Regulating NF-κB Signaling Research Articles

Abstract T P225: Loss of Pregnane X Receptor Impairs Stroke Recovery

Introduction: Inflammation plays an important role in exacerbating injury following stroke, but current understanding of the factors regulating this inflammation is incomplete. Pregnane X Receptor (PXR) is a nuclear receptor which binds many xenobiotic targets including many commonly prescribed medications. Recent work has shown that PXR is crucial for maintaining the integrity of the intestinal epithelial barrier as well repressing inflammatory responses mediated by NFκB. Since both these pathways can modify the peripheral immune response, we hypothesized that PXR would play an important role in limiting inflammation following stroke. Using PXR knockout mice, we show that loss of PXR is detrimental for stroke recovery. Materials & Methods: Male PXR knockout mice and littermate controls underwent sham surgery or 60min middle cerebral artery occlusion (MCAO). Weight loss was monitored post-stroke and mice were sacrificed after 7 days for splenic weights, infarct analysis by cresyl violet staining, and bacterial translocation as assessed by culture of homogenized mesenteric lymph nodes on blood agar plates. Results: Following MCAO, PXR-/- mice lost significantly more weight than wild type littermate controls (n=6 vs. 4 respectively; p<0.05) and also showed greater splenic atrophy 7 days following MCAO (2.14mg/g body weight vs. 3.40mg/g body weight; p<0.05). However, preliminary studies suggest PXR-/- mice do not have larger infarcts (n=3 vs. 2) and did not exhibit more bacterial translocation 7 days following MCAO (5.4 vs. 7 CFU). Discussion: PXR is known to negatively regulate NFκB signaling and maintain intestinal barrier integrity. Both of these mechanisms may contribute to delayed recovery following stroke with PXR acting as a master switch. In summary, this early work begins to explore the importance of a xenobiotic sensor, PXR, in stroke recovery.

CYLD and SUMO in neuroblastoma therapy.

Cylindromatosis gene, CYLD is a tumor suppressor gene that was originally identified as a gene mutated in familial cylindromatosis and related diseases [1]. CYLD belongs to the deubiquitination enzymes family, which function by removing of ubiquitin chains from different proteins. The ubiquitin-specific protease domain of CYLD is located at the C-terminus and mutations in this region inactivate the capacity of CYLD to cleave polyubiquitin chains from specific substrates. CYLD contains three cytoskeleton-associated protein-glycine-conserved (CAP-Gly) in its amino terminus [1]. The CAP-Gly domains consists of about 70 amino acid residues important for the re-localisation of CYLD to the perinuclear region or as a scaffold for substrates [5]. CYLD negatively regulates the NFκB pathway by deubiquitinating important factors such as NFκB essential modulator (NEMO, also known as IκB kinase γ), TRAF2 (TNF Receptor-Associated Factor 2) and TRAF6 (TNF Receptor-Associated Factor 6). Beside NFκB, CYLD has been show to modulate other signaling pathways such as Wnt, MAPK, and Bcl-3. Regulation of these signaling pathways are important for the tumor suppressor function of CYLD in different types of human cancer [5]. Recently, we have demonstrated a significant role of CYLD in differentiation of neuroblastoma [3]. Neuroblastoma is the most common extracranial tumor of childhood and it is originating from primordial neural crest cells that give rise to sympathetic neural ganglia and adrenal medulla. The age of patients, genetic aberrations, and metastatic spread are important factors with regard to treatment decisions and prognosis. Surgery, chemotherapy, radiotherapy, and autologous stem cell transplantation are commonly used as treatment therapy for neuroblastoma patients. In addition, forced differentiation of neuroblastoma cells by retinoids treatment is used mostly as a therapy for high-risk neuroblastomas. Treatment with retinoids leads to elimination of residual neuroblastoma cells that survive after chemotherapy or stem cell transplantation [6, 7]. Recent work from our laboratory, demonstrated that all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells. It, operates by increasing in the expression of the small ubiquitin-like modifier (SUMO) protein that can affect downstream signaling inside the cells by quickly switch on or off different cellular processes, including survival, proliferation, and differentiation. The SUMOylation is reversible through the action of SUMO specific proteases (SENPs) (Figure ​(Figure1).1). We observed that a short ATRA treatment of neuroblastoma causes a transient SUMOylation of CYLD that reduces its deubiquitin activity, as well as activation of NF-κB signaling via ubiquitination of TRAF2/TRAF6. However, prolonged ATRA treatment reduced CYLD SUMOylation and promoted cell death instead. Therefore, it was proposed that the balance between non-SUMOylated and SUMOylated CYLD can direct neuroblastoma cancer cells against differentiation or cell death via regulation of NF-κB signaling [3]. Furthermore, high CYLD expression is associated with better overall and relapse-free survival and inversely correlated with the stage of neuroblastoma [3]. Figure 1 SUMOylation of any substrate is directed by the action of SUMO-activating (E1), SUMO-conjugation (E2), and SUMO-ligation enzymes (E3) In this context, maintaining high expression levels of CYLD and SUMO proteins together can be used promising tools for future therapeutic strategies for neuroblastoma patients. Indeed, cellular death induced by oxidative stress via overexpression of SUMO-1 was previously shown to be blocked by the activation of deSUMOylated enzyme SENP1 [2]. Beside cell death, inhibition of SENP1 reduces migration and invasion of cancer cells [8]. Thus, inhibitors against SENP can be a novel anticancer tool for treating neuroblastoma. Recently, a number of small molecule inhibitors against SENP isoforms have been developed and tested using in-vitro approaches [4]. Future studies need to exploit the therapeutic potential of SENP inhibitors in combination with chemotherapy, radiotherapy, and ATRA treatment for neuroblastoma.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) promotes glioma cell invasion through induction of NF-κB-inducing kinase (NIK) and noncanonical NF-κB signaling.

