WAVE3-NFκB Interplay Is Essential for the Survival and Invasion of Cancer Cells

The WAVE3 cytoskeletal protein promotes cancer invasion and metastasis. We have shown that the WAVE3-mediated activation of cancer cell invasion is due, in part, to its regulation of expression and activity of key metalloproteinases (MMPs), including MMP9, which is centrally involved in invadopodia-mediated degradation of the extracellular matrix (ECM). MMP9 is also a major NFκB target gene, suggesting a potential linkage of WAVE3 to this pathway, which we sought to investigate. Mechanistically, we found that loss of WAVE3 in cancer cells leads to inhibition of NFκB signaling as a result of a decrease in the nuclear translocation of NFκB and therefore loss of activation of NFκB target genes. Conversely, overexpression of WAVE3 was sufficient to enhance NFκB activity. Both pharmacologic and genetic manipulations of NFκB effector molecules show that the biological consequence of loss of WAVE3 function in the NFκB pathway result the inhibition of invadopodia formation and ECM degradation by cancer cells, and these changes are a consequence of decreased MMP9 expression and activity. Loss of WAVE3 also sensitized cancer cells to apoptosis and cell death driven by TNFα, through the inhibition of the AKT pro-survival pathway. Our results identify a novel function of WAVE3 in NFκB signaling, where its activity is essential for the regulation of invadopodia and ECM degradation. Therefore, targeted therapeutic inhibition of WAVE3 will sensitize cancer cells to apoptosis and cell death, and suppress cancer invasion and metastasis.     

Candida albicans induces cyclo-oxygenase 2 expression and prostaglandin E2 production in synovial fibroblasts through an extracellular-regulated kinase 1/2 dependent pathway

Introduction

Synovial cells are potential sources of inflammatory mediators in bacterial-induced arthritis but their involvement in the inflammatory response to Candida albicans-induced septic arthritis is largely unknown.

Methods

Primary cultures of rat synovial fibroblasts were infected with C. albicans (ATCC90028). Immunocytochemistry, western blotting, and RT-PCR were performed to assess cyclo-oxygenase 2 induction. Phosphorylation of extracellular-regulated kinase (ERK1/2) following infection in the absence or presence of U0126 was assessed by western blotting whilst prostaglandin E2 production was measured by ELISA. Nuclear factor κB (NFκB) translocation was evaluated by an electrophoretic mobility shift assay.

Results

Infection of synovial fibroblasts with C. albicans resulted in cyclo-oxygenase 2 expression and prostaglandin E2 production. Cyclo-oxygenase 2 expression and prostaglandin E2 production was dependent upon extracellular-regulated kinase 1/2 phosphorylation, associated with activation of NFκB and significantly elevated in the presence of laminarin, an inhibitor of dectin-1 activity. Synovial fibroblasts adjacent to C. albicans hyphae aggregates appeared to be the major contributors to the increased levels of cyclo-oxygenase 2 and phosphorylated extracellular-regulated kinase 1/2.

Conclusions

C. albicans infection of synovial fibroblasts in vitro results in upregulation of cyclo-oxygenase 2 and prostaglandin E2 by mechanisms that may involve activation of extracellular-regulated kinase 1/2 and are associated with NFκB activation.     

TWEAK Activates the Non-Canonical NFκB Pathway in Murine Renal Tubular Cells: Modulation of CCL21

