NIK is involved in constitutive activation of the alternative NF-κB pathway and proliferation of pancreatic cancer cells

Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-κB is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-κB activation. Here, we show that the alternative pathway is constitutively activated and NF-κB-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.     

Identification of TGF-β-activated kinase 1 as a possible novel target for renal cell carcinoma intervention

Renal cell carcinoma (RCC) is common renal malignancy within poor prognosis. TGF-β-activated kinase 1 (TAK1) plays vital roles in cell survival, apoptosis-resistance and carcinogenesis through regulating nuclear factor-κB (NF-κB) and other cancer-related pathways. Here we found that TAK1 inhibitors (LYTAK1, 5Z-7-oxozeanol (5Z) and NG-25) suppressed NF-κB activation and RCC cell (786-O and A489 lines) survival. TAK1 inhibitors induced apoptotic cytotoxicity against RCC cells, which was largely inhibited by the broad or specific caspase inhibitors. Further, shRNA-mediated partial depletion of TAK1 reduced 786-O cell viability whiling activating apoptosis. Significantly, TAK1 was over-expressed in human RCC tissues, and its level was correlated with phosphorylated NF-κB. Finally, kinase inhibition or genetic depletion of TAK1 enhanced the activity of vinblastine sulfate (VLB) in RCC cells. Together, these results suggest that TAK1 may be an important oncogene or an effective target for RCC intervention.     

The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF’s role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-κB and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-κB activity and phosphorylation of the inhibitor of kappa B (IκBα) increased in ischemic brains, but IRF3, inhibitor of κB kinase complex-ε (IKKε), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-κB activity or p-IκBα induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-κB signaling and brain injury after acute cerebral I/R.     

NF-κB targeting for overcoming tumor resistance and normal tissues toxicity

Radiotherapy and chemotherapy are two famous modalities in tumor-targeted therapy that lead to systemic and local toxicities for normal tissues. Moreover, several studies have confirmed that exposure of the tumor to radiation or chemotherapy drugs stimulate some signaling pathways in the tumor microenvironment (TME), leading to resistance of cancer cells to apoptosis, as well as promoting angiogenesis and tumor growth. Nuclear factor kappa B (NF-κB) plays a central role in the regulation of inflammatory responses in both normal tissues and tumors via the release of several cytokines, regulation of prostaglandins, reduction/oxidation (redox) reactions, angiogenesis, and cell death. Upregulation of NF-κB in normal tissues causes an appearance of inflammatory reactions and oxidative stress, whereas it regulates angiogenesis and suppresses apoptosis, leading to resistance to subsequent doses of radiation or chemotherapy. Selective inhibition of NF-κB in experimental studies has shown promising results for tumor sensitization via apoptosis induction, inhibition of angiogenesis, and increasing delay of tumor growth. The use of some agents for NF-κB inhibition has been shown to alleviate radiation/chemotherapy toxicities in normal cells/ tissues. In this current review, we explained the pivotal role of NF-κB in both normal tissue toxicity and tumor resistance. We also discussed the promising strategies for overcoming these problems with regard to chemotherapy and radiotherapy.     

Radiation response and regulation of apoptosis induced by a combination of TRAIL and CHX in cells lacking mitochondrial DNA: a role for NF-κB-STAT3-directed gene expression

Mitochondrial DNA depleted (ρ⁰) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-κB and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental ρ⁺ HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in ρ⁰ cells compared to ρ⁺ HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, ΙL17Β, ΙL18, ΙL19, and ΙL28Β) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-κB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-κB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in ρ⁺ HSF, but this response was substantially decreased in ρ⁰ HSF. Suppression of the IKK–NF-κB pathway by the small molecular inhibitor BMS-345541 and of the JAK2–STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated ρ⁺ HSF. Inhibitory antibodies against IL6, the main activator of JAK2–STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-κB activation was partially lost in ρ⁰ HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-κB targets, further suppressing IL6–JAK2–STAT3 that in concert with NF-κB regulated protection against TRAIL-induced apoptosis.     

Lysophosphatidylcholine-induced apoptosis in H19-7 hippocampal progenitor cells is enhanced by the upregulation of Fas Ligand

Lysophospholipids regulate a wide array of biological processes including apoptosis and neutrophil migration. Fas/Apo-1 and its ligand (FasL) participate in neuronal cell apoptosis causing various neurological diseases. Here, we use hippocampal neuroprogenitor cells to investigate how lysophosphatidylcholine (LPC) induces apoptosis in H19-7 hippocampal progenitor cells via Fas/Fas ligand-mediated apoptotic signaling pathway. Exposed cells with LPC presented on apoptotic morphology, positive TUNEL staining, and DNA fragmentation. We found that the expression of FasL was increased after LPC treatment. Furthermore, LPC-induced H19-7 cell apoptosis was decreased by agonistic anti-FasL antibody. In addition to promotion of caspase cascade activity by LPC, the administration of the caspase inhibitor, DEVD-fmk, prevented H19-7 cell apoptosis. LPC also increased the activation of nuclear factor-κB (NF-κB), which in turn, significantly increased FasL mRNA level. The increase in FasL mRNA level by NF-κB transfection was significantly decreased in the presence of IκB-SR, a super-repressor of IκB. Taken together, these results demonstrate that LPC has the ability to induce apoptosis in H19-7 cells through the upregulation of FasL expression via NF-κB activation.     

miR-146a suppresses the sensitivity to interferon-α in hepatocellular carcinoma cells

