Nuclear NF-κB Activity Research Articles

Identification of an NF-κB p50/p65-responsive site in the human MIR155HG promoter

BackgroundMicroRNA-155 (miR-155) is the diced product of the MIR155HG gene. miR-155 regulates the expression of many immune-specific transcripts, is overexpressed in many human lymphomas, and has oncogenic activity in mouse transgenic models. MIR155HG has been proposed to be a target gene for transcription factor NF-κB largely due to the positive correlation between high nuclear NF-κB activity and increased miR-155 expression following treatment with NF-κB inducers or in subsets of hematopoietic cancers. Nevertheless, direct regulation of the human MIR155HG promoter by NF-κB has not been convincingly demonstrated previously.ResultsThis report shows that induction of NF-κB activity rapidly leads to increased levels of both primary MIR155HG mRNA and mature miR-155 transcripts. We have mapped an NF-κB-responsive element to a position approximately 178 nt upstream of the MIR155HG transcription start site. The -178 site is specifically bound by the NF-κB p50/p65 heterodimer and is required for p65-induced reporter gene activation. Moreover, the levels of miR-155 in nine human B-lymphoma cell lines generally correlate with increased nuclear NF-κB proteins.ConclusionOverall, the identification of an NF-κB-responsive site in the MIR155HG proximal promoter suggests that MIR155HG is a direct NF-κB target gene in vivo. Understanding NF-κB-mediated regulation of miR-155 could lead to improved immune cell-related diagnostic tools and targeted therapies.

Gelam Honey Attenuates Carrageenan-Induced Rat Paw Inflammation via NF-κB Pathway

The activation of nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of a number of inflammatory diseases. In this study, we investigated the anti-inflammatory mechanism of Gelam honey in inflammation induced rats via NF-κB signalling pathway. Rats paw edema was induced by subplantar injection of 1% carrageenan into the right hind paw. Rats were pre-treated with Gelam honey at different doses (1 or 2 g/kg, p.o.) and NSAID Indomethacin (10 mg/kg, p.o.), in two time points (1 and 7 days). Our results showed that Gelam honey at both concentrations suppressed the gene expressions of NF-κB (p65 & p50) and IκBα in inflamed rats paw tissues. In addition, Gelam honey inhibited the nuclear translocation and activation of NF-κB and decreased the cytosolic degradation of IκBα dose dependently in inflamed rats paw tissues. The immunohistochemical expressions of pro-inflammatory mediators COX-2 and TNF-α were also decreased in inflamed rats paw tissues when treated with Gelam honey. The results of our findings suggest that Gelam honey exhibits its inhibitory effects by attenuating NF-κB translocation to the nucleus and inhibiting IκBα degradation, with subsequent decrease of inflammatory mediators COX-2 and TNF-α.

Sodium butyrate inhibits interferon-gamma induced indoleamine 2,3-dioxygenase expression via STAT1 in nasopharyngeal carcinoma cells

AimsIndoleamine 2,3-dioxygenase (IDO) inhibits T-cell proliferation by catalyzing the conversion of l-tryptophan to l-kynurenine. IDO-induced immune tolerance weakens the clinical outcomes of immunotherapies. Sodium butyrate (NaB), one of the histone deacetylase inhibitors (HDACIs), has potential anti-tumor effects. Our previous studies revealed that NaB could inhibit IFN-γ induced IDO expression in nasopharyngeal carcinoma cells, CNE2. In the present study, we aim to investigate to the mechanism of NaB interfering with the interferon-gamma (IFN-γ)-mediated IDO expression signaling transduction. Main methodsIDO expression and STAT1 phosphorylation in CNE2 cells were analyzed by western blotting and STAT1 acetylation was evaluated by immunoprecipitation. STAT1 nuclear translocation and NF-κB activity were detected by transient transfection and reporter gene assay. Key findingsWe found that NaB inhibited IFN-γ-induced IDO expression in CNE2 cells via decreasing phosphorylation and nuclear translocation of STAT1, but not via down-regulation of IFN-γ-receptor (IFNGR). Immunoprecipitation assays revealed that NaB increased STAT1 acetylation. Furthermore, NaB elevated the activity of NF-κB in CNE2 cells, and blocking the NF-κB activity had no effect on the IFN-γ-induced IDO expression. SignificanceThese results suggest that NaB inhibited IFN-γ-induced IDO expression via STAT1 increased acetylation, decreased phosphorylation, and reduced nuclear translocation. These provided new evidence for the anti-tumor action of NaB and potential drug targets to reduce the IDO-induced immune tolerance.

