Nuclear Localization Of NF-κB Research Articles

First Responders Shape a Prompt and Sharp NF-κB-Mediated Transcriptional Response to TNF-α.

SummaryNuclear factor (NF)-κB controls the transcriptional response to inflammatory signals by translocating into the nucleus, but we lack a single-cell characterization of the resulting transcription dynamics. Here we show that upon tumor necrosis factor (TNF)-α transcription of NF-κB target genes is heterogeneous in individual cells but results in an average nascent transcription profile that is prompt (i.e., occurs almost immediately) and sharp (i.e., increases and decreases rapidly) compared with NF-κB nuclear localization. Using an NF-κB-controlled MS2 reporter we show that the single-cell nascent transcription is more heterogeneous than NF-κB translocation dynamics, with a fraction of synchronized “first responders” that shape the average transcriptional profile and are more prone to respond to multiple TNF-α stimulations. A mathematical model combining NF-κB-mediated gene activation and a gene refractory state is able to reproduce these features. Our work shows how the expression of target genes induced by transcriptional activators can be heterogeneous across single cells and yet time resolved on average.

Abstract 1403: HOTAIR functions through NF-κB pathway in regulating ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the fifth leading cause of cancer-related death among American women. Persistence of OC stem cells (OCSCs) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse. Long non-coding RNA HOXC transcript antisense RNA (HOTAIR) has been shown to be associated with chemoresistance and overexpressed in many types of cancers, including high-grade serous OC (HGSOC). Previously published work has demonstrated that NF-κB was activated by HOTAIR through I-κB inhibition via trimethylation of histone H3 lysine K27, which contributes to chemoresistance in HGSOC. NF-κB-medicated signaling pathways involved in maintaining characteristics of CSCs, such as targeting stem cell markers, CD44, CD133, and ALDH1 has been demonstrated but not well defined in OC. The goals of this study are to understand the mechanism of HOTAIR-mediated NF-κB signaling pathway in regulating OCSCs and develop novel strategies to target OCSCs and overcome OC recurrence and drug resistance. Quantitative RT-PCR analysis revealed that HOTAIR was overexpressed in OCSCs compared to non-OCSCs. In order to produce loss-of-function phenotypes of HOTAIR and investigate the function of this gene, we utilized the paired CRISPR guide RNA design to delete the functional sites of HOTAIR without affecting nearby protein-coding gene. Knockout of HOTAIR re-sensitized OC cells to platinum treatment and significantly decreased (P<0.001) OCSC population and stemness-related phenotypes, including spheroid formation and colony formation ability. ALDH1A1 and expression of other stemness-related genes, including Notch3, PROM1 were significantly decreased by HOTAIR knockout. Overexpression of HOTAIR in OC cells significantly increased (P<0.05) these stem-like characteristics. Furthermore, we showed NF-κB nuclear accumulation and activation in OCSCs compared to non-OCSCs. Knockout of HOTAIR decreased (P<0.01) nuclear localization of NF-κB in OCSCs. Inhibiting NF-κB in using a commercially available pharmacological approach as well as NF-κB knockout both significantly decreased OCSC population and stemness-related phenotypes, as well as ALDH1 protein expression (P< 0.01). However, inhibition of NF-κB has no effect on OCSC characteristics in HOTAIR knockout cells, indicating that HOTAIR may function through constitutively activated NF-κB in regulating OCSCs. We suggest a better understanding of HOTAIR- NF-κB axis in regulating OCSCs will facilitate identifying the key therapeutic target to eliminate residual tumor cells after conventional chemotherapy and prevent OC recurrence and drug resistance. Citation Format: Weini Wang, Fang Fang, Ali R. Özeş, Kenneth P. Nephew. HOTAIR functions through NF-κB pathway in regulating ovarian cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1403.

Alangium longiflorum Merr. Leaf Extract Induces Apoptosis in A549 Lung Cancer Cells with Minimal NFκB Transcriptional Activation

