NF-κB Binding Research Articles

Dabigatran reduces thrombin-induced neuroinflammation and AD markers in vitro: Therapeutic relevance for Alzheimer's disease.

Vascular risk factors such as atherosclerosis, diabetes, and elevated homocysteine levels are strongly correlated with onset of Alzheimer's disease (AD). Emerging evidence indicates that blood coagulation protein thrombin is associated with vascular and non-vascular risk factors of AD. Here, we examined the effect of thrombin and its direct inhibitor dabigatran on key mediators of neuro-inflammation and AD pathology in the retinoic acid (RA)-differentiated human neuroblastoma cell line SH-SY5Y. SH-SY5Y cells exposed to thrombin concentrations (10-100nM) +/- 250nM dabigatran for 24h were analyzed for protein and gene expression. Electrophoretic mobility shift assay (EMSA) was used to determine DNA binding of NFkB. Western blotting, qRT-PCR and ELISA were used to measure the protein, mRNA, and activity levels of known AD hallmarks and signaling molecules. Dabigatran treatment attenuated thrombin-induced increase in DNA binding of NFκB by 175% at 50nM and by 77% at 100nM thrombin concentration. Thrombin also augmented accumulation of Aβ protein expression and phosphorylation of p38 MAPK, a downstream molecule in the signaling cascade, expression of pro-apoptotic mediator caspase 3, APP, tTau and pTau. Additionally, thrombin increased BACE1 activity, GSK3β expression, and APP, BACE1, Tau and GSK3β mRNA levels. Co-incubation with dabigatran attenuated thrombin-induced increases in the protein, mRNA, and activities of the aforesaid molecules to various extents (between -31% and -283%). Our data demonstrates that thrombin promotes AD-related pathological changes in neuronal cultures and suggests that use of direct oral anticoagulants may provide a therapeutic benefit against thrombin-driven neuroinflammation and downstream pathology in AD.

Cardiac-specific overexpression of HIF-1α during acute myocardial infarction ameliorates cardiomyocyte apoptosis via differential regulation of hypoxia-inducible pro-apoptotic and anti-oxidative genes

HIF-1α acts as the cellular rheostat for oxygen sensing in cardiomyocytes. Overexpression of HIF-1α in the heart during acute myocardial infarction (MI) is known to attenuate cardiac dysfunction by upregulating pro-angiogenic HIF-1α target genes. However, the effect of HIF-1α overexpression on hypoxic cardiomyocyte apoptosis still remains obscure. In this study, we report for the first time that myocardium-targeted nanotized HIF-1α overexpression during MI downregulates cardiomyocyte apoptosis. HIF-1α overexpression attenuates bnip3-mediated apoptosis indirectly by promoting HO-1-induced anti-oxidant response. Chromatin immunoprecipitation experiment revealed that HIF-1α overexpression in hypoxic cardiomyocytes increases binding of HIF-1α to the hypoxia-responsive element in the promoter of its target anti-oxidant gene ho-1 which is known to attenuate ROS accumulation. ROS accumulation in hypoxic cardiomyocytes causes cysteine oxidation of the DNA-binding p50 subunit of NFκB, which hampers NFκB binding to κB-responsive genes like bnip3. Downregulated oxidative stress due to HIF-1α overexpression leads to decline in cysteine oxidation of NFκBp50, causing NFκB to bind to the promoter of bnip3 as a transcriptional repressor. Therefore HIF-1α overexpression-mediated attenuation of cardiomyocyte apoptosis occurs by transcriptional repression of bnip3 by NFκB. Our study thus reveals that downregulation of bnip3-mediated cardiomyocyte apoptosis occurs via ho-1 upregulation upon HIF-1α overexpression during MI, despite both being HIF-1α target genes. The cross-regulation of HIF-1α and NFκB-mediated pathways effectively downregulates apoptosis due to HIF-1α overexpression during MI, which can be exploited for possible therapeutic intervention.

The negative feedback loop of NF-κB/miR-376b/NFKBIZ in septic acute kidney injury.