Modulation of Rack-1/PKCβII Signalling By Soluble AβPPα in SH-SY5Y Cells

The soluble amyloid β precursor protein α (sAβPPα) released after α-secretase cleavage of the amyloid β precursor protein (AβPP) has several functions including modulation of neuronal excitability and synaptic plasticity; it has been suggested that some of these effects are mediated by activation of NF-κB via induction of PI3K/Akt signaling pathway. We have recently described the presence of several consensus binding sites of c-Rel transcription factor in the promoter region of the GNB2L1 gene, coding for the Receptor for Activated C Kinase -1 (RACK-1). We investigated whether sAβPPα could influence the expression of RACK-1 through NF-κB involvement. Our data demonstrate that sAβPPα regulates RACK-1 gene expression through PI3K/Akt-dependent pathway, inducing c-Rel nuclear translocation and NF-κB activation. Since RACK-1 is the scaffold of protein kinase C βII (PKCβII), we turned our attention to this kinase in order to evaluate whether sAβPPα could also influence PKCβII signalling demonstrating that sAβPPα induces PKCβII translocation and interaction with its scaffold with consequent RACK-1/PKCβII complex increase in membrane. Altogether these results suggest the existence of an interesting loop between the functions of the metabolic products of AβPP and the role of PKC and that the impact of a dysregulated AβPP metabolism occurring in several conditions (from physiological aging to injury response) may have consequences on the potential protective functions of the non amyloidogenic sAβPPα.

Surfactin exhibits neuroprotective effects by inhibiting amyloid β-mediated microglial activation

Microglial-mediated neuroinflammation and neurotoxicity contribute to the pathogenesis of neurodegenerative diseases including Alzheimer's disease; therefore, control of microglial activation and subsequent suppression of neurotoxic pro-inflammatory molecules could provide a potential therapeutic approach for the treatment of such diseases. In this study, we investigated the effects of surfactin, a surfactant from Bacillus subtilis, on oligomeric amyloid β (Aβ)-induced microglial activation and neurotoxicity. Surfactin significantly suppressed expression of MMP-9, iNOS and COX-2, as well as production of ROS, NO, PGE2, TNF-α, IL-1β, IL-6 and MCP-1 in Aβ-stimulated BV-2 microglial cells. Moreover, surfactin markedly inhibited Aβ-induced nuclear translocation and activation of NF-κB as well as phosphorylation of JNK and p38 MAPK. Furthermore, surfactin protected hippocampal HT22 cells from indirect neuronal toxicity mediated by Aβ-treated microglial cells, but had no effect on Aβ-induced direct toxicity to HT22 cells. These results suggest that surfactin impairs the Aβ-induced inflammatory response of microglial cells and confers protection against indirect neurotoxicity to hippocampal cells. Our findings indicate that surfactin may have therapeutic potential for ameliorating Alzheimer's disease as well as other neurodegenerative disorders which involve neuroinflammation.

Exposure to inhaled particulate matter activates early markers of oxidative stress, inflammation and unfolded protein response in rat striatum

To study central nervous system airborne PM related subchronic toxicity, SD male rats were exposed for eight weeks to either coarse (32μg/m3), fine (178μg/m3) or ultrafine (107μg/m3) concentrated PM or filtered air. Different brain regions (olfactory bulb, frontal cortex, striatum and hippocampus), were harvested from the rats following exposure to airborne PM. Subsequently, prooxidant (HO-1 and SOD-2), and inflammatory markers (IL-1β and TNFα), apoptotic (caspase 3), and unfolded protein response (UPR) markers (XBP-1S and BiP), were also measured using real-time PCR. Activation of nuclear transcription factors Nrf-2 and NF-κB, associated with antioxidant and inflammation processes, respectively, were also analyzed by GSMA. Ultrafine PM increased HO-1 and SOD-2 mRNA levels in the striatum and hippocampus, in the presence of Nrf-2 activation. Also, ultrafine PM activated NF-κB and increased IL-1β and TNFα in the striatum. Activation of UPR was observed after exposure to coarse PM through the increment of XBP-1S and BiP in the striatum, accompanied by an increase in antioxidant response markers HO-1 and SOD-2. Our results indicate that exposure to different size fractions of PM may induce physiological changes (in a neuroanatomical manner) in the central nervous system (CNS), specifically within the striatum, where inflammation, oxidative stress and UPR signals were effectively activated.

