Expression Of NF-κB Target Genes Research Articles

Endothelial PPAR-γ provides vascular protection from IL-1β-induced oxidative stress.

Loss of peroxisome proliferator-activated receptor (PPAR)-γ function in the vascular endothelium enhances atherosclerosis and NF-κB target gene expression in high-fat diet-fed apolipoprotein E-deficient mice. The mechanisms by which endothelial PPAR-γ regulates inflammatory responses and protects against atherosclerosis remain unclear. To assess functional interactions between PPAR-γ and inflammation, we used a model of IL-1β-induced aortic dysfunction in transgenic mice with endothelium-specific overexpression of either wild-type (E-WT) or dominant negative PPAR-γ (E-V290M). IL-1β dose dependently decreased IκB-α, increased phospho-p65, and increased luciferase activity in the aorta of NF-κB-LUC transgenic mice. IL-1β also dose dependently reduced endothelial-dependent relaxation by ACh. The loss of ACh responsiveness was partially improved by pretreatment of the vessels with the PPAR-γ agonist rosiglitazone or in E-WT. Conversely, IL-1β-induced endothelial dysfunction was worsened in the aorta from E-V290M mice. Although IL-1β increased the expression of NF-κB target genes, NF-κB p65 inhibitor did not alleviate endothelial dysfunction induced by IL-1β. Tempol, a SOD mimetic, partially restored ACh responsiveness in the IL-1β-treated aorta. Notably, tempol only modestly improved protection in the E-WT aorta but had an increased protective effect in the E-V290M aorta compared with the aorta from nontransgenic mice, suggesting that PPAR-γ-mediated protection involves antioxidant effects. IL-1β increased ROS and decreased the phospho-endothelial nitric oxide synthase (Ser(1177))-to-endothelial nitric oxide synthase ratio in the nontransgenic aorta. These effects were completely abolished in the aorta with endothelial overexpression of WT PPAR-γ but were worsened in the aorta with E-V290M even in the absence of IL-1β. We conclude that PPAR-γ protects against IL-1β-mediated endothelial dysfunction through a reduction of oxidative stress responses but not by blunting IL-1β-mediated NF-κB activity.

Inflammation activation and resolution in human tendon disease.

Improved understanding of the role of inflammation in tendon disease is required to facilitate therapeutic target discovery. We studied supraspinatus tendons from patients experiencing pain before and after surgical subacromial decompression treatment. Tendons were classified as having early, intermediate, or advanced disease, and inflammation was characterized through activation of pathways mediated by interferon (IFN), nuclear factor κB (NF-κB), glucocorticoid receptor, and signal transducer and activator of transcription 6 (STAT-6). Inflammation signatures revealed expression of genes and proteins induced by IFN and NF-κB in early-stage disease and genes and proteins induced by STAT-6 and glucocorticoid receptor activation in advanced-stage disease. The proresolving proteins FPR2/ALX and ChemR23 were increased in early-stage disease compared to intermediate- to advanced-stage disease. Patients who were pain-free after treatment had tendons with increased expression of CD206 and ALOX15 mRNA compared to tendons from patients who continued to experience pain after treatment, suggesting that these genes and their pathways may moderate tendon pain. Stromal cells from diseased tendons cultured in vitro showed increased expression of NF-κB and IFN target genes after treatment with lipopolysaccharide or IFNγ compared to stromal cells derived from healthy tendons. We identified 15-epi lipoxin A4, a stable lipoxin isoform derived from aspirin treatment, as potentially beneficial in the resolution of tendon inflammation.

Synergistic activity of BET protein antagonist-based combinations in mantle cell lymphoma cells sensitive or resistant to ibrutinib