BackgroundHigh-grade gliomas are one of the most invasive and therapy-resistant cancers. We have recently shown that noncanonical NF-κB/RelB signaling is a potent driver of tumorigenesis and invasion in the aggressive, mesenchymal subtype of glioma. However, the relevant signals that induce activation of noncanonical NF-κB signaling in glioma and its function relative to the canonical NF-κB pathway remain elusive.MethodsThe ability of tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) to regulate NF-κB signaling and promote tumor progression was investigated in both established and primary high-grade glioma tumor lines using a three-dimensional (3-D) collagen invasion assay. The roles of specific NF-κB proteins in regulating glioma cell invasion and expression of Matrix Metalloproteinase 9 (MMP9) in response to TWEAK were evaluated using shRNA-mediated loss-of-function studies. The ability of NF-κB-inducing kinase (NIK) to promote glioma growth in vivo was investigated using an orthotopic xenograft mouse model.ResultsIn glioma cells that display elevated noncanonical NF-κB signaling, loss of RelB attenuates invasion without affecting RelA expression or phosphorylation and RelB is sufficient to promote invasion in the absence of RelA. The cytokine TWEAK preferentially activates the noncanonical NF-κB pathway through induction of p100 processing to p52 and nuclear accumulation of both RelB and p52 without activating the canonical NF-κB pathway. Moreover, TWEAK, but not TNFα, significantly increases NIK mRNA levels. TWEAK also promotes noncanonical NFκB-dependent MMP9 expression and glioma cell invasion. Finally, expression of NIK is sufficient to increase gliomagenesis in vivo.ConclusionsOur data establish a key role for NIK and noncanonical NF-κB in mediating TWEAK-induced, MMP-dependent glioma cell invasion. The findings also demonstrate that TWEAK induces noncanonical NF-κB signaling and signal-specific regulation of NIK mRNA expression. Together, these studies reveal the important role of noncanonical NF-κB signaling in regulating glioma invasiveness and highlight the therapeutic potential of targeting activation of NIK in this deadly disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-014-0273-1) contains supplementary material, which is available to authorized users.

Regulation of NF-κB signaling in osteoclasts and myeloid progenitors.

The transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is crucial for immune responses and skeletal development. Work in recent years has shown that various members of the NF-κB family are viable targets to regulate activity and survival of bone cells and hence bone metabolism. In this regard, deletion of upstream kinases or distal NF-κB subunits resulted with bone deformities. Thus, it has become increasingly apparent that detailed investigation of NF-κB in bone cells may provide opportunities to design new therapeutic modalities. In this chapter we present modified methodology describing efficient approaches to regulate the NF-κB pathway in vitro and in vivo to assess its function in bone cells and tissues.