Sepsis is the leading cause of acute kidney injury (AKI). However, the pathogenesis of septic AKI remains largely unclear. Here, we demonstrate a significant decrease of microRNA-376b (miR-376b) in renal tubular cells in mice with septic AKI. Urinary miR-376b in these mice was also dramatically decreased. Patients with sepsis with AKI also had significantly lower urinary miR-376b than patients with sepsis without AKI, supporting its diagnostic value for septic AKI. LPS treatment of renal tubular cells led to the activation of NF-κB, and inhibition of NF-κB prevented a decrease of miR-376b. ChIP assay further verified NF-κB binding to the miR-376b gene promoter upon LPS treatment. Functionally, miR-376b mimics exaggerated tubular cell death, kidney injury, and intrarenal production of inflammatory cytokines, while inhibiting miR-376b afforded protective effects in septic mice. Interestingly, miR-376b suppressed the expression of NF-κB inhibitor ζ (NFKBIZ) in both in vitro and in vivo models of septic AKI. Luciferase microRNA target reporter assay further verified NFKBIZ as a direct target of miR-376b. Collectively, these results illustrate the NF-κB/miR-376b/NFKBIZ negative feedback loop that regulates intrarenal inflammation and tubular damage in septic AKI. Moreover, urinary miR-376b is a potential biomarker for the diagnosis of AKI in patients with sepsis.

Constitutive Activation of NF-κB in Primary Adult T-Cell Leukemia Cells

AbstractHuman T-cell leukemia virus type I (HTLV-I) is an etiologic agent of adult T-cell leukemia (ATL). The viral protein Tax induces the activation and nuclear translocalization of transcription factor NF-κB, which is proposed to play a crucial role in the transformation of T cells by HTLV-I. However, the HTLV-I genes including Tax are not expressed significantly in primary leukemic cells from ATL patients. In this study, we examined the basis for NF-κB activation in freshly isolated leukemic cells from ATL patients. We found that leukemic cells from ATL patients, like HTLV-I–infected T-cell lines, display constitutive NF-κB DNA binding activity and increased degradation of IκBα (an inhibitor of NF-κB). Whereas the NF-κB binding activity in Tax-expressing T-cell lines consisted mostly of p50/c-Rel, fresh ATL samples contained p50/p50 and p50/p65 heterodimers. One T-cell line derived from ATL leukemic cells, TL-Om1, displayed constitutive NF-κB activity, as well as enhanced degradation of IκBα, despite the lack of detectable Tax expression. Interestingly, the NF-κB in TL-Om1 consists of p50/p50 and p50/p65 like that in fresh primary leukemic cells. Our results suggest that activation of NF-κB occurs through a Tax-independent mechanism in leukemic cells of ATL patients, possibly due to differential NF-κB subunit activation.

BRD4 inhibition and FXR activation, individually beneficial in cholestasis, are antagonistic in combination.

Activation of farnesoid X receptor (FXR) by obeticholic acid (OCA) reduces hepatic inflammation and fibrosis in patients with primary biliary cholangitis (PBC), a life-threatening cholestatic liver failure. Inhibition of bromodomain-containing protein 4 (BRD4) also has antiinflammatory, antifibrotic effects in mice. We determined the role of BRD4 in FXR function in bile acid (BA) regulation and examined whether the known beneficial effects of OCA are enhanced by inhibiting BRD4 in cholestatic mice. Liver-specific downregulation of BRD4 disrupted BA homeostasis in mice, and FXR-mediated regulation of BA-related genes, including small heterodimer partner and cholesterol 7 alpha-hydroxylase, was BRD4 dependent. In cholestatic mice, JQ1 or OCA treatment ameliorated hepatotoxicity, inflammation, and fibrosis, but surprisingly, was antagonistic in combination. Mechanistically, OCA increased binding of FXR, and the corepressor silencing mediator of retinoid and thyroid hormone receptor (SMRT) decreased NF-κB binding at inflammatory genes and repressed the genes in a BRD4-dependent manner. In patients with PBC, hepatic expression of FXR and BRD4 was significantly reduced. In conclusion, BRD4 is a potentially novel cofactor of FXR for maintaining BA homeostasis and hepatoprotection. Although BRD4 promotes hepatic inflammation and fibrosis in cholestasis, paradoxically, BRD4 is required for the antiinflammatory, antifibrotic actions of OCA-activated FXR. Cotreatment with OCA and JQ1, individually beneficial, may be antagonistic in treatment of liver disease patients with inflammation and fibrosis complications.

Regulation of dimethylarginine dimethylaminohydrolase 2 expression by NF-κB acetylation.