AbstractMantle cell lymphoma (MCL) cells exhibit increased B-cell receptor and nuclear factor (NF)-κB activities. The bromodomain and extra-terminal (BET) protein bromodomain 4 is essential for the transcriptional activity of NF-κB. Here, we demonstrate that treatment with the BET protein bromodomain antagonist (BA) JQ1 attenuates MYC and cyclin-dependent kinase (CDK)4/6, inhibits the nuclear RelA levels and the expression of NF-κB target genes, including Bruton tyrosine kinase (BTK) in MCL cells. Although lowering the levels of the antiapoptotic B-cell lymphoma (BCL)2 family proteins, BA treatment induces the proapoptotic protein BIM and exerts dose-dependent lethality against cultured and primary MCL cells. Cotreatment with BA and the BTK inhibitor ibrutinib synergistically induces apoptosis of MCL cells. Compared with each agent alone, cotreatment with BA and ibrutinib markedly improved the median survival of mice engrafted with the MCL cells. BA treatment also induced apoptosis of the in vitro isolated, ibrutinib-resistant MCL cells, which overexpress CDK6, BCL2, Bcl-xL, XIAP, and AKT, but lack ibrutinib resistance-conferring BTK mutation. Cotreatment with BA and panobinostat (pan-histone deacetylase inhibitor) or palbociclib (CDK4/6 inhibitor) or ABT-199 (BCL2 antagonist) synergistically induced apoptosis of the ibrutinib-resistant MCL cells. These findings highlight and support further in vivo evaluation of the efficacy of the BA-based combinations with these agents against MCL, including ibrutinib-resistant MCL.

H. pylori-Induced DNA Strand Breaks Are Introduced by Nucleotide Excision Repair Endonucleases and Promote NF-κB Target Gene Expression.

The human bacterial pathogen Helicobacter pylori exhibits genotoxic properties that promote gastric carcinogenesis. H. pylori introduces DNA double strand breaks (DSBs) in epithelial cells that trigger host cell DNA repair efforts. Here, we show that H. pylori-induced DSBs are repaired via error-prone, potentially mutagenic non-homologous end-joining. A genome-wide screen for factors contributing to DSB induction revealed a critical role for the H. pylori type IV secretion system (T4SS). Inhibition of transcription, as well as NF-κB/RelA-specific RNAi, abrogates DSB formation. DSB induction further requires β1-integrin signaling. DSBs are introduced by the nucleotide excision repair endonucleases XPF and XPG, which, together with RelA, are recruited to chromatin in a highly coordinated, T4SS-dependent manner. Interestingly, XPF/XPG-mediated DNA DSBs promote NF-κB target gene transactivation and host cell survival. In summary, H. pylori induces XPF/XPG-mediated DNA damage through activation of the T4SS/β1-integrin signaling axis, which promotes NF-κB target gene expression and host cell survival.

Abstract 001: Mutation in the PPARγ Ligand Binding Domain Impairs the Anti-inflammatory Action of PPARγ

Peroxisome proliferator-activated receptor gamma (PPARγ) has been proposed to antagonize the activities of nuclear factor kappa B (NFκB) to regulate inflammation. Transgenic mice expressing dominant negative (DN) PPARγ specifically in vascular smooth muscle cells (SMC) exhibited exacerbated atherosclerosis but the mechanism remains unknown. We hypothesized that DN PPARγ promotes NFκB-induced inflammation in SMC. To test this, we cultured mesenteric SMCs from transgenic mice that would conditionally express wild-type (WT) or DN PPARγ (P467L) in response to adenovirus expressing Cre-recombinase (AdCRE). PPARγ expression remained silent in control SMC infected with AdGFP. TNF-α (0.05 ng/ml, 6 hr) induced NFκB target gene (MCP-1, iNOS and MMP9) expression to a greater extent in P467L-Cre compared to P467L-GFP. For example, MMP9 expression was induced 6.3±0.2-fold in P467L-Cre vs 3.2±0.5-fold in P467L-GFP (p<0.01). The NFκB subunit, p65, mRNA level was not altered in these cells. There was no induction of the PPARγ target aP2 in P467L-Cre, but it was induced 6.5±1.7-fold in WT-SMC infected with AdCRE (WT-Cre). The ability of TNF-α to induce NFκB target gene expression was blunted or abrogated in WT-Cre cells, and their expression was significantly reduced in TNF-α-treated WT-Cre compared to WT-GFP (MMP9: 0.7±1.2 vs 6.0±0.3, p<0.01). To examine mechanisms in vivo, we crossbred transgenic mice expressing WT PPARγ specifically in SMC (S-WT) with mice expressing luciferase under control of a NFκB-responsive promoter. TNF-α (500 ng/ml, 24 hr)-induced NFκB activity was decreased in aorta and carotid artery from S-WT mice compared to control mice (aorta: 4.7±1.1 v s 6.7±0.7, p<0.05, carotid artery: 4.0±0.6 vs 8.7±1.2, p<0.01). Finally, to assess the mechanism preventing anti-inflammatory activity by DN PPARγ, we assessed its interaction with p65 protein when co-expressed in HEK293T cells. WT PPARγ co-precipitated with p65, but the interaction between p65 and P467L PPARγ was severely impaired (n=6). Other mutants (R165T, V290M) could bind to p65, suggesting that loss of the ability is specific to P467L PPARγ. We conclude that SMC-PPARγ has anti-inflammatory effects mediated through inhibition of NFκB activity, which is abolished by P467L mutation.