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1β induced miRs-21-3p and -27a-5p in human aortic endothelial cells

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3' untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.

Dissecting the Potential Interplay of DEK Functions in Inflammation and Cancer.

There is a long-standing correlation between inflammation, inflammatory cell signaling pathways, and tumor formation. Understanding the mechanisms behind inflammation-driven tumorigenesis is of great research and clinical importance. Although not entirely understood, these mechanisms include a complex interaction between the immune system and the damaged epithelium that is mediated by an array of molecular signals of inflammation—including reactive oxygen species (ROS), cytokines, and NFκB signaling—that are also oncogenic. Here, we discuss the association of the unique DEK protein with these processes. Specifically, we address the role of DEK in chronic inflammation via viral infections and autoimmune diseases, the overexpression and oncogenic activity of DEK in cancers, and DEK-mediated regulation of NFκB signaling. Combined, evidence suggests that DEK may play a complex, multidimensional role in chronic inflammation and subsequent tumorigenesis.

Differential Regulation of NF-κB Signaling during Human Cytomegalovirus Infection

NF-κB transcription factors are key regulators of immune and stress responses, apoptosis, and differentiation. Human cytomegalovirus (HCMV) activates or represses NF-κB signaling at different times during infection. An initial increase in NF-κB activity occurs within a few hours of infection. The virus appears to adapt to this change since initial viral gene expression is promoted by the elevated NF-κB activity. Because NF-κB upregulates innate immune responses and inflammation, it has also been suggested that HCMV needs to downregulate NF-κB signaling. Recent studies have shown that HCMV has various mechanisms that inhibit NF-κB signaling. HCMV reduces cell surface expression of tumor necrosis factor receptor 1 (TNFR1) and blocks the DNA binding activity of NF-κB. Furthermore, some HCMV tegument proteins antagonize NF-κB activation by targeting the key components of NF-κB signaling at late stages of infection. In this review, we summarize the recent findings on the relationship between HCMV and NF-κB signaling, focusing, in particular, on the viral mechanisms by which the NF-κB signaling pathway is inhibited.

Intracellular annexin A2 regulates NF-κB signaling by binding to the p50 subunit: implications for gemcitabine resistance in pancreatic cancer

Annexin A2 (ANXA2) expression is highly upregulated in many types of cancer. Although cell surface localization of ANXA2 has been reported to have a critical role in the progression and metastasis of a variety of tumors, including pancreatic cancer, the biological role of intracellular ANXA2 is not fully understood. Herein the role of intracellular ANXA2 was investigated in a pancreatic cancer cell line. We first determined whether ANXA2 is involved in NF-κB signaling pathways. ANXA2 bound to the p50 subunit of NF-κB in a calcium-independent manner, and the ANXA2–p50 complex translocated into the nucleus. Furthermore, ANXA2 increased the transcriptional activity of NF-κB in both the resting and activated states and upregulated the transcription of several target genes downstream of NF-κB, including that encoding interleukin (IL)-6, which contributes to anti-apoptotic signaling. In Mia-Paca2 cells, we determined the effects of wild-type ANXA2 and an ANXA2 mutant, Y23A, which suppresses the cell surface localization, on upregulation of NF-κB transcriptional activity and secretion of IL-6. Both wild-type and Y23A ANXA2 induced anti-apoptotic effects in response to treatment with tumor necrosis factor-α or gemcitabine. Based on these results, we suggest that ANXA2 mediates resistance to gemcitabine by directly increasing the activity of NF-κB. Collectively, these data may provide additional information about the biological role of ANXA2 in pancreatic cancer and suggest that ANXA2 is a potential biomarker for the drug resistance phenotype and a candidate therapeutic target for the treatment of pancreatic cancer.

Noncoding RNAs regulate NF-κB signaling to modulate blood vessel inflammation.