TWEAK is a member of the TNF superfamily of cytokines that contribute to kidney tubulointerstitial injury. It has previously been reported that TWEAK induces transient nuclear translocation of RelA and expression of RelA-dependent cytokines in renal tubular cells. Additionally, TWEAK induced long-lasting NFκB activation suggestive of engagement of the non-canonical NFκB pathway. We now explore TWEAK-induced activation of NFκB2 and RelB, as well as expression of CCL21, a T-cell chemotactic factor, in cultured murine tubular epithelial cells and in healthy kidneys in vivo. In cultured tubular cells, TWEAK and TNFα activated different DNA-binding NFκB complexes. TWEAK-induced sustained NFκB activation was associated with NFκB2 p100 processing to p52 via proteasome and nuclear translocation and DNA-binding of p52 and RelB. TWEAK, but not TNFα used as control), induced a delayed increase in CCL21a mRNA (3.5±1.22-fold over control) and CCL21 protein (2.5±0.8-fold over control), which was prevented by inhibition of the proteasome, or siRNA targeting of NIK or RelB, but not by RelA inhibition with parthenolide. A second NFκB2-dependent chemokine, CCL19, was upregulates by TWEAK, but not by TNFα. However, both cytokines promoted chemokine RANTES expression (3-fold mRNA at 24 h). In vivo, TWEAK induced nuclear NFκB2 and RelB translocation and CCL21a mRNA (1.5±0.3-fold over control) and CCL21 protein (1.6±0.5-fold over control) expression in normal kidney. Increased tubular nuclear RelB and tubular CCL21 expression in acute kidney injury were decreased by neutralization (2±0.9 vs 1.3±0.6-fold over healthy control) or deficiency of TWEAK (2±0.9 vs 0.8±0.6-fold over healthy control). Moreover, anti-TWEAK treatment prevented the recruitment of T cells to the kidney in this model (4.1±1.4 vs 1.8±1-fold over healthy control). Our results thus identify TWEAK as a regulator of non-canonical NFκB activation and CCL21 expression in tubular cells thus promoting lymphocyte recruitment to the kidney during acute injury.     

The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NFκB Transcription Factors

Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells.     

Estradiol Reduces Ferrous Citrate Complex-Induced NOS2 Up-Regulation in Cerebral Endothelial Cells by Interfering the Nuclear Factor Kappa B Transactivation through an Estrogen Receptor β–Mediated Pathway

Hemorrhagic stroke caused leakage of red blood cells which converts to hemoglobin, heme, and iron accumulated at the lesions. High concentration of ferrous iron from subarachnoid hemorrhage (SAH) induced cerebral vasospasm. Using the two-hemorrhage SAH model in rats, we previously demonstrated that estradiol (E2) significantly attenuated the SAH-induced vasospasm by inhibiting the NOS2 expression. Adding ferrous citrate (FC) complexes to the primary cultured mouse cerebral endothelial cells (CEC) to mimic the SAH conditions, we also showed that FC up-regulates NOS2 through nuclear translocation of NFκB induced by free radicals generation. Here, we further studied the molecular mechanism underlying E2-mediated reduction of the FC-induced up-regulation of NOS2. Treatment with E2 (100 nM) reduced the FC (100 µM)-induced increases of free radical generation and the levels of NOS2 mRNA and protein in the CEC. Moreover, E2 also prevented the FC-induced increases of IκBα phosphorylation, NFκB nuclear translocation, NFκB binding onto the NOS2 promoter, and the NOS2 promoter luciferase activity. However, knock-down the estrogen receptor β (ERβ), but not ERα, abolished the E2-mediated prevention on the FC-induced increases of NOS2 mRNA and protein. The data from the present study suggest that E2 inhibited NOS2 gene expression by interfering with NFκB nuclear translocation and NFκB binding onto the NOS2 through an ERβ-mediated pathway. Our results provide the molecular basis for designing the applicable therapeutic or preventive strategies in the treatment SAH patients.     

Interleukin-1/Toll-Like Receptor-Induced Nuclear Factor Kappa B Signaling Participates in Intima Hyperplasia after Carotid Artery Balloon Injury in Goto-Kakizaki Rats: A Potential Target Therapy Pathway

The value of restenosis after percutaneous coronary intervention (PCI) is recognized worldwide, especially for diabetic patients. Interleukin-1/Toll-like receptor (IL-1/TLR) signaling is involved in innate and adaptive immune responses, but whether and how the IL-1/TLR-induced nuclear factor kappa B (NFκB) pathway plays key roles in intimal formation is unclear. The underlying mechanism of intima hyperplasia was investigated with a model of carotid balloon injury in Goto-Kakizaki (GK) and Wistar rats and with lipopolysaccharide-stimulated macrophages. Elastic-van Gieson staining showed the medial area peakedon Day 3 post-injury and decreased by Day 7 post-injury in both GK and Wistar rats. The N/M at Day 7 in GK rats was significantly higher than in Wistar rats (p<0.001). The percent of 5-ethynyl-2′-deoxyuridine (EdU) staining-positive cells on Day 3 post-injury was greater than seen on Day 7 post-injury in GK and Wistar rats. The percent of EdU-positive cells on Days 3 and 7 post-injury in Wistar rats was less than that found in GK rats (p<0.01; p<0.05). NFκBp65 immunostaining had increased by Day 7 post-injury. Agilent Whole Genome Oligo Microarray verified that the IL-1/TLR-induced NFκB pathway was activated by carotid balloon injury. TLR4, IL-1 receptor associated kinase, inhibitors α of NFκB, human antigen R, c-Myc (Proto-Oncogene Proteins), EGF-like module-containing mucin-like hormone receptor-like 1 and Interleukin-6 were up-regulated or down-regulated according to immunochemistry, quantitative real-time PCR, Western blotting and Enzyme linked immunosorbent assay. Overall, we conclude that the IL-1/TLR-induced NFκB pathway participates in the intimal hyperplasia after carotid injury in GK and Wistar rats and that GK rats respond more intensely to the inflammation than Wistar rats.     