Vitamin D Receptor Inhibits Nuclear Factor κB Activation by Interacting with IκB Kinase β Protein

1,25-Dihydroxyvitamin D (1,25(OH)2D3) is known to suppress NF-κB activity, but the underlying mechanism remains poorly understood. Here we show that the vitamin D receptor (VDR) physically interacts with IκB kinase β (IKKβ) to block NF-κB activation. 1,25(OH)2D3 rapidly attenuates TNFα-induced p65 nuclear translocation and NF-κB activity in a VDR-dependent manner. VDR overexpression inhibits IKKβ-induced NF-κB activity. GST pull-down assays and coimmunoprecipitation experiments demonstrated that VDR physically interacts with IKKβ and that this interaction is enhanced by 1,25(OH)2D3. Protein mapping reveals that VDR-IKKβ interaction occurs between the C-terminal portions of the VDR and IKKβ proteins. Reconstitution of VDR(-/-) cells with the VDR C terminus restores the ability to block TNFα-induced NF-κB activation and IL-6 up-regulation. VDR-IKKβ interaction disrupts the formation of the IKK complex and, thus, abrogates IKKβ phosphorylation at Ser-177 and abolishes IKK activity to phosphorylate IκBα. Consequently, stabilization of IκBα arrests p65/p50 nuclear translocation. Together, these data define a novel mechanism whereby 1,25(OH)2D3-VDR inhibits NF-κB activation.

RETRACTED: Signaling through hepatocellular A2B adenosine receptors dampens ischemia and reperfusion injury of the liver

Ischemia and reperfusion significantly contributes to the morbidity and mortality of liver surgery and transplantation. Based on studies showing a critical role for adenosine signaling in mediating tissue adaptation during hypoxia, we hypothesized that signaling events through adenosine receptors (ADORA1, ADORA2A, ADORA2B, or ADORA3) attenuates hepatic ischemia and reperfusion injury. Initial screening studies of human liver biopsies obtained during hepatic transplantation demonstrated a selective and robust induction of ADORA2B transcript and protein following ischemia and reperfusion. Subsequent exposure of gene-targeted mice for each individual adenosine receptor to liver ischemia and reperfusion revealed a selective role for the Adora2b in liver protection. Moreover, treatment of wild-type mice with an Adora2b-selective antagonist resulted in enhanced liver injury, whereas Adora2b-agonist treatment was associated with attenuated hepatic injury in wild-type, but not in Adora2b(-/-) mice. Subsequent studies in mice with Adora2b deletion in different tissues--including vascular endothelia, myeloid cells, and hepatocytes--revealed a surprising role for hepatocellular-specific Adora2b signaling in attenuating nuclear factor NF-κB activation and thereby mediating liver protection from ischemia and reperfusion injury. These studies provide a unique role for hepatocellular-specific Adora2b signaling in liver protection during ischemia and reperfusion injury.

Macrophage-stimulating protein attenuates hydrogen peroxide-induced apoptosis in human renal HK-2 cells

Macrophage-stimulating protein (MSP) and its receptor, recepteur d’origine nantais (RON), play an important role in cell proliferation and migration. We have investigated the role of MSP in hydrogen peroxide (H2O2)-induced renal tubular apoptosis. Human renal proximal tubular (HK-2) cells were incubated with H2O2 for 24h in the presence of different concentrations of MSP, and cell viability was measured by MTT assay. The protein expression of Bax, Bcl-2, caspase-3, mitogen-activated protein kinases (MAPKs), phosphatidylinositol-3-kinase (PI3K)/Akt, and nuclear factor-kappa B (NF-κB) was determined by semiquantitative immunoblotting. Apoptosis was assessed by flow cytometry analysis after HK-2 cells were stained with fluorescein isothiocyanate-conjugated annexin V protein and propidium iodide. H2O2 treatment decreased cell viability in HK-2 cells; this was counteracted by MSP pretreatment. H2O2 treatment induced an increased ratio of Bax/Bcl-2, cleaved caspase-3, and the number of condensed nuclei, which was also counteracted by MSP. Flow cytometry analysis showed H2O2-induced apoptosis, and its prevention by MSP treatment. Increased protein expression of phospho-p38 MAPK was attenuated by MSP, while phospho-extracellular signal-regulated kinase and c-Jun-N-terminal kinase were not affected. H2O2 induced NF-κB activation and IκB-α degradation, but the increased nuclear NF-κB activation was counteracted by MSP or by a p38 MAPK inhibitor. H2O2 treatment decreased expression of phospho-PI3K and phospho-Akt, which was reversed by MSP pretreatment. These findings suggest that MSP attenuates H2O2-induced apoptosis in HK-2 cells by modulating the p38 and NF-κB, as well as PI3K/Akt, signaling pathways.