Blockade of TNF-α-induced NF-κB signaling pathway and anti-cancer therapeutic response of dihydrotanshinone I

The nuclear factor-κB (NF-κB) transcription factors control many physiological processes including inflammation, immunity, apoptosis, and angiogenesis. We identified dihydrotanshinone I as an inhibitor of NF-κB activation through our research on Salvia miltiorrhiza Bunge. In this study, we found that dihydrotanshinone I significantly inhibited the expression of NF-κB reporter gene induced by TNF-α in a dose-dependent manner. And dihydrotanshinone I also inhibited TNF-α induced phosphorylation and degradation of IκBα, phosphorylation and nuclear translocation of p65. Furthermore, pretreatment of cells with this compound prevented the TNF-α-induced expression of NF-κB target genes, such as anti-apoptosis (cIAP-1 and FLIP), proliferation (COX-2), invasion (MMP-9), angiogenesis (VEGF), and major inflammatory cytokines (TNF-α, IL-6, and MCP1). We also demonstrated that dihydrotanshinone I potentiated TNF-α-induced apoptosis. Moreover, dihydrotanshinone I significantly impaired activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK/SAPK). In vivo studies demonstrated that dihydrotanshinone I suppressed the growth of HeLa cells in a xenograft tumor model, which could be correlated with its modulation of TNF-α production. Taken together, dihydrotanshinone I could be a valuable candidate for the intervention of NF-κB-dependent pathological conditions such as inflammation and cancer.

TLR4-MyD88-TRAF6-TAK1 Complex-Mediated NF-κB Activation Contribute to the Anti-Inflammatory Effect of V8 in LPS-Induced Human Cervical Cancer SiHa Cells.

The synthetic compound 7-4-[Bis-(2-hydroxyethyl)-amino]-butoxy-5-hydroxy-8-methoxy-2-phenylchromen-4-one (V8) is a novel flavonoid-derived compound. In this study, we investigated the effects of V8 on Toll-like receptor 4 (TLR4)-mediated inflammatory reaction in human cervical cancer SiHa cells and lipopolysaccharide (LPS)-induced TLR4 activity in cervical cancer SiHa (HPV16+) cells, but not in HeLa (HPV18+) and C33A (HPV-) cells. In addition, V8 inhibited LPS-induced expression of TLR4, MyD88, TRAF6 and phosphorylation of TAK1, and their interaction with TLR4 in SiHa cells, resulting in an inhibition of TLR4-MyD88-TRAF6-TAK1 complex. Moreover, V8 blocked LPS-induced phosphorylation of IκB and IKK, resulting in inhibition of the nuclear translocation of P65-NF-κB in SiHa cells. We also found that V8 reduced the expression of NF-κB target genes, such as those for COX-2, iNOS, IL-6, IL-8, CCL-2, and TNF-α in LPS-stimulated SiHa cells. These results suggested that V8 exerted an anti-inflammatory effect on SiHa cells by inhibiting the TLR4-MyD88-TRAF6-TAK1 complex-mediated NF-κB activation.

The hormone-bound vitamin D receptor enhances the FBW7-dependent turnover of NF-κB subunits.