The Nexus between VEGF and NFκB Orchestrates a Hypoxia-Independent Neovasculogenesis

Nuclear Factor-Kappa B [NFκB] activation triggers the elevation of various pro-angiogenic factors that contribute to the development and progression of diabetic vasculopathies. It has been demonstrated that vascular endothelial growth factor [VEGF] activates NFκB signaling pathway. Under the ischemic microenvironments, hypoxia-inducible factor-1 [HIF-1] upregulates the expression of several proangiogenic mediators, which play crucial roles in ocular pathologies. Whereas YC-1, a soluble guanylyl cyclase [sGC] agonist, inhibits HIF-1 and NFκB signaling pathways in various cell and animal models. Throughout this investigation, we examined the molecular link between VEGF and NFκB under a hypoxia-independent microenvironment in human retinal microvascular endothelial cells [hRMVECs]. Our data indicate that VEGF promoted retinal neovasculogenesis via NFκB activation, enhancement of its DNA-binding activity, and upregulating NFκB/p65, SDF-1, CXCR4, FAK, αVβ3, α5β1, EPO, ET-1, and MMP-9 expression. Conversely, YC-1 impaired the activation of NFκB and its downstream signaling pathways, via attenuating IκB kinase phosphorylation, degradation and activation, and thus suppressing p65 phosphorylation, nuclear translocation, and inhibiting NFκB-DNA binding activity. We report for the first time that the nexus between VEGF and NFκB is implicated in coordinating a scheme that upregulates several pro-angiogenic molecules, which promotes retinal neovasculogenesis. Our data may suggest the potential use of YC-1 to attenuate the deleterious effects that are associated with hypoxia/ischemia-independent retinal vasculopathies.     

CFTR Is a Negative Regulator of NFκB Mediated Innate Immune Response

Background

Dysfunctional CFTR in the airways is associated with elevated levels of NFκB mediated IL-8 signaling leading to neutrophil chemotaxis and chronic lung inflammation in cystic fibrosis. The mechanism(s) by which CFTR mediates inflammatory signaling is under debate.

Methodology/Principal Findings

We tested the hypothesis that wt-CFTR down-regulates NFκB mediated IL-8 secretion. We transiently co-expressed wt-CFTR and IL-8 or NFκB promoters driving luciferase expression in HEK293 cells. Wt-CFTR expression in HEK293 cells suppresses both basal and IL1β induced IL-8, and NFκB promoter activities as compared to the control cells transfected with empty vector (p<0.05). We also confirmed these results using CFBE41o- cells and observed that cells stably transduced with wt-CFTR secrete significantly lower amounts of IL-8 chemokine as compared to non-transfected control cells. To test the hypothesis that CFTR must be localized to cell surface lipid rafts in polarized airway epithelial cells in order to mediate the inflammatory response, we treated CFBE41o- cells that had been stably transduced with wt-CFTR with methyl-β-cyclodextrin (CD). At baseline, CD significantly (p<0.05) induced IL-8 and NFκB reporter activities as compared to control cells suggesting a negative regulation of NFκB mediated IL-8 signaling by CFTR in cholesterol-rich lipid rafts. Untreated cells exposed to the CFTR channel blocker CFTR-172 inhibitor developed a similar increase in IL-8 and NFκB reporter activities suggesting that not only must CFTR be present on the cell surface but it must be functional. We verified these results in vivo by comparing survival, body weight and pro-inflammatory cytokine response to P. aeruginosa LPS in CFTR knock out (CFKO) mice as compared to wild type controls. There was a significant (p<0.05) decrease in survival and body weight, an elevation in IL-1β in whole lung extract (p<0.01), as well as a significant increase in phosphorylated IκB, an inducer of NFκB mediated signaling in the CFKO mice.