Nitric oxide (NO) serves a crucial role in the kidney and is synthesized by NO synthase (NOS). Asymmetrical dimethylarginine is an endogenous inhibitor of NOS that is metabolized by dimethylarginine dimethylaminohydrolase (DDAH). To investigate the role of acetylation in DDAH2 expression, 293 cells were treated with trichostatin A (TSA), a deacetylase inhibitor and the mRNA and protein levels were assessed using quantitative PCR and western blotting respectively. Its promoter activity was detected using a luciferase assay. The effect of TSA on NF-κB acetylation was tested after immunoprecipitation. The binding of NF-κB to the DDAH2 promoter was analyzed using an electrophoretic mobility shift assay and chromatin immunoprecipitation. TSA upregulated DDAH2 expression and transcriptional activity of the DDAH2 promoter through a NF-κB responsive element, which is located at the -1582 to -1573 position of the DDAH2 promoter. Furthermore, TSA treatment promoted NF-κB acetylation, resulting in enhanced NF-κB binding affinity to its binding site both in vitro and in vivo. Taken together, the present study demonstrated that NF-κB acetylation upregulated DDAH2 expression by enhancing the binding ability of NF-κB to the DDAH2 promoter, resulting in increased promoter activity. The results provided a possible mechanism underlying the regulation of NO production in renal cells and a potential target for treating certain NO-associated renal disorders.

Hepatitis B virus core protein promotes the expression of neuraminidase 1 to facilitate hepatocarcinogenesis

AbstractNeuraminidase 1 (NEU1) has been reported to be associated with hepatocellular carcinoma (HCC). However, the function and associated molecular mechanisms of NEU1 in hepatitis B virus (HBV)-related HCC have not been well investigated. In the present study, the expression of NEU1 mediated by HBV and HBV core protein (HBc) was measured in hepatoma cells. The expression of NEU1 protein was detected via immunohistochemical analysis in HBV-associated HCC tissues. The role of NEU1 in the activation of signaling pathways and epithelial-mesenchymal transition (EMT) and the proliferation and migration of hepatoma cells mediated by HBc was assessed. We found that NEU1 was upregulated in HBV-positive hepatoma cells and HBV-related HCC tissues. HBV promoted NEU1 expression at the mRNA and protein level via HBc in hepatoma cells. Mechanistically, HBc was able to enhance the activity of the NEU1 promoter through NF-κB binding sites. In addition, through the increase in NEU1 expression, HBc contributed to activation of downstream signaling pathways and EMT in hepatoma cells. Moreover, NEU1 facilitated the proliferation and migration of hepatoma cells mediated by HBc. Taken together, our findings provide novel insight into the molecular mechanism underlying the oncogenesis mediated by HBc and demonstrate that NEU1 plays a vital role in HBc-mediated functional abnormality in HCC. Thus, NEU1 may serve as a potential therapeutic target in HBV-associated HCC.

A Synthetic CPP33-Conjugated HOXA9 Active Domain Peptide Inhibits Invasion Ability of Non-Small Lung Cancer Cells

Homeobox A9 (HOXA9) expression is associated with the aggressive growth of cancer cells and poor prognosis in lung cancer. Previously, we showed that HOXA9 can serve as a potential therapeutic target for the treatment of metastatic non-small cell lung cancer (NSCLC). In the present study, we have carried out additional studies toward the development of a peptide-based therapeutic agent. Vectors expressing partial DNA fragments of HOXA9 were used to identify a unique domain involved in the inhibition of NSCLC cell invasion. Next, we performed in vitro invasion assays and examined the expression of EMT-related genes in transfected NSCLC cells. The C-terminal fragment (HOXA9-C) of HOXA9 inhibited cell invasion and led to upregulation of CDH1 and downregulation of SNAI2 in A549 and NCI-H1299 cells. Reduced SNAI2 expression was consistent with the decreased binding of transcription factor NF-kB to the SNAI2 promoter region in HOXA9-C overexpressing cells. Based on the above results, we synthesized a cell-permeable peptide, CPP33-HADP (HOXA9 active domain peptide), for lung-specific delivery and tested its therapeutic efficiency. CPP33-HADP effectively reduced the invasion ability of NSCLC cells in both in vitro and in vivo mouse models. Our results suggest that CPP33-HADP has significant potential for therapeutic applications in metastatic NSCLC.

Rice Bran Extract Protected against LPS-Induced Neuroinflammation in Mice through Targeting PPAR-γ Nuclear Receptor.