Signaling by hormonal vitamin D, 1,25-dihydroxyvitamin D (1,25D) has attracted increasing interest because of its non-classical actions, particularly its putative anticancer properties and its role in controlling immune system function. Notably, the hormone-bound vitamin D receptor (VDR) suppresses signaling by pro-inflammatory NF-κB transcription factors, although the underlying mechanisms have remained elusive. Recently, the VDR was shown to enhance the turnover of the oncogenic transcription factor cMYC mediated by the E3 ligase and tumor suppressor FBW7. As FBW7 also controls the turnover of the p100 (NF-κB2) subunit of the family, we determined whether the 1,25D enhanced FBW7-dependent turnover of NF-κB subunits p100, p105 (NF-κB1) and p65 (RELA). Protein levels of all three subunits declined markedly in the presence of 1,25D in multiple cell lines in the absence of substantial changes in mRNA expression. The VDR coimmunoprecipitated with all three subunits, and 1,25D treatment accelerated subunit turnover in cycloheximide-treated cells. Importantly, we observed an association of FBW7 with p105 and p65, as well as p100, and knockdown of FBW7 eliminated 1,25D-dependent subunit turnover. Moreover, expression of NF-κB target genes was elevated in FBW7-depleted cells. These results reveal that 1,25D signaling suppresses NF-κB function by enhancing FBW7-dependent subunit turnover.

Abstract 4370: GSK3α/β inhibition as a drug target in prostate cancer

Abstract Despite what its namesake suggests, Glycogen Synthase Kinase 3 (GSK3) is a kinase that is implicated in a myriad of signalling pathways and has recently received considerable interest due to its conflicting roles oncogenesis. Though GSK3 has classically been regarded as a tumour suppressor due to in role in supressing WNT signalling, emerging evidence suggests that GSK3 also functions as a tumor promoter by facilitating tumor cell survival and conferring drug resistance in cancer. These tumor promoting effects have been partly linked to the expression of certain NFκB target genes, whose expression in some cancers are influenced by direct GSK3 phosphorylation of NFκB's p65 subunit. Given the complex interplay between GSK3 and the androgen receptor in prostate cancer (PCa), and the inverse correlation between the androgen receptor status and NFκB, we investigate the potential of GSK3 inhibitors to treat and overcome drug resistance in PCa. A kinase inhibitor screen for 80 compounds and additional chemical inhibitors for GSK3 was carried out using the JANUS Automated Workstation at the robotics facility at NUI Galway, Ireland. The cellular fractions for treated cells were separated and assessed for differences in expression between the cytoplasmic and nuclear compartments using western blotting. Flow cytometry and western blotting using antibodies for markers of apoptosis were used to assess for mode of cell death. Differences in gene expression of NFκB target genes were assessed using gene specific primers for SYBR green RT-PCR. RNAi using siRNA directed against each of the GSK3 isoforms, α and β, was utilised for target validation The kinase inhibitor screen demonstrated significant growth inhibition among all the tested PCa cell lines to the GSK3 inhibitor, BIO. This result was validated using two additional chemical GSK3 inhibitors, LiCl and CHIR99021. Expression analysis demonstrated aberrant NFκB and GSK3 expression in the cytoplasmic and nuclear compartments of the cell lines that varied upon treatment with GSK3 inhibitors. siRNA directed against GSK3 confirmed that the effects of the inhibitors were GSK3 specific and demonstrated that both isoforms distinguished between the effects of each of the two isoforms. Our results suggest that GSK3 has a role in the pathogenesis of PCa. This notion is supported by the findings that GSK3 inhibition reduces the viability of PCa cells via a mechanism that is consistent with apoptosis. Furthermore, both NFκB and GSK3 are shown to be active and aberrantly co-expressed in our PCa model. Collectively, these results suggest that GSK3 has an effect on NFκB that may be independent of WNT signalling. Citation Format: Husnain Ali, Amy Burke, Enda O'Connell, Frank Sullivan, Frank Giles, Sharon Glynn. GSK3α/β inhibition as a drug target in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4370. doi:10.1158/1538-7445.AM2015-4370

Abstract 2886: Epigenetic regulation of MUC1 in NF-kB target gene expression of pro-inflammatory cytokines in colon cancer