Cardiovascular diseases such as atherosclerosis are one of the leading causes of morbidity and mortality worldwide. The clinical manifestations of atherosclerosis, which include heart attack and stroke, occur several decades after initiation of the disease and become more severe with age. Inflammation of blood vessels plays a prominent role in atherogenesis. Activation of the endothelium by inflammatory mediators leads to the recruitment of circulating inflammatory cells, which drives atherosclerotic plaque formation and progression. Inflammatory signaling within the endothelium is driven predominantly by the pro-inflammatory transcription factor, NF-κB. Interestingly, activation of NF-κB is enhanced during the normal aging process and this may contribute to the development of cardiovascular disease. Importantly, studies utilizing mouse models of vascular inflammation and atherosclerosis are uncovering a network of noncoding RNAs, particularly microRNAs, which impinge on the NF-κB signaling pathway. Here we summarize the literature regarding the control of vascular inflammation by microRNAs, and provide insight into how these microRNA-based pathways might be harnessed for therapeutic treatment of disease. We also discuss emerging areas of endothelial cell biology, including the involvement of long noncoding RNAs and circulating microRNAs in the control of vascular inflammation.

DREAM, a Transcription Repressor, Is Critical for Calcium Signaling and Platelet Thrombus Formation Independently of Its Transcriptional Activity

DREAM (Downstream Regulatory Element Antagonistic Modulator) is a neuronal calcium sensor and acts as a transcriptional repressor for pain modulation. Recent studies showed that endothelial cell DREAM transcriptionally regulates NF-κB signaling during inflammation (Tiruppathi et al. Nat Immunol. 2014). However, it is unknown whether DREAM plays a role during thrombosis and hemostasis. Using real-time fluorescence intravital microscopy with DREAM knockout (KO) mice, we demonstrated that intravascular cell DREAM is required for platelet thrombus formation and fibrin generation at the site of laser-induced cremaster arteriolar injury. Studies of bone marrow chimeric mice further revealed that both hematopoietic and endothelial cell DREAM regulate thrombus formation following arteriolar injury. Using in vitro flow chamber assays, we found that DREAM is important for platelet thrombus formation on collagen-coated surfaces under shear conditions. Further, tail bleeding time and blood loss significantly increased in DREAM KO mice, compared with wild-type (WT) mice, suggesting the importance of DREAM for hemostasis as well as thrombus formation in vivo. We observed that DREAM KO platelets show markedly decreased aggregation, ATP secretion, P-selectin exposure, and αIIbβ3 integrin activation induced by thrombin, collagen-related peptide, ADP, and A23187 (calcium ionophore). Interestingly, DREAM deletion did not affect phorbol 12-myristate 13-acetate-induced platelet functions. Consistently, DREAM KO platelets exhibited decreased phosphorylation of phosphoinositide-3-kinase, AKT, and ERK, but not protein kinase C (PKC) isoforms during cell activation, suggesting that DREAM does not regulate protein kinase C signaling pathway. Electron microscopic analysis demonstrated that ultrastructural features of DREAM KO platelets are indistinguishable from those of WT platelets. Since DREAM is a transcription factor that binds to calcium in four EF-hand motifs, we investigated whether platelet DREAM regulates calcium signaling. Indeed, DREAM deletion in platelets resulted in significant reduction in both intracellular calcium release and store-operated calcium entry (SOCE) following agonist stimulation. When we transiently knocked down DREAM in a human megakaryoblastic leukemia cell line (MEG-01) using siRNA, we found that A23187-induced calcium release and SOCE are impaired by DREAM knockdown in MEG-01 cells without altering the expression of calcium signaling molecules, such as inositol trisphosphate receptor, stromal interaction molecule 1, and ORAI1. These defects were rescued by overexpression of siRNA-resistant WT but not EF-hand domain mutant DREAM. Therefore, our results provide novel evidence that platelet DREAM regulates calcium signaling independently of its transcriptional factor activity, thereby playing a critical role during thrombus formation and hemostasis in vivo. DisclosuresNo relevant conflicts of interest to declare.

Attenuation of inflammatory-mediated neurotoxicity by Saururus chinensis extract in LPS-induced BV-2 microglia cells via regulation of NF-κB signaling and anti-oxidant properties.