PPAR-γ anti-inflammatory functions have received significant attention since its agonists have been shown to exert a wide range of protective effects in many experimental models of neurologic diseases. Rice bran is very rich in polyunsaturated fatty acids, which are reported to act as PPAR-γ partial agonists. Herein, the anti-inflammatory effect of rice bran extract (RBE) through PPAR-γ activation was evaluated in LPS-induced neuroinflammatory mouse model in comparison to pioglitazone (PG) using 80 Swiss albino mice. RBE (100mg/kg) and PG (30mg/kg) were given orally for 21days and LPS (0.25mg/kg) was injected intraperitoneally for the last 7days. TNF-α and COX-2 brain contents were evaluated by real-time PCR and immunohistochemical analysis. In addition, NFκB binding to its response element was evaluated alongside with the effect of treatments on IκB gene expression. Furthermore, PPAR-γ sumoylation was also studied. Finally, histopathological examination was performed for different brain areas. RBE administration was found to protect against the LPS-induced inflammatory effects by decreasing the inflammatory mediator expression in mice brains. It also decreased PPAR-γ sumoylation without significant effect on IκB expression or NFκB binding to its response element. The majority of the effects were attenuated in presence of PPAR-γ antagonist (GW9662). Level of significance was set to P < 0.05. Such findings highlight the agonistic effect of RBE component(s) on PPAR-γ and support the hypothesis of involvement of PPAR-γ activation in its neuroprotective effect.

Sustained TNF-α stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness.

Transcriptional memory allows certain genes to respond to previously experienced signals more robustly. However, whether and how the key proinflammatory cytokine TNF-α mediates transcriptional memory are poorly understood. Using HEK293F cells as a model system, we report that sustained TNF-α stimulation induces transcriptional memory dependent on TET enzymes. The hypomethylated status of transcriptional regulatory regions can be inherited, facilitating NF-κB binding and more robust subsequent activation. A high initial methylation level and CpG density around κB sites are correlated with the functional potential of transcriptional memory modules. Interestingly, the CALCB gene, encoding the proven migraine therapeutic target CGRP, exhibits the best transcriptional memory. A neighboring primate-specific endogenous retrovirus stimulates more rapid, more strong, and at least 100-fold more sensitive CALCB induction in subsequent TNF-α stimulation. Our study reveals that TNF-α-mediated transcriptional memory is governed by active DNA demethylation and greatly sensitizes memory genes to much lower doses of inflammatory cues.

Immunogenomics of Aplastic Anemia: The Role of HLA Somatic Mutations and the HLA Evolutionary Divergence

Downregulation of class I human leukocyte antigen (HLA)-restricted antigen presentation has been identified as mechanism of immune-escape in many malignant and non-malignant disorders. In idiopathic aplastic anemia (AA), evolution of immune-privileged paroxysmal nocturnal hemoglobinuria (PNH) clones has been attributed to immune escape due to deficiency of GPI-anchored protein in the context of T-cell mediated autoimmunity. However, other mechanisms of clonal selection may also operate with or independently of PNH. Our group first described the presence of both somatic uniparental disomy (UPD) and microdeletions of the HLA region leading to loss of heterozygozity (LOH) and/or haploinsuffciency.1 Later the proof-of-concept of somatic mutations in HLA class I was provided.2 Mechanistically, HLA LOH leads to loss of an allele involved in the presentation of immune-dominant peptides, while haploinsufficiency may decrease the presentation threshold. Moreover, the general level of individual structural diversity of HLA molecules may determine the ability to present diverse targets, eventually derived from auto-antigens, and functionally would operate in the opposite direction to HLA LOH. In this scenario, we hypothesize that defects in both class I and II HLA loci may constitute different patterns of immune escape, reducing respectively CD8+ and CD4+ related activation and thus contributing to rescue hematopoietic stem cells from the immune attack. Furthermore, our idea is that the immune-escape environment may be related to the grade of HLA evolutionary divergence (HED), a metric that, accounting for the degree of structural diversity within a particular locus, represents an indirect measure of the antigenic landscape that the hematopoietic target cell is able to present (see abstract #:142693). Using a deep targeted HLA NGS panel and a newly developed in-house bioinformatic pipeline (characterized by stringent criteria for alignment, preprocessing and variant calling in the HLA region, based on the IPD IMGT/HLA database, Fig.A), we studied a large cohort of patients with idiopathic bone marrow failures (AA n=75, AA/MDS=10). In addition, we determined the impact of inter-loci HED on the probability to acquire somatic hits in HLA genes. Overall, 29 somatic HLA mutations were found in 16 patients (18%) at a median VAF of 11% (range: 2-93%):12 in class I (41%) and 17 in class II (59%), with 5 patients carrying mutations in both classes (Fig.B, C, D). The majority of those events (N=21, 72%) occurred in subjects also harbouring a PNH clone of small size (12 out 16 patients, median PNH clone size 1% [range:1-46%]). Most mutated loci were A and C for class I and DQB1 for class II (Fig. C, D); 9 mutations were identified as missense, with disruptive changes, 7 were intronic indels while 13 hits were localized in 5' or 3' untranslated regions (UTRs) (Fig.E, F). Through a computational prediction of the HLA regulatory domains involved in the UTR aberrations, we identified domains essential for the binding of GATA-1, RXRbeta, SP-1 and NFKB. The impairment of those regions may affect the transcription of HLA complexes. AA HLA mutant cases had more frequently a severe disease at diagnosis (severe AA: 81% vs. 60%, respectively in HLA mutated vs non mutated cases) and were in most part responders to immunosuppressive therapy (complete/partial responses: 75% vs 50% in HLA mutated vs non mutated patients). Within the AA/MDS group instead HLA mutations were found in 4 out of 10 patients (40%), including of note three -7/del7q cases. Using Pierini and Lenz algorithm3 to determine inter-class HED, we found that HLA mutations tended to occur more often in patients with a high inter-class mean HED (94% vs 72% in non mutated group, p=.001, Fig. G), consistent with the idea that higher structural diversity of HLA molecules may induce more pervasive auto-immune responses, stronger immune pressure and ultimately the establishment of immune-escape mechanisms. In summary, our results indicate the importance of class-I and -II HLA loci somatic hits as markers of autoimmunity and thereby the severity of the immune selection pressure, configuring possibly alternative mechanisms of immune-escape, in addition to immune privileged PNH clones. This environment may ultimately facilitate leukemic clonal expansion in AA-MDS setting. Disclosures Patel: Alexion: Other: educational speaker. Peffault De Latour:Apellis: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Research Funding. Maciejewski:Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau.