The chemotherapy drug doxorubicin (DOX) is effective in treating many types of cancers. However, due to its pro-inflammatory and cardiotoxic side effects, other remedies have also been explored as alternative treatments. The plant Alangium longiflorum was reported to contain cytotoxic activity against cancer cells, but it is unclear whether this plant would also yield side effects similar to doxorubicin. Hence, this study investigated cytotoxic activity of A. longiflorum leaf extract against lung cancer cells and compared its pro-inflammatory and cardiotoxic side effects with those of DOX. Methods:Cytotoxic activity of A. longiflorum in human lung (A549) and breast (MCF-7) cancer cells was initially assessed by MTT assay and then was compared with doxorubicin. Presence of secondary metabolites in the leaf extract was examined by phytochemical screening. The ability of the plant extract to induce apoptosis was determined by measuring caspase-3/7 activity and apoptosis-related gene expression. Pro-inflammatory response was assessed by quantifying NFκB transcriptional activity and nuclear translocation with dual luciferase reporter and immunofluorescence assays, respectively. Cardiotoxicity was measured using zebrafish as a model organism. Results: A. longiflorum leaf extract displayed high cytotoxic activity against A549 versus MCF-7, which led this study to focus further on A549. Phytochemical screening showed that the extract contained terpenoids, alkaloids, phenols, cardiac glycosides, and tannins. The extract induced apoptosis through activation of caspase-3/7 and upregulation of pro-apoptotic genes without causing NFκB transcriptional activation and nuclear localization. The extract also did not significantly reduce heart function in zebrafish. Conclusion:Overall, our data suggested that extract from leaves of A. longiflorum can have the potential to serve as apoptotic agent towards lung cancer without inducing significant cardiotoxicity.

Suppression of Ocular Vascular Inflammation through Peptide-Mediated Activation of Angiopoietin-Tie2 Signaling.

Persistent inflammation is a complication associated with many ocular diseases. Changes in ocular vessels can amplify disease responses and contribute to vision loss by influencing the delivery of leukocytes to the eye, vascular leakage, and perfusion. Here, we report the anti-inflammatory activity for AXT107, a non-RGD, 20-mer αvβ3 and α5β1 integrin-binding peptide that blocks vascular endothelial growth factor (VEGF)-signaling and activates tyrosine kinase with immunoglobulin and EGF-like domains 2 (Tie2) using the normally inhibitory ligand angiopoietin 2 (Ang2). Tumor necrosis factor α (TNFα), a central inflammation mediator, induces Ang2 release from endothelial cells to enhance its stimulation of inflammation and vascular leakage. AXT107 resolves TNFα-induced vascular inflammation in endothelial cells by converting the endogenously released Ang2 into an agonist of Tie2 signaling, thereby disrupting both the synergism between TNFα and Ang2 while also preventing inhibitor of nuclear factor-κB α (IκBα) degradation directly through Tie2 signaling. This recovery of IκBα prevents nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nuclear localization, thereby blocking NF-κB-induced inflammatory responses, including the production of VCAM-1 and ICAM-1, leukostasis, and vascular leakage in cell and mouse models. AXT107 also decreased the levels of pro-inflammatory TNF receptor 1 (TNFR1) without affecting levels of the more protective TNFR2. These data suggest that AXT107 may provide multiple benefits in the treatment of retinal/choroidal and other vascular diseases by suppressing inflammation and promoting vascular stabilization.

TFEB-NF-\u03baB inflammatory signaling axis: a novel therapeutic pathway of Dihydrotanshinone I in doxorubicin-induced cardiotoxicity

BackgroundDoxorubicin is effective in a variety of solid and hematological malignancies. Unfortunately, clinical application of doxorubicin is limited due to a cumulative dose-dependent cardiotoxicity. Dihydrotanshinone I (DHT) is a natural product from Salvia miltiorrhiza Bunge with multiple anti-tumor activity and anti-inflammation effects. However, its anti-doxorubicin-induced cardiotoxicity (DIC) effect, either in vivo or in vitro, has not been elucidated yet. This study aims to explore the anti-inflammation effects of DHT against DIC, and to elucidate the potential regulatory mechanism.MethodsEffects of DHT on DIC were assessed in zebrafish, C57BL/6 mice and H9C2 cardiomyocytes. Echocardiography, histological examination, flow cytometry, immunochemistry and immunofluorescence were utilized to evaluate cardio-protective effects and anti-inflammation effects. mTOR agonist and lentivirus vector carrying GFP-TFEB were applied to explore the regulatory signaling pathway.ResultsDHT improved cardiac function via inhibiting the activation of M1 macrophages and the excessive release of pro-inflammatory cytokines both in vivo and in vitro. The activation and nuclear localization of NF-κB were suppressed by DHT, and the effect was abolished by mTOR agonist with concomitant reduced expression of nuclear TFEB. Furthermore, reduced expression of nuclear TFEB is accompanied by up-regulated phosphorylation of IKKα/β and NF-κB, while TFEB overexpression reversed these changes. Intriguingly, DHT could upregulate nuclear expression of TFEB and reduce expressions of p-IKKα/β and p-NF-κB.ConclusionsOur results demonstrated that DHT can be applied as a novel cardioprotective compound in the anti-inflammation management of DIC via mTOR-TFEB-NF-κB signaling pathway. The current study implicates TFEB-IKK-NF-κB signaling axis as a previously undescribed, druggable pathway for DIC.