Celastrol induces apoptosis of gastric cancer cells by miR-146a inhibition of NF-κB activity

BackgroundCelastrol, a plant triterpene, is known to play important role in inhibiting proliferation and inducing apoptosis of gastric cancer cells. In the present study, the mechanism of celastrol on gastric cancer cells apoptosis was examined.MethodsWe assessed effect of celastrol on NF-κB signaling pathway in gastric cancer cells using western blot and luciferase reporter assay. The real-time PCR was used to evaluate the effect of celastrol on miR-146a expression, and miR-146a mimic to evaluate whether over-expression of miR-146a can affect NF-κB activity. Finally, the effect of miR-146a on celastrol-induced anti-tumor activity was assessed using miR-146a inhibitor.ResultsCelastrol decreased gastric cancer cells viability in a dose-dependent. Celastrol also reduced IκB phosphorylation, nuclear P65 protein levels and NF-κB activity. Furthermore, Celastrol could increase miR-146a expression and up-regulation of miR-146a expression could suppress NF-κB activity. More important, down-regulation of miR-146a expression can reverse the effect of celastrol on NF-κB activity and apoptosis in gastric cancer cells.ConclusionsIn this study, we demonstrated that the effect of celastrol on apoptosis is due to miR-146a inhibition of NF-κB activity.

The Anti-Inflammatory and Immunomodulatory Activity of Thymulin Peptide is NF-κB-Dependent and Involves the Downregulation of IκB-α

The immunomodulatory activity of thymulin and related analogues <i>in vivo</i> is not well characterized in the CNS. We have previously provided evidence for an anti-inflammatory potential of thymulin in downregulating proinflammatory cytokines in a NF-κB-dependent mechanism<i> </i><i>in vitro</i>. Furthermore, we have shown that intracerebroventricular (ICV) treatment with thymulin in the hippocampus (HC) reduced the nuclear localization and activation of NF-κB, an effect mediated by the IκB-α/<i>p</i>IκB- α pathway <i>in vivo</i>. ICV stereotaxic injection of endotoxin (ET) differentially upregulated the nuclear translocation /expression of NF-κB<SUB>1</SUB> (p50), NF-κB<SUB>2</SUB> (p52), RelA (p65), RelB (p68) and c-Rel (p75) in the HC. Pretreatment with thymulin followed by ET exposure reduced the nuclear translocation of NF-κB subunits. The anti-inflammatory effect of thymulin seems to be mediated via the IκB-α pathway since thymulin downregulated ET-induced phosphorylation of IκB-α. Stereotaxic pretreatment with synthetic peptide analogue of thymulin (PAT) reduced the nuclear translocation of NF-κB subunits, an effect mediated by downregulating the phosphorylation of IκB-α. EMSA revealed dose-dependent inhibition of NF-κB/DNA activation mediated by ICV ET. These results indicate that the anti-inflammatory effect of thymulin/PAT, mediated by IκB-α, is NF-κB-dependent and involves the downregulation of the nuclear translocation of various NF-κB subunits and their subcellular activation.