MiR-673/menin/JunD axis modulates hyperglycemia-induced oxidative stress and inflammation in the diabetic heart

Abstract Background Hyperglycemia-induced reactive oxygen species generation in diabetic heart contributes to myocardial dysfunction. JunD, a member of the activated protein 1 (AP-1) family of transcription factors, is emerging as a major gatekeeper against oxidative stress. Previous studies have shown that downregulation of AP-1 transcription factor JunD is involved in vascular aging and heart failure. However, the role of JunD in diabetes-induced myocardial dysfunction is unknown. Purpose The present study was designed to investigate whether hyperglycemia-driven epigenetic regulation of JunD contributes to oxidative stress, inflammation and myocardial dysfunction in the diabetic heart. Methods Diabetes (DB) was induced in C57BL/6 wild-type (WT) mice by streptozotocin. After four weeks of DB, left ventricular (LV) function was assessed by standard and 2D speckle-tracking echocardiography in both groups (n=10). Then, the animals were euthanized and LV specimens were collected to determine JunD mRNA and protein expression as well as superoxide anion production by ESR spectroscopy. Chromatin modifications of JunD gene promoter were assessed by chromatin immunoprecipitation. Isolated DNA was analyzed for promoter methylation following Methylminer kit. Cardiac biopsies were collected from age-matched patients with and without diabetes. Results DB mice showed LV dysfunction with reduced ejection fraction and fractional shortening. JunD mRNA and protein expression were reduced in the myocardium of DB as compared to control mice. JunD downregulation was associated with oxidative stress, increased NF-kB binding activity and expression of inflammatory mediators. Accordingly, expression of free radical scavenger superoxide dismutase 1 and aldehyde dehydrogenase 2 was reduced, whereas nicotinamide adenine dinucleotide phosphate oxidase subunits NOX2 and NOX4 were upregulated in DB. A reduction of JunD mRNA and protein expression was confirmed in LV specimens obtained from patients with diabetes. The downregulation of JunD was epigenetically regulated by promoter hypermethylation and histone modifications. Post-translational repression by tumor suppressor menin also contributed to JunD downregulation. Indeed, menin was significantly upregulated in DB hearts and co-immunoprecipitation experiments confirmed the binding of menin to JunD. Furthermore, rat ventricular myocytes exposed to high glucose (HG) showed increased menin expression. We found that miR-673 targeting menin was downregulated in hearts of DB mice. Reprogramming miR-673 in HG-treated myocytes was able to restore both menin and JunD expression to control levels. Conclusions Our findings show that downregulation of AP-1 transcription factor JunD contributes to diabetes-induced myocardial dysfunction and miR-673/menin/JunD represents a novel molecular axis involved in hyperglycemia-induced ROS-driven cardiac damage. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): European Society of Cardiology (ESC) Research Grant 2017

Protective effect of corynoline in a murine allergic rhinitis model via inhibition of caspase-1/NF-κB.