Previous studies have demonstrated expression of Toll-like receptors (TLRs) in the surface epithelium of normal ovaries (OSE) and in epithelial ovarian tumors. Most notably, OSE-derived cancers express TLR4, which activates the nuclear factor-kappa B (NF-κB) signaling cascade as a mediator of inflammatory response. Currently, there is considerable interest in elucidating the role of TLR-mediated signaling in cancers. Nevertheless, the expression of TLRs in granulosa cell tumors (GCTs) of the ovary, and the extent to which GCT expression of TLRs may influence cell-signaling pathways and/or modulate the efficacy of chemotherapeutics, has yet to be determined. In the present study, human GCT lines (COV434 and KGN) were utilized to evaluate expression of functional TLR4. TLR4 is expressed in GCT cell lines and ligation of TLR4 with bacterial lipopolysaccharide (LPS) led to IκB degradation and activation of NF-κB. NF-κB activation was confirmed by nuclear localization of NF-κB p65 following treatment with LPS and the naturally occurring ligand, HSP60. Notably, immunoneutralization of TLR4 blocked nuclear localization, and inhibition of NF-κB signaling attenuated LPS-induced TNFα plus increased doubling time in both cell lines. Contradictory to reports using human OSE cell lines, inhibition of NF-κB signaling failed to sensitize GCT lines to TRAIL or cisplatin. In summary, findings herein are the first to demonstrate a functional TLR-signaling pathway specifically in GCTs, and indicate that in contrast to OSE-derived cancers, inhibition of NF-κB does not sensitize GCTs to TRAIL or cisplatin.     

1,25-dihydroxyvitamin D3 impairs NF-κB activation in human naïve B cells

1α,25-dihydroxyvitamin D₃ (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-κB p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-κB mediated activation of human naïve B cells. Naïve B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human naïve B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-κB activation by interference with NF-κB p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in naïve B cells, namely by reducing CD40 signaling.     

核因子-κB分子生物学特性及其在口腔疾病中的作用

核转录因子-κB（NF-κB）是一种广泛存在于体内多种细胞的核转录因子。静息状态下,NF-κB二聚体与其抑制蛋白IκB结合而存在于胞质中。当细胞受到外界刺激时,IκB磷酸化,使NF-κB活化进入细胞核,调节相应靶细胞的表达。本文对NF-κB家族、分子生物学特性及其在口腔疾病中发生和治疗中分子机制进行探讨,为口腔疾病致病机理以及探寻其＂干预治疗＂的关键靶点提供理论依据和新思路。     

大气可吸入颗粒物造成肺损伤的NF-κB研究进展

NF-κB信号通路在大气可吸入颗粒物（PM10）对肺的损伤过程中起到重要的作用,但NF-κB信号通路参与的损伤机理尚不清楚。本文对NF-κB的结构与组成、颗粒物中NF-κB的激活因素、激活过程和抑制剂的相关研究等内容做简要综述。     

Identification and characterization of receptor-interacting protein 2 as a TNFR-associated factor 3 binding partner

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a highly versatile immune regulator that positively controls type I interferon production, but negatively regulates the activation of mitogen-activated protein kinase and alternative nuclear factor-κB signaling. The precise function of TRAF3 in different signaling pathways remains unclear. Thus, in a yeast two-hybrid assay, TRAF3 was used as the bait to screen a human spleen cDNA library for TRAF3 interactors that may potentially mediate TRAF3-regulated functions. Receptor-interacting protein 2 (RIP2) was identified as a TRAF3 binding partner. The interaction between TRAF3 and RIP2 was further confirmed by mammalian two-hybrid, co-immunoprecipitation and GST pull-down assays, and this interaction was also verified by immunoprecipitation of endogenous proteins in Ramos cells, a human B lymphoma cell line. RIP2 is an activator of NF-κB. We therefore examined the effect of TRAF3 in RIP2-induced NF-κB activation. The result showed that TRAF3 could inhibit RIP2-induced NF-κB activation. Given the high expression of RIP2 in the B lymphoma cell line and endogenous interaction between TRAF3 and RIP2 in Ramos cells, the role of RIP2 was further studied. The result demonstrated that RIP2 knockdown was capable of increasing the expression of TRAF3 and suppressing the activation of alternative NF-кB pathway in Ramos cells. These findings suggest that functional interactions between RIP2 and TRAF3 may provide some clues to the mechanisms of TRAF3-involvement in both positive and negative regulatory functions.     

Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20

Nuclear factor κB (NF-κB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-κB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-κB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.     

Thiophenacetamide as a potential modulator to NF-κB: structure and dynamics study using in silico and molecular biology assays

Abstract

Nuclear factor kappa B (NF-κB) plays critical roles in the regulation of many pathophysiological processes, including inflammation and immune responses, cell growth and apoptosis. This DNA-binding protein receptor is considered an important molecular target to treat many diseases through host-directed therapy. In this line, several drugs containing thiophene cores have been extensively evaluated due to their ability to interfere on NF-κB translocation to the nucleus. In this work, assays using drug affinity responsive target stability (DARTS) revealed that the parent compound N-(Aryl)-2-thiophen-2-ylacetamide referred to as thiophenacetamide (TAA) specifically binds to the p65 subunit of the NF-κB. Since no experimental binding mode of TAA with p65 is available, we explored TAA within putative sites in silico to gain insights into its possible binding mode and behavior. The binding mode of TAA found in Site 1 formed hydrogen bonds with Lys37 and Asp125 on p65, important residues near DNA-binding region. Molecular dynamics simulations showed the stability of this mode of binding in contrast to the other also tested modes. Our results suggest that TAA binding could occur in regions close to residues responsible for DNA binding, increasing NF-κB protein rigidity and affecting the association between DNA and NF-κB.

Communicated by Ramaswamy H. Sarma