Molecular cloning and immune function study of an oyster IκB gene in the NF-κB signaling pathway

Mass mortality caused by pathogens has severely affected the oyster cultivation industry, but our knowledge about the innate immune system of oysters is still limited. The nuclear factor-κB (NF-κB) signaling pathway plays a vital role in many cellular, developmental, and biological processes. And inhibitor of NF-κB (IκB) proteins can prevent NF-κB nuclear localization and DNA binding, thereby inhibiting NF-κB functions. However, little is known about their existence and function in invertebrates. In this study, a novel homolog of the IκB gene was identified in the Pacific oyster Crassostrea gigas (designated as CgIκB4). The open reading frame of CgIκB4 is 1029 bp and encodes a putative protein of 342 amino acids. Comparative and phylogenetic analyses revealed that the CgIκB4 belongs to the invertebrate IκB family. The results of quantitative real-time PCR showed that CgIκB4 transcripts are expressed in all test tissues, and with the highest expression level in the stomach and labial palpus, and that the expression is significantly induced after LPS, PGN, and poly(I:C) challenges. Importantly, Co-IP assays showed that CgIκB4 can interact with oyster NF-κB family proteins (i.e., CgRel1 and CgRel2) directly. The results of dual-luciferase reporter assays showed an inhibition of CgIκB4 on CgRel1-dependent NF-κB and TNFα activation. These findings demonstrate that CgIκB4 may play an important role in the innate immunity of C. gigas through regulation of NF-κB activity. And the research results will be helpful for deciphering the mechanisms of innate immunity in invertebrates and provide theoretical basis for molecular breeding of disease-resistance oysters.

Zinc Deficiency‐Induced Renal NFkB Activation drives Sodium Chloride Co‐transporter Upregulation.

BackgroundZn2+ deficiency (ZnD) is a worldwide problem. In the United States, 14% of Americans are Zn2+ deficient, which represents 1 out of 7 people. In ZnD populations, the prevalence of hypertension is higher. In our recent studies, we demonstrated that ZnD induces hypertension by promoting renal Na+ reabsorption by the sodium chloride co‐transporter (NCC). However, the exact molecular mechanisms involved in NCC upregulation were undefined. Nuclear factor‐κB (NFκB) is a transcription factor found to play a role in ZnD‐mediated detrimental effects throughout the body.HypothesisAs such, we hypothesized that ZnD drives renal NFκB activation.Experimental DesignTo examine the effects of ZnD on renal NFκB activation, adult, male C57Bl/6 mice were fed a ZnA‐ or ZnD‐diet for 6 weeks. NFκB expression and nuclear translocation were examined by immunohistochemistry. To confirm the role of Zn2+ in NFκB regulation, mouse distal convoluted tubular (mDCT) cells were treated with the Zn2+ chelator ‐ N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethane‐1,2‐diamine (TPEN) or vehicle ± Zn2+ supplementation.ResultsIn ZnD mice, NFκB protein expression and nuclear localization were increased compared to ZnA mice. Consistently, in mDCT cells, TPEN‐induced ZnD stimulated NFκB expression and nuclear translocation. However, Zn2+ supplementation reversed TPEN‐induced NFκB upregulation.ConclusionThese results indicate that 1) NFκB is a Zn2+‐regulated nuclear transcription factor, and 2) ZnD drives renal NFkB activation.SignificanceNFκB represents a potential mediator that drives ZnD‐induced NCC upregulation and consequently renal Na+ reabsorption and hypertension.Support or Funding InformationAmerican Heart Association ‐ Scientist Development Grant #16SDG27080009 National Institutes of Health ‐ R21 Exploratory/Development Grant #R21DK119879

Abstract P6-05-02: Estrogen receptor beta repurposes EZH2 to inhibit oncogenic NFκB signaling in triple negative breast cancer