2:10 PM Abstract No. 97 - Reduced murine hind limb ischemia-reperfusion injury in toll-like receptor mutant mice is associated with decreased nets

Purpose Mechanism of ischemia-reperfusion (IR) injury in peripheral vascular disease is unknown. The pattern recognition receptors, Toll-like receptor (TLR) family of proteins, are key players in IR associated with stroke and myocardial infarction. Our aim was to: 1) create a murine model of hind limb injury to evaluate the role of TLR4 in IR, and 2) determine whether inactive TLR4 led to a decrease in the detection of neutrophil extracellular traps (NETs), which is known to be highly thrombogenic and may mediate microvascular injury. Materials and Methods A calibrated tension tourniquet was applied to unilateral hind limb of wild type (WT) and TLR4 knockout mice (TLR4m) for 1.5 hours to induce ischemia and then immediately removed to initiate reperfusion. At the end of 48 hours of reperfusion, mice were sacrificed and hind limb tissue as well as serum specimens were collected for analysis. For immunohistochemistry, mouse monoclonal anti-histone H2A/H2B/DNA complex antibody to detect NETs, rabbit polyclonal anti-H2A and rabbit polyclonal anti-myeloperoxidase were used. Muscle ATP levels were determined using ATP-lite luminescence assay. Nuclear NF-κB activity was determined using a Trans-AM™ NF-κB p65 transcription factor assay. IκBα expression was quantitated using Western blot analysis. ELISA assay was performed to compare levels of KC, MCP-1 and VEGF in the serum samples isolated from both the WT and TLR4m groups. Results IR injury in the hind limb of WT demonstrated significant levels of muscle fiber injury, decreased energy substrates, increased NF-κB activation and decreased I-κBα levels when compared to the TLR4 knockout mice samples. There was marked decrease in the level of neutrophil and monocyte infiltration in the TLR4 mutant mice, marked decrease in the levels of NETs detection. In situ nuclease treatment of wild-type tissue sections significantly diminished the level of NETs immunostaining demonstrating the specificity of our antibody to detect NETs and suggesting a potential role for nuclease treatment in IR injury. Conclusion These results suggest a pivotal role for TLR4 in mediating hind limb IR injury and suggest that NETs may potentially exacerbate the extent of muscle fiber injury.

Inhibition of c-Jun NH2-terminal kinase stimulates mu opioid receptor expression via p38 MAPK-mediated nuclear NF-κB activation in neuronal and non-neuronal cells

Despite its potential side effects of addiction, tolerance and withdrawal symptoms, morphine is widely used for reducing moderate and severe pain. Previous studies have shown that the analgesic effect of morphine depends on mu opioid receptor (MOR) expression levels, but the regulatory mechanism of MOR is not yet fully understood. Several in vivo and in vitro studies have shown that the c-Jun NH2-terminal kinase (JNK) pathway is closely associated with neuropathic hyperalgesia, which closely resembles the neuroplastic changes observed with morphine antinociceptive tolerance. In this study, we show that inhibition of JNK by SP600125, its inhibitory peptide, or JNK-1 siRNA induced MOR at both mRNA and protein levels in neuronal cells. This increase in MOR expression was reversed by inhibition of the p38 mitogen-activated protein kinase (MAPK) pathway, but not by inhibition of the mitogen-activated protein/extracellular signal-regulated kinase (MEK) pathway. Further experiments using cell signaling inhibitors showed that MOR upregulation by JNK inhibition involved nuclear factor-kappa B (NF-κB). The p38 MAPK dependent phosphorylation of p65 NF-κB subunit in the nucleus was increased by SP600125 treatment. We also observed by chromatin immunoprecipitation (ChIP) analysis that JNK inhibition led to increased bindings of CBP and histone-3 dimethyl K4, and decreased bindings of HDAC-2, MeCP2, and histone-3 trimethyl K9 to the MOR promoter indicating a transcriptional regulation of MOR by JNK inhibition. All these results suggest a regulatory role of the p38 MAPK and NF-κB pathways in MOR gene expression and aid to our better understanding of the MOR gene regulation.

Phosphorylation of IκBα at Serine 32 by T-lymphokine-activated Killer Cell-originated Protein Kinase Is Essential for Chemoresistance against Doxorubicin in Cervical Cancer Cells