Allergic rhinitis (AR) is a serious public health concern worldwide. Therefore, the present study was conducted to scrutinize the protective effect of corynoline (COR) against ovalbumin (OVA)-induced AR in BALB/c mice. The effect of COR was investigated on various parameters, such as nose-rub score, histamine intensity, level of cytokines, and NF-κB binding activity. It was found that COR causes a significant reduction in the nose-rub score with a reduction in histamine intensity. It also causes reductions in cytokines, such as TNF-α, IL-1β, and MIP-2, in comparison to OVA-challenged mice. COR reduces the gene expression of active caspase-1 in Western blot analysis, together with inhibition of NF-κB binding activity. The inhibitory effect on NF-κB binding was further substantiated by docking analysis, where COR excellently docked into the active site of NF-κB via the creation of H-bond and π-cation interactions with Lys145. Taken altogether, our results demonstrated that COR could be used as a potential therapeutic agent against AR.

Production of fever mediator PGE2 in human monocytes activated with MDP adjuvant is controlled by signaling from MAPK and p300 HAT: Key role of T cell derived factor

Fever and inflammatory responses were observed in some subjects in early clinical trials of vaccines adjuvanted with muramyl dipeptide (MDP), a NOD2 agonist. Biosynthesis of Prostaglandin E2 (PGE2) that transmits febrile signals to the brain is controlled by an inducible enzyme, Cyclooxygenase 2 (COX-2). MDP alone was not sufficient to induce expression of COX-2 and PGE2 production in vitro. Conditioned medium prepared from Peripheral Blood Mononuclear Cells (PBMCs)-derived CD3-bead purified human T cells (TCM) dramatically increased COX2 gene transcription, COX-2 protein expression, and PGE2 production in MDP-treated monocytes. We explored epigenetic changes at the COX2 promoter using Chromatin Immunoprecipitation assay (ChIP). Increase in COX2 transcription correlated with increased recruitment of RNA polymerase II (Pol II) and p300 histone acetyl transferase (HAT) to the COX2 promoter in monocytes activated with MDP and TCM. The role of p300 HAT was confirmed by using C646, an inhibitor of p300, that reduced binding of acetylated H3 and H4 histones at the COX2 promoter, COX2 transcription, and PGE2 production in monocytes. Binding of p300, Nuclear Factor Kappa B (NF-κB), and Pol II to the COX2 promoter was also sensitive to inhibitors of Mitogen-Activated Protein Kinase (MAPK) pathway and to antibodies against Macrophage-1 (Mac-1) integrin in MDP/TCM-treated monocytes. Importantly, recombinant Glycoprotein Ib alfa (GPIbα), the recently identified factor in TCM, increased binding of NF-κB, p300, and of Pol II to the COX2 promoter and COX2 transcription in MDP-treated monocytes. Our findings suggest that a second signal through Mac-1 and MAPK is triggered by a T cell derived soluble GPIbα protein leading to the assembly of the transcription machinery at the COX2 promoter and production of PGE2 in human monocytes in response to MDP/NOD2 activation.

Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

Forkhead box M1 (FOXM1) and nuclear factor kappa B (NF-ĸB) are oncogenic drivers in liver cancer that positively regulate each other. We showed that methionine adenosyltransferase 1A (MAT1A) is a tumor suppressor in the liver and inhibits NF-ĸB activity. Here, we examined the interplay between FOXM1/NF-κB and MAT1A in liver cancer. We examined gene and protein expression, effects on promoter activities and binding of proteins to promoter regions, as well as effects of FOXM1 inhibitors T0901317 (T0) and forkhead domain inhibitory-6 (FDI-6) in vitro and in xenograft and syngeneic models of liver cancer. We found, in both hepatocellular carcinoma and cholangiocarcinoma, that an induction in FOXM1 and NF-κB expression is accompanied by a fall in MATα1 (protein encoded by MAT1A). The Cancer Genome Atlas data set confirmed the inverse correlation between FOXM1 and MAT1A. Interestingly, FOXM1 directly interacts with MATα1 and they negatively regulate each other. In contrast, FOXM1 positively regulates p50 and p65 expression through MATα1, given that the effect is lost in its absence. FOXM1, MATα1, and NF-κB all bind to the FOX binding sites in the FOXM1 and MAT1A promoters. However, binding of FOXM1 and NF-κB repressed MAT1A promoter activity, but activated the FOXM1 promoter. In contrast, binding of MATα1 repressed the FOXM1 promoter. MATα1 also binds and represses the NF-κB element in the presence of p65 or p50. Inhibiting FOXM1 with either T0 or FDI-6 inhibited liver cancer cell growth in vitro and in vivo. However, inhibiting FOXM1 had minimal effects in liver cancer cells that do not express MAT1A. We have found a crosstalk between FOXM1/NF-κB and MAT1A. Up-regulation in FOXM1 lowers MAT1A, but raises NF-κB, expression, and this is a feed-forward loop that enhances tumorigenesis.