BackgroundA Saururus chinensis Baill (SC) has been used by Native Americans, early colonists and practitioners of Korean traditional medicine for treating several diseases including cancer, rheumatoid arthritis and edema. The objective of this study was to evaluate the effects of SC extract in lipopolysaccharide (LPS)-stimulated neuroinflammatory responses in BV-2 microglial cells.MethodsThe effects of SC on the LPS–induced neuroinflammatory responses in BV-2 microglial cells were assessed by Western blotting, RT-PCR and immunofluorescence labeling techniques. DPPH and alkyl radical scavenging assay was performed to evaluate the anti-oxidant effects. Comparisons between groups were analyzed using one-way analysis of variance followed by Dunnett’s multiple comparisons test using GraphPad Prism V5.01 software.ResultsPre-treatment with SC extract (1, 5 and 10 μg/mL) significantly (p < 0.001 at 10 μg/mL) and concentration dependently inhibited LPS-induced production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and suppressed the inflammatory cytokine levels such as tumor necrosis factor-alpha and interleukin (IL)-6 in BV-2 microglial cells (p < 0.001 at 10 μg/mL). Further, SC suppressed the nuclear factor-kappa B (NF-κB) activation by blocking the degradation of IκB-α. SC also exhibited profound anti-oxidant effects by scavenging 1, 1-diphenyl-2-picrylhydrazyl (DPPH) (IC50: 0.055 mg/mL) and alkyl radicals (IC50: 0.349 mg/mL). High performance liquid chromatography finger printing analysis of SC revealed quercetin (QCT) as one of the major constituents compared with reference standard. QCT also inhibited the excessive release of NO, and inhibited the increased expressional levels of IL-6, iNOS and COX-2 in LPS-stimulated BV-2 cells.ConclusionsOur results indicated that SC inhibited the LPS-stimulated neuroinflammatory responses in BV-2 microglia via regulation of NF-κB signaling. The antioxidant active constituents of SC might be partly involved in delivering such effects. Based on the traditional claims and our present results SC can be potentially used in treating inflammatory-mediated neurodegenerative diseases.

Apigenin Inhibits Tumor Necrosis Factor-α-Induced Production and Gene Expression of Mucin through Regulating Nuclear Factor-Kappa B Signaling Pathway in Airway Epithelial Cells.

In the present study, we investigated whether apigenin significantly affects tumor necrosis factor-α (TNF-α)-induced production and gene expression of MUC5AC mucin in airway epithelial cells. Confluent NCI-H292 cells were pretreated with apigenin for 30 min and then stimulated with TNF-α for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription - polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Apigenin significantly inhibited MUC5AC mucin production and down-regulated MUC5AC gene expression induced by TNF-α in NCI-H292 cells. To elucidate the action mechanism of apigenin, effect of apigenin on TNF-α-induced nuclear factor kappa B (NF-κB) signaling pathway was also investigated by western blot analysis. Apigenin inhibited NF-κB activation induced by TNF-α. Inhibition of inhibitory kappa B kinase (IKK) by apigenin led to the suppression of inhibitory kappa B alpha (IκBα) phosphorylation and degradation, p65 nuclear translocation. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Apigenin also has an influence on upstream signaling of IKK because it inhibited the expression of adaptor protein, receptor interacting protein 1 (RIP1). These results suggest that apigenin can regulate the production and gene expression of mucin through regulating NF-κB signaling pathway in airway epithelial cells.

Abstract 12641: Endothelial Cell Hspa12B Plays a Cardioprotective Role in Sepsis Induced Cardiac Dysfunction via the Release of MicroRNA Containing Exosomes