Abstract Background: Triple Negative Breast Cancer (TNBC) affects approximately 15-20% of BC patients, yet accounts for a disproportionately higher rate of BC morbidity and mortality, in part due to lack of targeted therapies. Using well-validated antibodies, we have found that estrogen receptor beta (ERβ) protein is expressed in approximately 20% of TN breast tumors. We have demonstrated that ligand-mediated activation of ERβ with estradiol (E2) or ERβ-selective agonists decreases tumor cell proliferation, invasion, and migration in vitro, as well as primary tumor growth and metastatic spread in vivo. As such, we aimed to elucidate the mechanisms by which ERβ elicits its anti-cancer effects in TNBC. Methods: RNAseq and ChIPseq were used to identify ERβ and NFκB regulomes and cistromes in TNBC cells. The global effects of ERβ on H3K27Ac and H3K27Me3 were also assessed via ChIPseq. The influence of ERβ on NFκB signaling was determined using co-immunoprecipitation, luciferase assays transcriptional readouts and ChIPseq. The involvement of EZH2 in mediating ERβ’s suppression of NFκB signaling and cancer cell phenotypes was determined using pharmacological approaches. Results: Pathway analysis of ERβ-regulated genes identified NFκB and inflammatory signaling as being highly suppressed in response to E2 treatment. Numerous NFκB target genes were among the most down-regulated genes by ERβ. ChIPseq revealed that ERβ primarily associated with estrogen response elements (EREs), but was also highly enriched at NFκB binding sites following E2 treatment. However, ERβ had no impact on TNFα-mediated NFκB phosphorylation or nuclear localization, but was shown to physically associate with NFκB protein. Using an NFκB reporter construct and qPCR, ERβ was shown to block TNFα-mediated induction of NFκB signaling and NFκB target gene expression. Globally, RNAseq identified 373 genes to be significantly regulated by TNFα in TNBC cells, of which 107 were blocked in the presence of E2+TNFα. Interestingly, E2+TNFα treatment induced novel NFκB binding sites compared to TNFα alone as identified by ChIPseq for NFκB. ChIPseq also demonstrated that ligand-mediated activation of ERβ significantly diminished an activating histone mark (H3K27Ac) at many of these NFκB target genes while enhancing a repressive mark (H3K27Me3). These modifications were shown to occur through the recruitment of the histone methyltransferase, EZH2, to enhancer elements of these NFκB target genes. Drug-mediated blockade of EZH2 activity reversed suppression of NFκB target gene expression by ERβ. Finally, knockdown of NFκB expression inhibited the anti-cancer effects of ERβ while expression of a constitutively active form of NFκB enhanced ERβ’s inhibitory effects. Conclusions: Our data suggest that ERβ elicits its anti-cancer effects, in part, via formation of a novel co-repressor complex consisting of ERβ, NFκB, and EZH2. These data are in keeping with prior observations of the importance of NFκB signaling as it relates to TNBC cell proliferation and invasion, and that decreased expression of NFκB target genes is associated with improved outcomes in TNBC patients. Currently, a Mayo Breast SPORE prospective study is underway to investigate the efficacy of estradiol for the treatment of metastatic ERβ+TNBC and to further evaluate the cross-talk between ERβ and NFκB signaling. Citation Format: Kirsten GM Aspros, Adam W Nelson, Zhenqing Ye, Zhifu Sun, Igor Chernukhin, Jason S Carroll, James N Ingle, Matthew P Goetz, John R Hawse. Estrogen receptor beta repurposes EZH2 to inhibit oncogenic NFκB signaling in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-02.

TF-343 Alleviates Diesel Exhaust Particulate-Induced Lung Inflammation via Modulation of Nuclear Factor-κB Signaling.

Inhalation of diesel exhaust particulate (DEP) causes oxidative stress-induced lung inflammation. This study investigated the protective effects of TF-343, an antioxidant and anti-inflammatory agent, in mouse and cellular models of DEP-induced lung inflammation as well as the underlying molecular mechanisms. Mice were intratracheally instilled with DEP or vehicle (0.05% Tween 80 in saline). TF-343 was orally administered for 3 weeks. Cell counts and histological analysis of lung tissue showed that DEP exposure increased the infiltration of neutrophils and macrophages in the peribronchial/perivascular/interstitial regions, with macrophages harboring black pigments observed in alveoli. TF-343 pretreatment reduced lung inflammation caused by DEP exposure. In an in vitro study using alveolar macrophages (AMs), DEP exposure reduced cell viability and increased the levels of intracellular reactive oxygen species and inflammatory genes (IL-1β, inhibitor of nuclear factor- (NF-) κB (IκB), and Toll-like receptor 4), effects that were reduced by TF-343. A western blot analysis showed that the IκB degradation-induced increase in NF-κB nuclear localization caused by DEP was reversed by TF-343. In conclusion, TF-343 reduces DEP-induced lung inflammation by suppressing NF-κB signaling and may protect against adverse respiratory effects caused by DEP exposure.