T-lymphokine-activated killer cell-originated protein kinase (TOPK) is known to be up-regulated in cancer cells and appears to contribute to cancer cell proliferation and survival. However, the molecular mechanism by which TOPK regulates cancer cell survival still remains elusive. Here we show that TOPK directly interacted with and phosphorylated IκBα at Ser-32, leading to p65 nuclear translocation and NF-κB activation. We also revealed that doxorubicin promoted the interaction between nonphosphorylated or phosphorylated TOPK and IκBα and that TOPK-mediated IκBα phosphorylation was enhanced in response to doxorubicin. Also, exogenously overexpressed TOPK augmented transcriptional activity driven by either NF-κB or inhibitor of apoptosis protein 2 (cIAP2) promoters. On the other hand, NF-κB activity including IκBα phosphorylation and p65 nuclear translocation, as well as cIAP2 gene expression, was markedly diminished in TOPK knockdown HeLa cervical cancer cells. Moreover, doxorubicin-mediated apoptosis was noticeably increased in TOPK knockdown HeLa cells, compared with control cells, which resulted from caspase-dependent signaling pathways. These results demonstrate that TOPK is a molecular target of doxorubicin and mediates doxorubicin chemoresistance of HeLa cells, suggesting a novel mechanism for TOPK barrier of doxorubicin-mediated cervical cancer cell apoptosis.

IL-17A Induces Pro-Inflammatory Cytokines Production in Macrophages via MAPKinases, NF-κB and AP-1

Background: Interleukin (IL)-17A, a newly identified cytokine, may participate in the transition of a stable plaque into an unstable plaque. Macrophages play a critical role in the destabilization of atherosclerotic plaque. Methods: RAW 264.7 cells were stimulated with IL-17A. The mRNA expression of inflammatory cytokines was determined by RT-PCR. The cytokines production in the supernatants was measured by ELISA. Small interfering RNA (siRNA) was used to confirm that IL-17A-induced pro-inflammatory cytokines production via IL-17RA signaling. The western blot assay was used to detect the phosphorylation of MAPKinases including p38 and ERK1/2. The DNA binding activity of nuclear factor NF-κB and AP-1 were detected by EMSA. Results: IL-17A induced the production of pro-inflammatory cytokines in macrophages in a time- and dose-dependent manner, such as tumor necrosis factor (TNF)-a, IL-1ß, and IL-6. Meanwhile, IL-17A resulted in the phosphorylation of p38 and ERK1/2 and increased DNA-binding activity of NF-κB and AP-1. Pharmacological inhibitors of p38 and ERK1/2 partly attenuated IL-17A-induced TNF-a, IL-1ß, and IL-6 production. Either NF-κB inhibitor or AP-1 inhibitor also partly decreased the IL-17A-induced cytokine production. Conclusions: IL-17A induces pro-inflammatory cytokines production in macrophages via MAPKinases, NF-κB and AP-1 pathway.

Palmitic Acid Induces Production of Proinflammatory Cytokines Interleukin-6, Interleukin-1β, and Tumor Necrosis Factor-αvia a NF-κB-Dependent Mechanism in HaCaT Keratinocytes

To investigate whether palmitic acid can be responsible for the induction of inflammatory processes, HaCaT keratinocytes were treated with palmitic acid at pathophysiologically relevant concentrations. Secretion levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), NF-κB nuclear translocation, NF-κB activation, Stat3 phosphorylation, and peroxisome proliferator-activated receptor alpha (PPARα) mRNA and protein levels, as well as the cell proliferation ability were measured at the end of the treatment and after 24 hours of recovery. Pyrrolidine dithiocarbamate (PDTC, a selective chemical inhibitor of NF-κB) and goat anti-human IL-6 polyclonal neutralizing antibody were used to inhibit NF-κB activation and IL-6 production, respectively. Our results showed that palmitic acid induced an upregulation of IL-6, TNF-α, IL-1β secretions, accompanied by NF-κB nuclear translocation and activation. Moreover, the effect of palmitic acid was accompanied by PPARα activation and Stat3 phosphorylation. Palmitic acid-induced IL-6, TNF-α, IL-1β productions were attenuated by NF-κB inhibitor PDTC. Palmitic acid was administered in amounts able to elicit significant hyperproliferation and can be attenuated by IL-6 blockage. These data demonstrate for the first time that palmitic acid can stimulate IL-6, TNF-α, IL-1β productions in HaCaT keratinocytes and cell proliferation, thereby potentially contributing to acne inflammation and pilosebaceous duct hyperkeratinization.