Abstract MUC1 is a transmembrane glycoprotein normally expressed at low levels in a fully glycosylated form on the apical surface of epithelial cells but it is found overexpressed and hypoglycosylated in human adenocarcinomas as well as sites of chronic epithelial inflammations. Aberrant glycosylation of MUC1 in cancer cells alters its normal function and affects the behavior of cancer cells. The MUC1 extracellular domain also participates in activation of the NF-kB family members such as phospho-IkBa and phospho-p65. Our data showed that the tumor form of MUC1, in association with p65, regulates the transcriptional activity of pro-inflammatory cytokines, including IL-6 and TNF-alpha, by binding to their promoter regions in both mouse and human cancer cells. Because proteomic analysis showed that MUC1 is able to interact directly with histone proteins, we hypothesized that MUC1 might influence the transcriptional activity of cytokines by inducing epigenetic modification. Here, we explored MUC1/p65-regulated transcription of IL-6 and TNF-alpha in colon cancer cells and discovered that MUC1/p65 complex modulated the expression and interaction of histone methyltransferase Enhancer of Zeste protein-2 (EZH2) with pro-inflammatory cytokine promoters. Interestingly, the contribution of EzH2 in controlling the MUC1/p65-modulated cytokines expression was independent of its histone H3K27 methyltransferase activity but it shows novel properties in regulating chromatin remodeling that are currently under detailed investigation To investigate the in vivo significance of MUC1/p65-induced cytokines expression in the tumor microenvironment, we used human MUC1 transgenic mice that express MUC1 on the colonic epithelial cells, treated with azoxymethane to initiate and dextran sulfate sodium (AOM/DSS) to promote colorectal carcinogenesis. MUC1.Tg mice showed higher tumor incidence and decreased survival when compared with wild-type mice. Consistent with the in vitro data, NF-kB family members and EzH2 expression were higher in MUC1.Tg mice when compared to wild-type mice. In addition, the number of infiltrating macrophages strongly increased in AOM/DSS-treated colon tissues of MUC1.Tg mice. These are important findings that may explain the mechanism by which MUC1 and NF-kB regulate cancer progression. Citation Format: Sandra Cascio, Joshua Sciurba, Olivera Finn. Epigenetic regulation of MUC1 in NF-kB target gene expression of pro-inflammatory cytokines in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2886. doi:10.1158/1538-7445.AM2015-2886

Xia-yu-xue decoction (XYXD) reduces carbon tetrachloride (CCl4)-induced liver fibrosis through inhibition hepatic stellate cell activation by targeting NF-κB and TGF-β1 signaling pathways.

BackgroundHepatic stellate cell (HSC) activation is activated mainly by endotoxin and transforming growth factor (TGF-β1) in chronic liver injury, consequently, can be important therapeutic targets. Xia-yu-xue decoction (XYXD), a classical recipe used in China to treat liver fibrosis, and has been revealed to inhibit hepatic fibrosis in animal models, the mechanism of action of XYXD remains elusive. In the present study, we evaluated whether XYXD reduced endotoxin and pro-fibrogenic pathways induced by lipopolysaccharide (LPS) and TGF-β1 in HSCs.MethodsThe in vivo effect of XYXD on fibrosis progression was assessed in mice model induced by carbon tetrachloride (CCl4), The in vitro effect of XYXD on mice GFP-Col-HSC cells was evaluated using LPS and TGF-β1 stimulation.ResultsXYXD treatment reduced CCl4-induced liver fibrosis and decreased hepatic hydroxyproline (Hyp) content, the mRNA levels of smooth muscle actin (α-SMA) and Col 1(α1) in fibrotic liver. XYXD suppressed nuclear factor-κB (NF-κB) activation induced by LPS and TGF-β1 assessed by using NF-κB-luciferase reporter. The expression of NF-κB target genes, chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-X-C motif) ligand 2 (CXCL2) induced by LPS was suppressed after XYXD treatment. The expression of TGF-β1 targets genes, Col1(α1) and tissue inhibitor of metalloproteinases (TIMP1) induced by TGF-β1 was inhibit after XYXD treatment.ConclusionXYXD treatment attenuates liver fibrosis by inhibiting HSC activation via inhibition of NF-κB and TGF-β1 signaling pathway, thereby blocking the synthesis of Col1 (α1) and TIMP-1. These findings from present study suggest that XYXD may be a therapeutic decoction for liver fibrosis in which NF-κB and TGF-β1 are thought to take part.