Heat shock protein A12B (HSPA12B) is predominately expressed in endothelial cells and has been reported to contribute to angiogenesis. We hypothesized that HSPA12B plays a protective role in sepsis-induced cardiac dysfunction. Endothelial HSPA12B -/- (n=6) and wild type (WT, n=6) mice were subjected to cecal ligation and puncture (CLP)-induced sepsis. Sham surgical operation served as sham control (n=6). Cardiac function was examined by echocardiography before and 6 hour after CLP. In WT septic mice, ejection fraction (EF%) and fractional shortening (%FS) were significantly reduced by 34.8% and 43.1% (p&lt;0.05), compared with control. HSPA12B -/- septic mice showed even greater decrements in EF% and FS% values (19.9% and 22.5%), when compared with WT septic mice. Serum levels of TNFα and IL-6 were higher in HSPA12B -/- septic mice than in WT septic mice. The expression of ICAM1 and VCAM1 in the myocardium of HSPA12B -/- septic mice was greater than in WT septic mice. Exosomes play an important role in cell-cell communication. In vitro data showed that exosomes isolated from the serum of HSPA12B -/- septic mice induced increased levels of TNFα and IL-6, NF-κB binding activity and TRAF6 ubiquitination in macrophages, when compared with WT septic exosomes. HSPA12B -/- septic exosomes contained significantly less microRNA-146a, which negatively regulates NF-κB signaling, when compared with WT septic exosomes. Endothelial cells (HUVEC) treated with HSPA12B -/- septic exosomes showed decreased expression of tight junction proteins (ZO-1and Occludin) and miR-126, which regulates the expression of adhesion molecules, when compared with WT septic exosomes treatment. In addition, the amounts of miR-146a and miR-126 in the exosomes from the serum of HSPA12B -/- septic mice were less than in the exosomes from WT septic mice. This is the first report that endothelial cell HSPA12B attenuates cardiac dysfunction in sepsis via secretion of exosomes and microRNA expression. We conclude that endothelial HSPA12B plays a cardioprotective role in sepsis via microRNA containing exosomes.

Crosstalk between JNK and NF-κB signaling pathways via HSP27 phosphorylation in HepG2 cells

The crosstalk of intracellular signaling pathways is extremely complex. Previous studies have shown that there is a potential crosstalk between MAPKs and NF-κB signaling pathways. It has been reported that JNK regulates cell survival under some conditions. But the molecular mechanism through which JNK regulates cell survival is still unclear. In the present study, we hypothesized that there was a crosstalk between JNK and NF-κB signaling pathway regulating cell survival and HSP27 phosphorylation mediates such a crosstalk. Our data showed that in HepG2 cells, suppression of JNK activation by a specific inhibitor or overexpression of JNK inactive mutant enhanced TNF-α-induced apoptosis. In addition, reduction of JNK activation attenuated HSP27 phosphorylation envoked by TNF-α, especially the phosphorylation of HSP27 at serine 78 residue. Our results also showed that suppression of JNK activation reduced the degradation of IκB-α, but did not affect IKK phosphorylation upon TNF-α stimulation. Co-immunoprecipitation experiments demonstrated that JNK regulated the degradation of IκB-α through promoting the formation of HSP27/IKK/IκB-α ternary complex in response to TNF-α. Suppression of JNK activation hindered HSP27 phosphorylation at Ser78 residue and subsequently reduced the interaction between IKK and IκB-α. Taken together, our study suggests that through modulation the phosphorylation of HSP27, JNK plays an important roles in cell survival via regulating NF-κB signaling pathway.

Down-regulation of LATS2 in non-small cell lung cancer promoted the growth and motility of cancer cells.

LATS2 (Large tumor suppressor) has been reported to be dys-regulated in several cancer types. However, its function in non-small cell lung cancer (NSCLC) remains poorly understood. Here, it was found that the expression level of LATS2 was decreased in NSCLC tissues. Moreover, forced expression of LATS2 in NSCLC cells inhibited cell growth and migration, while knockdown of the expression of LATS2 promoted the tumorigenicity of NSCLC cells. Mechanistically, LATS2 was found to negatively regulate NF-κB signaling in NSCLC cells. Taken together, our study suggested that down-regulation of LATS2 was very important in the progression of NSCLC, and restoring the function of LATS2 might be a promising therapeutic strategy for NSCLC.

The E3 ubiquitin ligase RNF121 is a positive regulator of NF-κB activation.