Ethyl 3′,4′,5′-trimethoxythionocinnamate modulates NF-κB and Nrf2 transcription factors

Recently, we identified a novel cinnamate analog, ethyl 3′,4′,5′-trimethoxythionocinnamate (ETMTC) as a potent inhibitor of cell adhesion molecules (CAMs), such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. However, its mechanism of action has not been elucidated so far. Since, nuclear factor-kappa B (NF-κB) is the major transcription factor involved in the regulation of ICAM-1, VCAM-1 and E-selectin expression, we determined the status of NF-κB activation in ETMTC treated human endothelial cells. Here, we demonstrate that ETMTC inhibits TNF-α-induced nuclear translocation and activation of NF-κB by inhibiting phosphorylation and degradation of IκBα. The inhibition of IκBα phosphorylation and degradation by ETMTC was found to be due to its ability to inhibit IκB kinase activity. In addition, oxidative stress is known to regulate NF-κB activation through TNF-α signaling cascade, therefore, we examined the effect of ETMTC on TNF-α-induced reactive oxygen species generation. We observed that ETMTC significantly inhibits TNF-α-induced reactive oxygen species generation in endothelial cells. To further elucidate the anti-oxidant potential of ETMTC, we examined its effect on induction of anti-oxidant genes viz. glutamate–cysteine ligase, modifier subunit (GCLM), heme oxygenase-1 (HO1) and NAD (P)H:quinone oxidoreductase 1 (NQO1) in human bronchial epithelial cells. Interestingly, ETMTC significantly induces the anti-oxidant genes viz. GCLM, HO1 and NQO1 by activating nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). Thus, ETMTC could be useful towards developing potent anti-inflammatory molecules.

Abstract P4-06-04: Gamma-secretase inhibitors suppress the activation of NFκB and the expression of TNFα, IL-6 and IL-8 in triple negative breast cancer cells

Abstract Background: Inflammation mediators, such as NF-kappa B (NFκB) and tumor necrosis factor alpha (TNFα), play major role in breast cancer pathogenesis, progression, and relapse. Triple negative breast cancer (TNBC) is a heterogeneous group of aggressive breast cancers and often has a poor outcome, in which Notch pathways are highly activated. However, role of crosstalk between Notch and inflammation mediators in TNBC progression and recurrence is poorly understood. The present study determines: 1) whether NFκB is highly activated in TNBC cells such as MDA-MB-231 (claudin-low-like) and MDA-MB-468 (basal-like), compared to ER-positive cells (MCF-7); 2) whether TNFα, IL-6 and IL-8 are highly expressed in TNBC cells, compared to MCF-7 cells; 3) whether Notch inhibition by gamma-secretase inhibitors (GSIs) significantly decrease NFκB activation and the expression of TNFα, IL-6 and IL-8 in TNBC cells; 4) whether GSI inhibits TNBC cell migration. Material and Methods: MDA-MB-231, MDA-MB-468, MCF-7 cells were cultured using RPMI 1640 media with 10% FBS. The cells were exposed to GSIs such as DAPT and RO4929097 for 18 hours. Nuclear NFκB activation was determined by using the TransAM NFκB p65 kit (Active Motif). The expressions of TNFα, IL-6, IL-8, and IFNγ were determined by the ELISA kits (R&amp;D Systems). Migration was determined using BD BioCoat Matrigel Invasion Chamber (BD Bioscience Discovery Labware, Sedford, MA). Results: Nuclear NFκB p65 activations (A450) were 0.292±0.015, 0.222±0.005, and 0.132±0.004 in cultured MDA-MB-231, MDA-MB-468, and MCF-7 cells, respectively, in which the negative control was 0.125±0.008 and the difference between the groups were significant (P &amp;lt; 0.01, n=6). TNFα protein level was 266±14 pg/mg in MDA-MB-468 cells, but it was not detected in MCF-7 and MDA-MB-231 cells. IL-8 protein levels were 42.8±3, 30.3±2, and 0.23±0.01 ng/mg in MDA-MB-468, MDA-MB-231, and MCF-7 cells, respectively (P &amp;lt; 0.01 between groups, n=6). IL-6 protein levels were 17.1±0.9, 13.9±0.7, and 0.11±0.02 ng/mg in MDA-MB-468, MDA-MB-231, and MCF-7 cells, respectively (P &amp;lt; 0.01 between groups, n=6). IFNγ was not detected in these three cell lines. DAPT (20 µmol/L) or RO4929097 (10 µmol/L) caused 47.7% or 30.6% reduction in NFκB activation, 34.6% or 32.1% reduction in TNFα expression, and 22.2% or 16.4% reduction in IL-8 expression in MDA-MB-468 cells, compared to DMSO group (P &amp;lt; 0.01, n=6). GSIs had no effect on IL-6 expression. DAPT (20 µmol/L) significantly reduced migration of MDA-MB-468 cells by 23%, compared to DMSO group (P &amp;lt; 0.01, n=6). Discussion: The novel findings are 1) NFκB is highly activated in TNBC cells, compared to ER-positive cells, which is associated with significantly increased the expression of inflammation mediators such as IL-6 and IL-8 in TNBC cells and TNFα in MDA-MB-468; and 2) Notch inhibition by GSIs significantly decrease NFκB activation and the expression of TNFα, and IL-8 in TNBC cells. These results support the hypothesis that the crosstalk between Notch and NFκB lead to activation of inflammation mediators such as TNFα and IL 8, which contribute to TNBC progression and recurrence. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-04.