Blockade of the Adenosine A3 Receptor Attenuates Caspase 1 Activation in Renal Tubule Epithelial Cells and Decreases Interleukins IL-1β and IL-18 in Diabetic Rats

Diabetic nephropathy (DN) is the main cause of end-stage renal disease, which remains incurable. The progression of DN is associated with progressive and irreversible renal fibrosis and also high levels of adenosine. Our aim was to evaluate the effects of ADORA3 antagonism on renal injury in streptozotocin-induced diabetic rats. An ADORA3 antagonist that was administered in diabetic rats greatly inhibited the levels of inflammatory interleukins IL-1β and IL-18, meanwhile when adenosine deaminase was administered, there was a non-selective attenuation of the inflammatory mediators IL-1β, IL-18, IL-6, and induction of IL-10. The ADORA3 antagonist attenuated the high glucose-induced activation of caspase 1 in HK2 cells in vitro. Additionally, ADORA3 antagonisms blocked the increase in caspase 1 and the nuclear localization of NFκB in the renal tubular epithelium of diabetic rats, both events that are involved in regulating the production and activation of IL-1β and IL-18. The effects of the A3 receptor antagonist resulted in the attenuation of kidney injury, as evidenced by decreased levels of the pro-fibrotic marker α-SMA at histological levels and the restoration of proteinuria in diabetic rats. We conclude that ADORA3 antagonism represents a potential therapeutic target that mechanistically works through the selective blockade of the NLRP3 inflammasome.

In vitro 6-hydroxydopamine-induced neurotoxicity: New insights on NFκB modulation

Neuronal exposure to 6-hydroxydopamine (6-OHDA), a hydroxylated analog of dopamine, constitutes a very useful strategy for studying the molecular events associated with neuronal death in Parkinson's disease. 6-OHDA increases oxidant levels and impairs mitochondrial respiratory chain, thus promoting neuronal injury and death. Despite the extensive use of 6-OHDA in animal models, the exact molecular events triggered by this neurotoxicant at the neuronal level have not been yet fully understood.Human IMR-32 neuroblastoma cells exposed to increasing concentrations of 6-OHDA displayed high levels of reactive oxygen species and increased plasma membrane permeability with concomitant cell viability diminution. As part of the neuronal response to 6-OHDA exposure, the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) p65 subunit was observed. NFκB nuclear localization was also accompanied by an increase of IκB phosphorylation as well as a rise in cyclooxygenase-2 (COX-2) and the prostaglandin receptor, EP4, mRNA levels.Even though the canonical pathways participating in the modulation of NFκB have been extensively described, here we tested the hypothesis that 6-OHDA-induced injury can activate lipid signaling and, in turn, modulate the transcriptional response. 6-OHDA challenge triggered the activation of lipid signaling pathways and increased phosphatidic acid (PA), diacylglycerol and free fatty acid levels in human neuroblastoma cells. The inhibition of PA production was able to prevent the decrease in cell viability triggered by 6-OHDA, the nuclear translocation of NFκB p65 subunit and the rise in COX-2 mRNA expression. Our results indicate that the onset of the inflammatory process triggered by 6-OHDA involves the activation of PA signaling that, in turn, governs NFκB subcellular localization and COX-2 expression.

Genetic inactivation of the phospholipase A2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury.

Peroxiredoxin 6 (Prdx6) is a multifunctional enzyme that serves important antioxidant roles by scavenging hydroperoxides and reducing peroxidized cell membranes. Prdx6 also plays a key role in cell signaling by activating the NADPH oxidase, type 2 (Nox2) through its acidic Ca2+-independent phospholipase A2 (aiPLA2) activity. Nox2 generation of O2·-, in addition to signaling, can contribute to oxidative stress and inflammation such as during sepsis-induced acute lung injury (ALI). To evaluate a possible role of Prdx6-aiPLA2 activity in the pathophysiology of ALI associated with a systemic insult, wild-type (WT) and Prdx6-D140A mice, which lack aiPLA2 but retain peroxidase activity were administered intraperitoneal LPS. LPS-treated mutant mice had increased survival compared with WT mice while cytokines in lung lavage fluid and lung VCAM-1 expression, nitrotyrosine levels, PMN infiltration, and permeability increased in WT but not in mutant mice. Exposure of mouse pulmonary microvascular endothelial cells in primary culture to LPS promoted phosphorylation of Prdx6 and its translocation to the plasma membrane and increased aiPLA2 activity as well as increased H2O2 generation, nitrotyrosine levels, lipid peroxidation, NF-κB nuclear localization, and nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome assembly; these effects were not seen in Nox2 null cells, Prdx6-D140A cells, or WT cells pretreated with MJ33, an inhibitor of aiPLA2 activity. Thus aiPLA2 activity is needed for Nox2-derived oxidant stress associated with LPS exposure. Since inactivation of aiPLA2 reduced mortality and prevented lung inflammation and oxidative stress in this animal model, the aiPLA2 activity of Prdx6 could be a novel target for prevention or treatment of sepsis-induced ALI.