Abstract P6-14-05: A novel link between anti-apoptotic signaling, NFκB, and SMAD7 in IBC pathobiology

Abstract Background: Inflammatory breast cancer (IBC) has the highest lethality amongst all subtypes of breast cancer and develops rapid therapeutic resistance. High NFκB activation has been identified as a distinct molecular determinant in IBC pathobiology; however, the precise sequence of its activation and functional consequence in IBC remains unknown. Our previous work identified increased expression of the X-linked inhibitor of apoptosis protein (XIAP) due to altered translation in IBC, while other studies have noted a crosstalk between XIAP and NFκB. We hypothesized that XIAP drives NFκB activation in IBC promoting therapeutic resistance and tumorigenesis. Methods: NFκB phosphorylation, nuclear translocation, and target gene expression were evaluated in triple-negative SUM149 IBC cells with targeted overexpression or knockdown of XIAP. Using specific point mutants, we assessed the domain and mechanism of XIAP-mediated NFκB activation in IBC. We evaluated proliferation and viability in 2D and 3D culture of SUM149 cells treated with JSH-23, a small molecule inhibitor of NFκB nuclear translocation. We monitored the effects of XIAP overexpression or knockdown on in vivo tumorigenicity in IBC xenograft models by measuring tumor growth and NFκB signaling. IHC analysis of XIAP and NFκB was performed on tumor microarrays containing both non-IBC and IBC. Results: Knockdown of XIAP significantly decreased NFκB activation in IBC cells. Domain analysis revealed the necessity of the BIR1 domain of XIAP and TAB1:IKKβ complex formation in activating NFκB. NFκB antagonism inhibited proliferation of cells and sensitized therapy-resistant, XIAP overexpressing cells to targeted therapy. Loss of XIAP inhibited tumor growth of SUM149 tumor cells, correlating with decreased ALDH activity and varied epithelial-mesenchymal characteristics in these cells, while overexpression of XIAP significantly enhanced tumor growth of SUM149 cells. Further analysis revealed altered SMAD7 expression in XIAP knockdown cells, revealing crosstalk between XIAP, NFκB, and TGFβ signaling in IBC. IHC analysis of XIAP expression in invasive non-IBC tumors correlated with triple-negative status as well as increased grade and stage of tumors. In IBC tumors, XIAP expression associated with increased NFκB. Conclusions: In summary, our studies reveal that XIAP expression is necessary for NFκB activation in IBC and is critical for IBC development and progression. This study provides a novel insight into how an anti-apoptotic protein may regulate survival signaling and disease progression and may guide further research into innovative inhibitors of this interaction. Citation Format: Myron K Evans, Scott J Sauer, Amy J Aldrich, Joseph Geradts, Peter Vermeulen, Luc Dirix, Steven Van Laere, Gayathri R Devi. A novel link between anti-apoptotic signaling, NFκB, and SMAD7 in IBC pathobiology [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-14-05.

The roles of endoplasmic reticulum overload response induced by HCV and NS4B protein in human hepatocyte viability and virus replication.

Hepatitis C virus (HCV) replication is associated with endoplasmic reticulum (ER) and its infection triggers ER stress. In response to ER stress, ER overload response (EOR) can be activated, which involves the release of Ca2+ from ER, production of reactive oxygen species (ROS) and activation of nuclear factor κB (NF-κB). We have previously reported that HCV NS4B expression activates NF-κB via EOR-Ca2+-ROS pathway. Here, we showed that NS4B expression and HCV infection activated cancer-related NF-κB signaling pathway and induced the expression of cancer-related NF-κB target genes via EOR-Ca2+-ROS pathway. Moreover, we found that HCV-activated EOR-Ca2+-ROS pathway had profound effects on host cell viability and HCV replication. HCV infection induced human hepatocyte death by EOR-Ca2+-ROS pathway, whereas activation of EOR-Ca2+-ROS-NF-κB pathway increased the cell viability. Meanwhile, EOR-Ca2+-ROS-NF-κB pathway inhibited acute HCV replication, which could alleviate the detrimental effect of HCV on cell viability and enhance chronic HCV infection. Together, our findings provide new insights into the functions of EOR-Ca2+-ROS-NF-κB pathway in natural HCV replication and pathogenesis.

The stretch responsive microRNA miR-148a-3p is a novel repressor of IKBKB, NF-κB signaling, and inflammatory gene expression in human aortic valve cells.