BackgroundThe nuclear factor κB (NF-κB) family members regulate several biological processes as cell proliferation and differentiation, inflammation, immunity and tumor progression. Ubiquitination plays a key role in NF-κB activation and the ubiquitylated transmitters of the NF-κB signaling cascade accumulate in close proximity to endomembranes.FindingsWe performed an unbiased siRNA library screen targeting the 46 E3 ubiquitin ligases bearing transmembrane domains to uncover new modulators of NF-κB activation, using tumor necrosis factor–α (TNF-α) receptor (TNFR) stimulation as a model. We report here the identification of a new Golgi Apparatus-resident protein, RNF121, as an enhancer of NF-κB promoter activity through the catalytic function of its RING domain. From a molecular standpoint, while knocking down RNF121 did not alter RIP1 ubiquitination and IKK activation, the proteasomal degradation of IκBα was impaired suggesting that this E3 ubiquitin ligase regulates this process. However, RNF121 did not directly ubiquitinate IκBα While they were found in the same complex. Finally, we discovered that RNF121 acts as a broad regulator of NF-κB signaling since its silencing also dampens NF-κB activation following stimulation of Toll-Like Receptors (TLRs), Nod-Like Receptors (NLRs), RIG-I-Like Receptors (RLRs) or after DNA damages.ConclusionsThese results unveil an unexpected role of Golgi Apparatus and reveal RNF121 as a new player involved in the signaling leading to NF-κB activation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0072-8) contains supplementary material, which is available to authorized users.

Caspase-1/ASC inflammasome-mediated activation of IL-1β-ROS-NF-κB pathway for control of Trypanosoma cruzi replication and survival is dispensable in NLRP3-/- macrophages.

In this study, we have utilized wild-type (WT), ASC−/−, and NLRP3−/− macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1β production in mφs in comparison to LPS-treated controls. When WT and ASC−/− macrophages were treated with inhibitors of caspase-1, IL-1β, or NADPH oxidase, we found that IL-1β production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1β regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3−/− macrophages, despite an inability to elicit IL-1β activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3−/− macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1β-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1β/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1β/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.

VEGF suppresses T-lymphocyte infiltration in the tumor microenvironment through inhibition of NF-κB-induced endothelial activation.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) signaling pathway is in clinical use, but its effect on vascular function and the tumor microenvironment is poorly understood. Here, we investigate cross-talk between VEGF and proinflammatory TNF-α signaling in endothelial cells and its impact on leukocyte recruitment. We found that cotreatment with VEGF decreased TNF-α-induced Jurkat cell adhesion to human microvascular endothelial cells by 40%. This was associated with inhibition of TNF-α-mediated regulation of 86 genes, including 2 T-lymphocyte-attracting chemokines, CXCL10 and CXCL11 [TNF-α concentration 1 ng/ml; 50% inhibition/inhibitory concentration (IC50) VEGF, 3 ng/ml]. Notably, VEGF directly suppressed TNF-α-induced gene expression through negative cross-talk with the NF-κB-signaling pathway, leading to an early decrease in IFN regulatory factor 1 (IRF-1) expression and reduced phosphorylation of signal transducer and activator of transcription 1 (p-Stat1) at later times. Inhibition of VEGF signaling in B16 melanoma tumor-bearing mice by sunitinib treatment resulted in up-regulation of CXCL10 and CXCL11 in tumor vessels, accompanied by up to 18-fold increased infiltration of CD3(+) T-lymphocytes in B16 tumors. Our results demonstrate a novel role of VEGF in negative regulation of NF-κB signaling and endothelial activation in the tumor microenvironment and provide evidence that pharmacological inhibition of VEGF signaling enhances T-lymphocyte recruitment through up-regulation of chemokines CXCL10 and CXCL11.

Attenuation of Neuroinflammatory Responses in Lipopolysaccharide-Induced BV-2 Microglia by &lt;i&gt;Suaeda asparagoides&lt;/i&gt; Miq. (&lt;i&gt;Chenopodiaceae&lt;/i&gt;)