Conclusions/Significance

Our data suggest that CFTR is a negative regulator of NFκB mediated innate immune response and its localization to lipid rafts is involved in control of inflammation.     

Novel Synthetic Monoketone Transmute Radiation-Triggered NFκB-Dependent TNFα Cross-Signaling Feedback Maintained NFκB and Favors Neuroblastoma Regression

Recently, we demonstrated that radiation (IR) instigates the occurrence of a NFκB-TNFα feedback cycle which sustains persistent NFκB activation in neuroblastoma (NB) cells and favors survival advantage and clonal expansion. Further, we reported that curcumin targets IR-induced survival signaling and NFκB dependent hTERT mediated clonal expansion in human NB cells. Herein, we investigated the efficacy of a novel synthetic monoketone, EF24, a curcumin analog in inhibiting persistent NFκB activation by disrupting the IR-induced NFκB-TNFα-NFκB feedback signaling in NB and subsequent mitigation of survival advantage and clonal expansion. EF24 profoundly suppressed the IR-induced NFκB-DNA binding activity/promoter activation and, maintained the NFκB repression by deterring NFκB-dependent TNFα transactivation/intercellular secretion in genetically varied human NB (SH-SY5Y, IMR-32, SK–PN–DW, MC-IXC and SK–N-MC) cell types. Further, EF24 completely suppressed IR-induced NFκB-TNFα cross-signaling dependent transactivation/translation of pro-survival IAP1, IAP2 and Survivin and subsequent cell survival. In corroboration, EF24 treatment maximally blocked IR-induced NFκB dependent hTERT transactivation/promoter activation, telomerase activation and consequent clonal expansion. EF24 displayed significant regulation of IR-induced feedback dependent NFκB and NFκB mediated survival signaling and complete regression of NB xenograft. Together, the results demonstrate for the first time that, novel synthetic monoketone EF24 potentiates radiotherapy and mitigates NB progression by selectively targeting IR-triggered NFκB-dependent TNFα-NFκB cross-signaling maintained NFκB mediated survival advantage and clonal expansion.     

Enhancing fractalkine/CX3CR1 signalling pathway can reduce neuroinflammation by attenuating microglia activation in experimental diabetic retinopathy

The concept of diabetic retinopathy (DR) has been extended from microvascular disease to neurovascular disease in which microglia activation plays a remarkable role. Fractalkine (FKN)/CX3CR1 is reported to regulate microglia activation in central nervous system diseases. To characterize the effect of FKN on microglia activation in DR, we employed streptozotocin‐induced diabetic rats, glyoxal‐treated R28 cells and hypoxia‐treated BV2 cells to mimic diabetic conditions and explored retinal neuronal apoptosis, reactive oxygen species (ROS), as well as the expressions of FKN, Iba‐1, TSPO, NF‐κB, Nrf2 and inflammation‐related cytokines. The results showed that FKN expression declined with diabetes progression and in glyoxal‐treated R28 cells. Compared with normal control, retinal microglia activation and inflammatory factors surged in both diabetic rat retinas and hypoxia‐treated microglia, which was largely dampened by FKN. The NF‐κB and Nrf2 expressions and intracellular ROS were up‐regulated in hypoxia‐treated microglia compared with that in normoxia control, and FKN significantly inhibited NF‐κB activation, activated Nrf2 pathway and decreased intracellular ROS. In conclusion, the results demonstrated that FKN deactivated microglia via inhibiting NF‐κB pathway and activating Nrf2 pathway, thus to reduce the production of inflammation‐related cytokines and ROS, and protect the retina from diabetes insult.     