Convergent findings for abnormalities of the NF-κB signaling pathway in schizophrenia.

Neurons exhibit a constitutive level of nuclear factor-κB (NF-κB) signaling and this pathway plays a significant role in neurite outgrowth, activity-dependent plasticity, and cognitive function. Transcription factor analysis was performed in a microarray data set profiled in four different brain regions (n=54 comparison group; n=53 schizophrenia (SZ)). An independent postmortem cohort was used for gene expression (n=24 comparison group; n=22 SZ), protein abundance (n=8 comparison group; n=8 SZ), and NF-κB nuclear activity (n=10 comparison group; n=10 SZ) quantification. Expression quantitative trait locus analysis was performed using publicly available data. Prepulse inhibition (PPI) of the acoustic startle reflex was tested in healthy individuals (n=690). Comparison of microarray data showed that NF-κB was among the transcription factors associated with the differential expression of genes in cases vs controls. NF-κB gene and protein levels and nuclear activation were significantly decreased in the superior temporal gyrus of patients with SZ. Upstream NF-κB genes related to translocation were significantly dysregulated in SZ. The gene expression levels of an NF-κB-associated importin (KPNA4: one of the proteins responsible for the translocation of NF-κB to the nucleus) was decreased in SZ and an SNP within the KPNA4 locus was associated with susceptibility to SZ, reduced KPNA4 expression levels and attenuated PPI of the startle reflex in healthy control subjects. These findings implicate abnormalities of the NF-κB signaling pathway in SZ and provide evidence for an additional possible mechanism affecting the translocation of NF-κB signaling to the nucleus.

Expression of IL-32 modulates NF-κB and p38 MAP kinase pathways in human esophageal cancer

BackgroundEsophageal cancer is the seventh leading cause of cancer death in males in USA, and there is a strong link has been demonstrated between inflammation and esophageal cancer, interleukin (IL)-32 is a recently described pro-inflammatory cytokine characterized by the induction of nuclear factor NF-κB activation, the p38MAPK also plays an important role in key cellular processes related to inflammation and cancer. We investigated whether the IL-32 expression may be involved in esophageal carcinogenesis through modulates the activity of NF-κB and p-p38 MAPK. MethodMalignant esophageal tissue and blood samples were obtained from 65 operated untreated patients, normal samples was obtained from 35 patients operated for other reasons as control. IL-32 expression visualized by immunohistochemistry, Real time RT–PCR for IL-32 mRNA expression, NF-κB phosphorylation and phosphorylated p38mapk were analyzed by immunoblotting, ELISA for further detection IL-32 and cytokines (TNF-α, IL-1β, IL-6 and IL-8) concentration in the patient’s sera. ResultsIL-32 expression was increased in immunohistochemical staining for malignant esophageal tissue and it’s correlated with the relative expression level of IL-32 mRNA P=0.007, the P-NF-κB level elevated in tumor tissue compared with control and no difference in the total NF-κB level P=0.003 while the IL-32 up-regulated the P-pNF-κB in the esophageal tumor P=0.005. There is increase in p-p38MAPK activation underlying IL-32 expression in tumor P=0.004, but no change in total p38 MAPK in malignant esophagus. The plasma level of IL-32 expression was increased in malignant esophageal patients P=0.01, with increased in the levels of the cytokines TNF-α, IL-6, and IL-1β P<0.05. ConclusionsUnderstanding the pathway of IL-32 expression to stimulate the secretion cytokines via the activation of NF-κB and up-regulation of p-p38MAPK may or may not prove to be a therapeutic target, or a biomarker, and future studies will finally answer this hypothesis generated.