Interleukin-10 Protects Schwann Cells against Advanced Glycation End Products-Induced Apoptosis via NF-κB Suppression.

Demyelination resulting from Schwann cell injury is a main pathological feature of diabetic neuropathy, and a key contributor to this process may be inflammation due to advanced glycation end products (AGEs). Therefore, protection by anti-inflammation agents is anticipated. In this study, we showed that interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits apoptosis of Schwann cells induced by AGEs in vitro. We isolated and cultured Schwann cells from rat sciatic nerves. As detected by flow cytometry, apoptosis of Schwann cells markedly increased following incubation with AGEs for 48 h. However, pretreatment with IL-10 inhibited AGE-induced apoptosis. The effect of IL-10 on NF-κB, which is a very important regulator of inflammation, was also evaluated, and results showed high levels of phospho-NF-κB and nuclear localization of NF-κB in cells incubated with AGEs but low levels of phospho-NF-κB and cytoplasmic localization in the cells incubated with IL-10, indicating the activation of NF-κB by AGEs and inhibition of NF-κB by IL-10. Moreover, incubating Schwann cells with an NF-κB inhibitor (caffeic acid phenethyl ester) for 30 min before adding AGEs mimicked IL-10, lowering the amount of reactive oxygen species and activity of caspase-3 and also decreasing apoptosis in Schwann cells. These results indicate that IL-10 may protect Schwann cells against AGE-induced apoptosis by attenuating oxidative stress via the inhibition of activation of NF-κB.

Schisandra Chinensis Lignans Suppresses the Production of Inflammatory Mediators Regulated by NF-κB, AP-1, and IRF3 in Lipopolysaccharide-Stimulated RAW264.7 Cells.

Schisandra Fructus (SF) is a traditional Chinese herb used in the treatment of inflammatory disorders like hepatitis. One of the main anti-inflammatory components of SF is the lignans. However, the underlying anti-inflammatory mechanism of Schisandra Chinensis lignans (SCL) remains unclear. This study aims to investigate the effects of SCL on inflammatory mediators in lipopolysaccharide-stimulated RAW264.7 cells and explore the underlying mechanism. The production of nitric oxide (NO) was determined by Griess reaction. ELISA was used to determine cytokine levels and chemokines secretion. To estimate protein levels and enzyme activities, we employed Western blotting. Nuclear localization of NF-κB, AP-1, and IRF3 was detected using immunofluorescence analyses. The results showed that SCL significantly reduced the release of inflammatory mediators, including NO and PGE2, which may be related to down-regulation of iNOS and COX-2 expression. The production of cytokines and chemokines was suppressed by SCL treatment. SCL also decreased the phosphorylation of IKKα/β, IκB-α, Akt, TBK1, ERK, p38, JNK, NF-κB (p65), AP-1 (c-Jun), and IRF3 in RAW264.7 macrophages activated with LPS. The nuclear protein levels and nuclear translocation of AP-1, NF-κB and IRF3 were suppressed by SCL. These results indicated that SCL suppressed the IKKα/β/NF-κB, MAPKs/AP-1 and TBK1/IRF3 signaling pathways in LPS-stimulated RAW264.7 macrophages.

Differential expression of NF-κB heterodimer RelA/p50 in human urothelial carcinoma.

BackgroundUrothelial carcinoma (UC) is the fifth most common malignancy that accounts for 5% of all cancers. Diagnostic markers that predict UC progressions are inadequate. NF-κB contributes towards disease progression upon constitutive activation in many solid tumors. The nuclear localization of NF-κB indicates increased transcriptional activity while cytoplasmic localization indicates the inactive protein repository that can be utilized readily by a malignant cell. This study delineates the nuclear and cytoplasmic differential expression of NF-κB heterodimers in UC progression.MethodsThe involvement of the NF-κB proteins in UC was analyzed in silico using cytoscape. The expression of NF-κB heterodimers was analyzed by immunohistochemistry.ResultsPINA4MS app in cytoscape revealed over expression of RelA and suppression of NF-κB1 (p50 precursor) in UC whereas the expression of NF-κB target proteins remained unhindered. Immunohistochemical localization showed nuclear RelA/p50 in low grade UC whereas in high grade only RelA expression was observed. Conversely, cytoplasmic expression of RelA/p50 remained extensive across high and low grade UC tissues (p < 0.005). RelA nuclear and cytoplasmic expression (p < 0.005) was directly proportional to the disease progression. In our study, some of the high-grade UC tissues with squamous differentiation and muscle invasion had extensive nuclear p50 localization. The phenomenon of RelA/p50 expression seen increased in low-grade UC than high grade UC might be due to their interaction with other members of NF-κB family of proteins. Thus, NF-κB RelA/p50 differential expression may play a unique role in UC pathogenesis and can serve as a biomarker for diagnosis.