Emodin Reverses Gemcitabine Resistance of Pancreatic Cancer Cell Lines Through Inhibition of IKKβ/NF-κB Signaling Pathway.

BackgroundPancreatic cancer is one of the most malignant tumors, and gemcitabine has been considered as the standard treatment and been widely utilized as a first-line drug for advanced pancreatic cancer, but gemcitabine-resistance always occurs after a short period of treatment.MethodsTwo pancreatic cancer cell lines Panc-1 and MIA-PaCa-2 were used as the study subject and their gemcitabine-resistant cells were established. Both drug-resistant cells were divided into four groups: blank, emodin, gemcitabine, and emodin+gemcitabine. Cell viability was detected by MTT assay. Flow cytometry was performed to detect cell apoptosis rate and P-gp function. Quantitative real-time polymerase chain reaction and Western blotting were used to detect Survivin, XIAP, Caspase-9/3, NF-κB p65, IKKβ and IκB-α mRNA/protein expressions, respectively. Electrophoretic mobility shift assay (EMSA) was performed to detect NF-κB binding activity. Rhodamine 123 efflux assay was used to detect P-gp function.ResultsEmodin could inhibit cell activity in all cell lines. Both emodin and gemcitabine can significantly increase the apoptosis rate, and the combination of the two drugs can further significantly increase the apoptosis rate in normal pancreatic cancer cell lines. In both drug-resistant pancreatic cancer cell lines, it can be observed that although gemcitabine can increase the apoptosis rate, the effect of promoting apoptosis is significantly lower than that of emodin; the drug combination can still significantly increase the apoptosis rate on the basis of emodin alone. Emodin can significantly reduce the mRNA and protein expression levels of Survivin, XIAP, NF-κB, and IKKβ, and significantly increase the mRNA and protein expression levels of Caspase-3/9 and IκB-α. Emodin significantly reduced NF-κB activity and emodin significantly promoted P-gp fluorescence intensity from Rhodamine 123 efflux assay.ConclusionEmodin inhibits the expression of IKKβ, thereby inhibiting the expression and activity of downstream NF-κB, and inhibits P-gp function at the same time, ultimately achieving the purpose of reversing the drug-resistance of pancreatic cancer cell lines.

Adenovirus 7 Induces Interlukin-6 Expression in Human Airway Epithelial Cells via p38/NF-κB Signaling Pathway.

Human Adenovirus (AdV) infection is very common and usually has a significant impact on children. AdV-induced inflammation is believed to be one of the main causes of severe symptoms. However, an inflammatory response profile in the airway in AdV-infected children is still lacking, and the mechanism underlying AdV-induced inflammation in the airway is also poorly understood. In the current study, we determined the expression of a panel of inflammation cytokines in the airway samples from AdV 7 infected children and further investigated the molecular mechanism underlying AdV 7-induced cytokine expression. Our results showed that eight out of 13 tested inflammatory cytokines were significantly increased in nasal washes of AdV 7-infected children comparing to healthy control, with IL-6 showing the highest enhancement. AdV 7 infection of bronchial epithelial cell line and primary airway epithelial cells confirmed that AdV 7 increased IL-6 mRNA and protein expression in an infection dose-dependent manner. Promoter analysis revealed that AdV 7 infection transactivated IL-6 promoter and a NF-κB binding site in IL-6 promoter was involved in the transactivation. Further analysis showed that upon AdV 7 infection, NF-κB p65 was phosphorylated and translocated into nucleus and bound onto IL-6 promoter. Signaling pathway analysis revealed that p38/NF-κB pathway was involved in AdV 7 infection induced IL-6 elevation. Taken together, our study shows that AdV 7 infection triggers the expression of a range of inflammatory cytokines including IL-6 in the airway of infected children, and AdV 7 enhances IL-6 expression by transactivating IL-6 promoter via p38/NF-κB signaling pathway. Findings of our current study have provided more information toward a better understanding of AdV-induced airway inflammation, which might also benefit the development of intervention strategies.