TRAF2 recruitment via T61 in CD30 drives NFκB activation and enhances hESC survival and proliferation

CD30 (TNFRSF8), a tumor necrosis factor receptor family protein, and CD30 variant (CD30v), a ligand-independent form encoding only the cytoplasmic signaling domain, are concurrently overexpressed in transformed human embryonic stem cells (hESCs) or hESCs cultured in the presence of ascorbate. CD30 and CD30v are believed to increase hESC survival and proliferation through NFκB activation, but how this occurs is largely unknown. Here we demonstrate that hESCs that endogenously express CD30v and hESCs that artificially overexpress CD30v exhibit increased ERK phosphorylation levels, activation of the canonical NFκB pathway, down-regulation of the noncanonical NFκB pathway, and reduced expression of the full-length CD30 protein. We further find that CD30v, surprisingly, resides predominantly in the nucleus of hESC. We demonstrate that alanine substitution of a single threonine residue at position 61 (T61) in CD30v abrogates CD30v-mediated NFκB activation, CD30v-mediated resistance to apoptosis, and CD30v-enhanced proliferation, as well as restores normal G₂/M-checkpoint arrest upon H₂O₂ treatment while maintaining its unexpected subcellular distribution. Using an affinity purification strategy and LC-MS, we identified TRAF2 as the predominant protein that interacts with WT CD30v but not the T61A-mutant form in hESCs. The identification of Thr-61 as a critical residue for TRAF2 recruitment and canonical NFκB signaling by CD30v reveals the substantial contribution that this molecule makes to overall NFκB activity, cell cycle changes, and survival in hESCs.     

6-Mercaptopurine reduces cytokine and Muc5ac expression involving inhibition of NFκB activation in airway epithelial cells

Background

Mucus hypersecretion and excessive cytokine synthesis is associated with many of the pathologic features of chronic airway diseases such as asthma. 6-Mercaptopurine (6-MP) is an immunosuppressive drug that is widely used in several inflammatory disorders. Although 6-MP has been used to treat asthma, its function and mechanism of action in airway epithelial cells is unknown.

Methods

Confluent NCI-H292 and MLE-12 epithelial cells were pretreated with 6-MP followed by stimulation with TNFα or PMA. mRNA levels of cytokines and mucins were measured by RT-PCR. Western blot analysis was performed to assess the phosphorylation of IκBα and luciferase assays were performed using an NFκB reporter plasmid to determine NFκB activity. Periodic Acid Schiff staining was used to assess the production of mucus.

Results

6-MP displayed no effect on cell viability up to a concentration of 15 μM. RT-PCR analysis showed that 6-MP significantly reduces TNFα- and PMA-induced expression of several proinflammatory cytokines in NCI-H292 and MLE-12 cells. Consistent with this, we demonstrated that 6-MP strongly inhibits TNFα-induced phosphorylation of IκBα and thus attenuates NFκB luciferase reporter activity. In addition, 6-MP decreases Rac1 activity in MLE-12 cells. 6-MP down-regulates gene expression of the mucin Muc5ac, but not Muc2, through inhibition of activation of the NFκB pathway. Furthermore, PMA- and TNFα-induced mucus production, as visualized by Periodic Acid Schiff (PAS) staining, is decreased by 6-MP.

Conclusions

Our data demonstrate that 6-MP inhibits Muc5ac gene expression and mucus production in airway epithelial cells through inhibition of the NFκB pathway, and 6-MP may represent a novel therapeutic target for mucus hypersecretion in airway diseases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0236-0) contains supplementary material, which is available to authorized users.     

TIFA has dual functions in Helicobacter pylori‐induced classical and alternative NF‐κB pathways

Helicobacter pylori infection constitutes one of the major risk factors for the development of gastric diseases including gastric cancer. The activation of nuclear factor‐kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) via classical and alternative pathways is a hallmark of H. pylori infection leading to inflammation in gastric epithelial cells. Tumor necrosis factor receptor‐associated factor (TRAF)‐interacting protein with forkhead‐associated domain (TIFA) was previously suggested to trigger classical NF‐κB activation, but its role in alternative NF‐κB activation remains unexplored. Here, we identify TRAF6 and TRAF2 as binding partners of TIFA, contributing to the formation of TIFAsomes upon H. pylori infection. Importantly, the TIFA/TRAF6 interaction enables binding of TGFβ‐activated kinase 1 (TAK1), leading to the activation of classical NF‐κB signaling, while the TIFA/TRAF2 interaction causes the transient displacement of cellular inhibitor of apoptosis 1 (cIAP1) from TRAF2, and proteasomal degradation of cIAP1, to facilitate the activation of the alternative NF‐κB pathway. Our findings therefore establish a dual function of TIFA in the activation of classical and alternative NF‐κB signaling in H. pylori‐infected gastric epithelial cells.