Purpose: To investigate the protective effect of Suaeda asparagoides (Chenopodiaceae) extract on neuroinflammatory responses induced by lipopolysaccharide (LPS) in BV-2 microglial cells and its antioxidant effects. Methods: Biochemical studies carried out include 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl-tetrazolium bromide (MTT) assay and 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) assay for cell viability and radical scavenging activities, respectively. To evaluate the anti-neuroinflammatory effects of S. asparagoides (SAE) extract, LPS (1 µg/ml)-stimulated BV-2 microglial cells were used and pro-inflammatory mediators and cytokines such as nitric oxide (NO), inducible NO (iNOS), cyclooxygenase (COX)-2, tumor necrosis factor-alpha (TNF-α) and nuclear factor-kappa B (NF-κB) were measured using Western blotting and enzyme-linked immunosorbent assay (ELISA). Results: LPS-stimulation of BV-2 cells increased the levels of NO (25.2 ± 2.15, p < 0.001) and proinflammatory mediators such as iNOS, COX-2 and TNF- α. However, treatment with SAE extract (20, 40 and 80 µg/ml) to LPS-stimulated BV-2 cells significantly inhibited the excessive release of NO (p < 0.05 at 20 µg/ml and p < 0.001 at 40 and 80 µg/ml, respectively) and suppressed the increased levels of iNOS, COX-2 and TNF-α. SAE also concentration dependently inhibited the NF-κB activation in LPSstimulated BV-2 microglia. Further, SAE significantly and concentration-dependently (p < 0.001 at 20 200 µg/ml, respectively) scavenged DPPH radicals with IC 50 of 36.33 ± 2.12 µg/ml. Conclusion: The results strongly suggest that SAE exhibits protective activity against LPS-stimulated neuroinflammatory responses. Mechanistic study reveals that SAE might by regulating NF-κB signaling. The antioxidant activity exhibited by SAE extract might also play a role in the plant’s significant antineuroinflammatory effect.

Abstract 4234: Identification of molecular targets for retinoid signaling to regulate human hepatocellular carcinoma development: contribution of OTUD7B, a negative regulator of NF-κB signaling, in antitumor effect of retinoic acid

Abstract BACKGROUND &amp; AIMS: The loss of retinoid content and diminished retinoid signaling and downstream gene function are closely associated with the progression of liver disease including hepatocellular carcinoma (HCC). We previously identified 26 retinoic acid (RA)-responsive genes through a gene screening based on genome-wide in silico analysis of RA response elements. To understand the clinical and biological significance of RA-responsive genes in HCC, we performed gene expression analysis using clinical HCC samples and functional analyses using HCC cell lines. METHODS: mRNA expressions of RA-responsive genes were investigated in tumor and non-tumor tissues of the liver from 171 HCC patients by qRT-PCR. The association of clinicopathological parameters and gene expression with survival time was evaluated using univariate and multivariate Cox regression modeling. Potential anti-tumor activity of the candidate genes were evaluated using siRNA-mediated knockdown and adenovirus-mediated overexpression experiments in HCC cells. Functional analyses of the genes in the regulation of intracellular signal pathways were performed using luciferase-based reporter assays. RESULTS: Among the 9 genes that showed ATRA responsiveness in HuH7 or HepG2 cells, 3 genes, OTU domain-containing 7B (OTUD7B), kinesin family member 21B (KIF21B), and odd-skipped related 1 (OSR1) were significantly upregulated in tumor tissue compared to non-tumor tissue, and 4 genes, colony stimulating factor 3 receptor (CSF3R), thioredoxin interacting protein (TXNIP), cytochrome P450 26A1 (CYP26A1), and olfactomedin-like 1 (OLFML1) were downregulated. Frizzled 4 (FZD4) mRNA level was not different between tumor and non-tumor tissue. In tumor tissue, low expression of OTUD7B gene, which was upregulated by ATRA, was significantly associated with a low cancer-specific survival of HCC patients. Functional analyses revealed that OTUD7B negatively regulates nuclear factor κB (NF-κB) signaling and decreased the cell viability by inducing apoptosis in HCC cells. Frizzled 4 (FZD4), which was downregulated by ATRA, was shown to positively regulate Wnt/β-catenin signaling and increased the cell viability in HCC cells. DISCUSSIONS AND CONCLUSION: Our results suggest that the downregulation of OTUD7B expression in tumors facilitates tumor growth and enhances the cancer phenotype, leading to malignancy and poor cancer-specific survival. Therefore, retinoid signaling, which upregulates OTUD7B, prevents tumor development by suppressing aberrant NF-κB signaling in HCC cells, being beneficial for antitumor therapy for HCC. Our results also suggested that Wnt/β-catenin signaling may be a potential target for retinoid signaling to regulate HCC development. Citation Format: Goshi Shiota, Keita Kanki. Identification of molecular targets for retinoid signaling to regulate human hepatocellular carcinoma development: contribution of OTUD7B, a negative regulator of NF-κB signaling, in antitumor effect of retinoic acid. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4234. doi:10.1158/1538-7445.AM2014-4234