TNF-Receptor-Associated Factor 3 in Litopenaeus vannamei Restricts White Spot Syndrome Virus Infection Through the IRF-Vago Antiviral Pathway

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are vital signaling adaptor proteins for the innate immune response and are involved in many important pathways, such as the NF-κB- and interferon regulatory factor (IRF)-activated signaling pathways. In this study, the TRAF3 ortholog from the shrimp Litopenaeus vannamei (LvTRAF3) was cloned and characterized. LvTRAF3 has a transcript of 3,865 bp, with an open reading frame (ORF) of 1,002 bp and encodes a polypeptide of 333 amino acids, including a conserved TRAF-C domain. The expression of LvTRAF3 in the intestine and hemocyte was up-regulated in response to poly (I:C) challenge and white spot syndrome virus (WSSV) infection. RNAi knockdown of LvTRAF3 in vivo significantly increased WSSV gene transcription, viral loads, and mortality in WSSV-infected shrimp. Next, we found that LvTRAF3 was not able to induce the activation of the NF-κB pathway, which was crucial for synthesis of antimicrobial peptides (AMPs), which mediate antiviral immunity. Specifically, in dual-luciferase reporter assays, LvTRAF3 could not activate several types of promoters with NF-κB binding sites, including those from WSSV genes (wsv069, wsv056, and wsv403), Drosophila AMPs or shrimp AMPs. Accordingly, the mRNA levels of shrimp AMPs did not significantly change when TRAF3 was knocked down during WSSV infection. Instead, we found that LvTRAF3 signaled through the IRF-Vago antiviral cascade. LvTRAF3 functioned upstream of LvIRF to regulate the expression of LvVago4 and LvVago5 during WSSV infection in vivo. Taken together, these data provide experimental evidence of the participation of LvTRAF3 in the host defense to WSSV through the activation of the IRF-Vago pathway but not the NF-κB pathway.

The Epstein-Barr virus-encoded G protein-coupled receptor BILF1 upregulates ICAM-1 through a mechanism involving the NF-қB pathway.

Although the Epstein-Barr virus (EBV) infection is usually asymptomatic, a primary encounter with the virus can cause mononucleosis. EBV infection is also strongly associated with lymphoma and epithelial cancers. The structure and infection mechanism of EBV have been well studied, but the EBV-encoded G protein-coupled receptor, BILF1, is not fully understood. Here, it was found that the EBV BILF1 was expressed early in the viral lytic cycle and its ectopic expression strikingly upregulated the ICAM-1 expression in Raji cells. The positive effect of BILF1 on the ICAM-1 promoter was observed and the BILF1 deficiency attenuated ICAM-1 promoter activity. Moreover, NF-κB binding sites were important for the regulation of ICAM-1 promoter by BILF1. Furthermore, BILF1 reduced the constitutive level of the IқB-a protein and increased the amount of nuclear NF-қB in Raji cells. In conclusion, this study determined that BILF1 upregulated ICAM-1 in a mechanism involving NF-қB.

Toll-like Receptor (TLR)-induced Rasgef1b expression in macrophages is regulated by NF-κB through its proximal promoter

Ras Guanine Exchange Factor (RasGEF) domain family member 1b is encoded by a Toll-like receptor (TLR)-inducible gene expressed in macrophages, but transcriptional mechanisms that govern its expression are still unknown. Here, we have functionally characterized the 5' flanking Rasgef1b sequence and analyzed its transcriptional activation. We have identified that the inflammation-responsive promoter is contained within a short sequence (-183 to +119) surrounding the transcriptional start site. The promoter sequence is evolutionarily conserved and harbors a cluster of five NF-κB binding sites. Luciferase reporter gene assay showed that the promoter is responsive to TLR activation and RelA or cRel, but not RelB, transcription factors. Besides, site-directed mutagenesis showed that the κB binding sites are required for maximal promoter activation induced by LPS. Analysis by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that the promoter is located in an accessible chromatin region. More important, Chromatin Immunoprecipitation sequencing (ChIP-seq) showed that RelA is recruited to the promoter region upon LPS stimulation of bone marrow-derived macrophages. Finally, studies with Rela-deficient macrophages or pharmacological inhibition by Bay11-7082 showed that NF-κB is required for optimal Rasgef1b expression induced by TLR agonists. Our data provide evidence of the regulatory mechanism mediated by NF-κB that facilitates Rasgef1b expression after TLR activation in macrophages.