Abstract 514: Focal Adhesion Kinase Inhibition Prevents Prolonged Nuclear Factor-κB Activation and Reduces Atherosclerosis in ApoE-/- Mice

A key event in atherogenesis is the expression of cell adhesion molecules on endothelial cells (ECs) that recruit leukocytes to the vessel wall, where they accumulate over time and form atherosclerotic plaques filled with fatty lipids. Although lipid-lowering drugs have been beneficial in slowing down atherosclerosis, there is currently no treatment available to reduce the underlying triggers that promote EC activation. Focal adhesion kinase (FAK) is an integrin-associated protein tyrosine kinase that plays a key role in cytokine-induced pro-inflammatory molecule expression; however, the molecular mechanism has not been elucidated. We have revealed that a small molecule FAK inhibitor (PF-271) in human ECs reduces tumor necrosis factor-α (TNF-α)-induced sustained activation of nuclear factor-κB (NF-kB), a major inflammatory transcription factor. While NF-κB is normally bound to inhibitor of NF-κB α (IκBα) in the cytosol, TNF-α leads to rapid activation and nuclear localization of NF-κB after degradation of IκBα by activation of IκB kinase (IKK). Chronic exposure to cytokines leads to oscillations in NF-κB activation as IκBα is continually made by active NF-κB and degraded by active IKK. Surprisingly, we discovered that FAK inhibition blocks prolonged NF-κB activation through a time-dependent increase in IκBα protein stability, suggesting decreased IKK activity. By performing in vitro IKK kinase assays, we found that FAK inhibition did indeed reduce sustained IKK activity as seen by reduced serine phosphorylation of IκBα 3hr after TNF-α stimulation. Since both cell adhesion molecule expression and NF-κB in ECs is important for atherosclerotic lesion development, we tested the efficacy of FAK inhibition on atherogenesis in vivo . We found that oral delivery of PF-271 (twice daily, 8 weeks) significantly reduced atherosclerotic lesion area in ApoE-/- mice fed a western diet. Taken together, our data demonstrates that FAK inhibition potentially reduces atherogenesis by blocking sustained NF-κB signaling through decreased IKK activity.

Icariin abrogates osteoclast formation through the regulation of the RANKL-mediated TRAF6/NF-κB/ERK signaling pathway in Raw264.7 cells

BackgroundIcariin is pharmacologically active prenylated flavonoid glycoside that has various biologic effects such as antioxidant, anticancer, and anti-inflammatory activities. In addition, icariin has been used in Chinese medicine for thousands of years to treat osteoporosis and it is still being used today. However, direct mechanism of icariin in the treatment of bone disease is not understood. PurposeThe purpose of this study is to investigate whether icariin influences RANKL-induced osteoclast formation in murine macrophages. MethodsOsteoclastogenesis was determined by TRAP staining and activity assay. Inhibition of signaling pathways and marker protein expression were evaluated by western blot analysis. The NF-κB (p65) nuclear localization was detected by immunofluorescence assay, and NF-κB/DNA-binding activity was detected by electrophoretic mobility shift assay (EMSA). ResultsIn our study, icariin inhibited the differentiation of pre-osteoclast cells into osteoclasts and suppressed expression of various genes involved in osteoclast formation and bone resorption. Also, icariin blocked the osteoclastogenesis induced by MCF7 and MDA-MB-231 breast cancer cells through inhibition of NF-κB activation. We found that icariin inhibited RANKL-stimulated TRAF-6 expression, and subsequently suppressed the phosphorylation of ERK, but icariin did not show an effect on p38, JNK, and Akt activation. ConclusionThese results indicate that icariin is likely to be a candidate for bone-related disease treatment and that icariin provides insights into the molecular mechanisms that influence RANKL-induced osteoclast differentiation.

CD4+ T Cell Fate Decisions Are Stochastic, Precede Cell Division, Depend on GITR Co-Stimulation, and Are Associated With Uropodium Development.

