated in the progression of various human cancers, including gliomas, whereas its role in glioma angiogenesis remains unclear. Our current study examined the effects of Bmi-1 on glioma angiogenesis in vitro as well as in vivo. We found that overexpression of Bmi-1 enhanced, whereas knockdown of Bmi-1 diminished, the capability of glioma cells to induce tubule formation and migration of endothelial cells and neovascularization in chicken chorioallantoic membrane. In vivo, Bmi-1 overexpression and knockdown, respectively, promoted and inhibited angiogenesis in orthotopically transplanted human gliomas. Furthermore, NF-κB activity and VEGF-C expression was induced by Bmi-1 overexpression, whereas Bmi-1 knockdown attenuated NF-κB signaling and decreased VEGF-C expression. Additionally suppression of NF-κB activity using a specific chemical inhibitor abrogated the NF-κB activation and the pro-angiogenic activities of glioma cells. Together, our data suggest that Bmi-1 plays an important role in glioma angiogenesis and therefore could represent a potential target for anti-angiogenic therapy against the disease.     

Activation of NF-κB by alloferon through down-regulation of antioxidant proteins and IκBα

Alloferon is a 13-amino acid peptide isolated from the bacteria-challenged larvae of the blow fly Calliphora vicina. The pharmaceutical value of the peptide has been well demonstrated by its capacity to stimulate NK cytotoxic activity and interferon (IFN) synthesis in animal and human models, as well as to enhance antiviral and antitumor activities in mice. Antiviral and the immunomodulatory effectiveness of alloferon have also been supported clinically proved in patients suffering with herpes simplex virus (HSV) and human papilloma virus (HPV) infections. To elucidate molecular response to alloferon treatment, we initially screened a model cell line in which alloferon enhanced IFN synthesis upon viral infection. Among the cell lines tested, Namalva was chosen for further proteomic analysis. Fluorescence difference gel electrophoresis (DIGE) revealed that the levels of a series of antioxidant proteins decreased after alloferon treatment, while at least three glycolytic enzymes and four heat-shock proteins were increased in their expression levels. Based on the result of our proteomic analysis, we speculated that alloferon may activate the NF-kappaB signaling pathway. IkappaB kinase (IKK) assay, Western blot analysis on IkappaBalpha and its phosphorylated form at Ser 32, and an NF-kappaB reporter assay verified our proteomics-driven hypothesis. Thus, our results suggest that alloferon potentiates immune cells by activating the NF-kappaB signaling pathway through regulation of redox potential. Since NF-kappaB activation is involved in IFN synthesis, our results provide further clues as to how the alloferon peptide may stimulate IFN synthesis.