During an immune response, naïve CD4+ T cells proliferate and generate a range of effector, memory, and regulatory T cell subsets, but how these processes are co-ordinated remains unclear. A traditional model suggests that memory cells use mitochondrial respiration and are survivors from a pool of previously proliferating and glycolytic, but short-lived effector cells. A more recent model proposes a binary commitment to either a memory or effector cell lineage during a first, asymmetric cell division, with each lineage able to undergo subsequent proliferation and differentiation. We used improved fixation and staining methods with imaging flow cytometry in an optimized in vitro system that indicates a third model. We found that cell fates result from stochastic decisions that depend on GITR co-stimulation and which take place before any cell division. Effector cell commitment is associated with mTORC2 signaling leading to uropodium development, while developing memory cells lose mitochondria, have a nuclear localization of NFκB and depend on TGFβ for their survival. Induced, T helper subsets and foxp3+ regulatory T cells were found in both the effector and memory cell lineages. This in vitro model of T cell differentiation is well suited to testing how manipulation of cytokine, nutrient, and other components of the microenvironment might be exploited for therapeutic purposes.

Auraptene regulates Th1/Th2/TReg balances, NF-κB nuclear localization and nitric oxide production in normal and Th2 provoked situations in human isolated lymphocytes

BackgroundAuraptene as member of dietary coumarins, is found in citrus fruits. Former studies have demonstrated its anti-inflammatory and anti-cancer activity. PurposeThe mechanism of action and immune-modulatory property of this compound on human lymphocytes are greatly unknown. Study Design/MethodsThe effect of three concentrations (10, 30 and 90 µM) of auraptene or dexamethasone (0.1 mM) were evaluated on percentage of cell proliferation and nitric oxide (NO) production as well as secretion and gene expression of cytokines, and NF-κB level in PHA-stimulated and non-stimulated lymphocytes. ResultsIn non-stimulated cells, all three concentrations of auraptene significantly increased the gene expression index of IL-10 (P < 0.05–0.001). The IFN-γ gene expression index, IFN-γ/IL-4 and IL-10/IL-4 gene expression ratio were significantly increased due to the high concentration (90 µM) of auraptene treatment compared to control group (P < 0.05–0.001). In PHA stimulation, all three concentrations of the extract significantly decreased proliferation, cytokines (IL-4, IL-10 and IFN-γ) and NF-κB level as well as NO production, but IFN-γ/IL-4 and IL-10/IL-4 ratio were significantly increased compared control group (P < 0.05–0.001). Gene expression of IL-10 and IL-4 was decreased but that of IFN-γ as well as FN-γ/IL-4 and IL-10/IL-4 ratio were significantly increased due to all three concentrations of auraptene. ConclusionThe results showed promoting effects of auraptene on T cell subsets toward Th1 (IFN-γ) and Treg (IL-10), which suggest its therapeutic value for treatment of Th2 cells predominant diseases including allergic disease such as asthma and atopic dermatitis as well as cancers.

Diabetes Activates Periodontal Ligament Fibroblasts via NF-κB In Vivo

Diabetes mellitus increases periodontitis and pathogenicity of the oral microbiome. To further understand mechanisms through which diabetes affects periodontitis, we examined its impact on periodontal ligament fibroblasts in vivo and in vitro. Periodontitis was induced by inoculation of Porphyromonas gingivalis and Fusobacterium nucleatum in normoglycemic and diabetic mice. Diabetes, induced by multiple low-dose injections of streptozotocin increased osteoclast numbers and recruitment of neutrophils to the periodontal ligament, which could be accounted for by increased CXC motif chemokine 2 (CXCL2) and receptor activator of nuclear factor kappa B ligand (RANKL) expression by these cells. Diabetes also stimulated a significant increase in nuclear factor kappa B (NF-κB) expression and activation in periodontal ligament (PDL) fibroblasts. Surprisingly, we found that PDL fibroblasts express a 2.3-kb regulatory unit of Col1α1 (collagen type 1, alpha 1) promoter typical of osteoblasts. Diabetes-enhanced CXCL2 and RANKL expression in PDL fibroblasts was rescued in transgenic mice with lineage-specific NF-κB inhibition controlled by this regulatory element. In vitro, high glucose increased NF-κB transcriptional activity, NF-κB nuclear localization, and RANKL expression in PDL fibroblasts, which was reduced by NF-κB inhibition. Thus, diabetes induces changes in PDL fibroblast gene expression that can enhance neutrophil recruitment and bone resorption, which may be explained by high glucose–induced NF-κB activation. Furthermore, PDL fibroblasts express a regulatory element in vivo that is typical of committed osteoblasts.