Bicuspid aortic valves calcify at a significantly higher rate than normal aortic valves, a process that involves increased inflammation. Because we have previously found that bicuspid aortic valve experience greater stretch, we investigated the potential connection between stretch and inflammation in human aortic valve interstitial cells (AVICs). Microarray, quantitative PCR (qPCR), and protein assays performed on AVICs exposed to cyclic stretch showed that stretch was sufficient to increase expression of interleukin and metalloproteinase family members by more than 1.5-fold. Conditioned medium from stretched AVICs was sufficient to activate leukocytes. microRNA sequencing and qPCR experiments demonstrated that miR-148a-3p was repressed in both stretched AVICs (43% repression) and, as a clinical correlate, human bicuspid aortic valves (63% reduction). miR-148a-3p was found to be a novel repressor of IKBKB based on data from qPCR, luciferase, and Western blot experiments. Furthermore, increasing miR-148a-3p levels in AVICs was sufficient to decrease NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling and NF-κB target gene expression. Our data demonstrate that stretch-mediated activation of inflammatory pathways is at least partly the result of stretch-repression of miR-148a-3p and a consequent failure to repress IKBKB. To our knowledge, we are the first to report that cyclic stretch of human AVICs activates inflammatory genes in a tissue-autonomous manner via a microRNA that regulates a central inflammatory pathway.

Age-related increase in IL-17 activates pro-inflammatory signaling in prostate cells.

A close relationship between aging, inflammation, and prostate cancer is widely accepted. Aging is accompanied by a progressive increase in pro-inflammatory cytokines, including interleukin 17 (IL-17), a key pro-inflammatory cytokine that becomes dysregulated with age. However, the contribution of IL-17 to age-related prostate tumorigenesis remains unclear. The aim of this study was to investigate the role of age-related IL-17 dysregulation in prostate tumorigenesis. Serum and splenic T-lymphocytes from young GPAT-1 knock-out aging-mimic T cell mice as well as young and aged wild-type mice were collected. shRNA was used to knock down the IL-17 receptor in LNCaP prostate cancer cells and RWPE-1 non-transformed prostate epithelial cells, which were then exposed to the mouse sera or conditioned media from stimulated T-lymphocytes. NF-κB activation, NF-κB target gene expression, and cell proliferation were all measured in these cells by luciferase assay, qPCR, Western blot analysis, and MTT assay, respectively. T-lymphocyte-secreted IL-17 from aging-mimic mice induced NF-κB activity and target gene expression in LNCaP and RWPE-1 cells. It also promoted proliferation of these cells. Aging-mimic T cell mice produce increased levels of IL-17, which stimulates the pro-inflammatory NF-κB pathway in prostate epithelial cells. NF-κB increases inflammation, carcinogenesis and metastatic potential in the prostate. These findings provide evidence that the dysregulation of cytokine production seen in aged T cells may directly contribute to the increased risk for prostate cancer in the elderly.

The Role of BMP Signaling and NF-κB Signaling on Osteoblastic Differentiation, Cancer Development, and Vascular Diseases--Is the Activation of NF-κB a Friend or Foe of BMP Function?

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-β family, were first identified as potent inducers of ectopic bone formation when implanted into muscle tissue. Subsequent studies have demonstrated that BMPs play important roles during developmental processes, including cell proliferation, differentiation, and apoptosis. Furthermore, recent studies have shown that BMPs are also involved in the initiation and/or progression of various diseases, such as skeletal diseases, cancer, and vascular diseases. Nuclear factor κ light-chain enhancer of activated B cells (NF-κB) was originally identified as a transcription factor that bound to the enhancer region of the immunoglobulin κ light-chain promoter in B cells. A wide range of stimuli, including inflammatory cytokines and bacterial and viral products, activate the NF-κB pathway, leading to the expression of NF-κB target genes. NF-κB also has functions in multiple biological processes, such as immune and inflammatory responses, cell differentiation, cellular stress responses, and cancer development. Recent findings have demonstrated that BMP and NF-κB signaling agonistically or antagonistically regulate bone development, cancer development, and vascular diseases. This review describes the role of BMPs and NF-κB in bone development, cancer development, and vascular diseases with special attention given to concepts that have emerged in recent years.

Diffuse large B-cell lymphoma: sub-classification by massive parallel quantitative RT-PCR

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity with remarkably variable clinical outcome. Gene expression profiling (GEP) classifies DLBCL into activated B-cell like (ABC), germinal center B-cell like (GCB), and Type-III subtypes, with ABC-DLBCL characterized by a poor prognosis and constitutive NF-κB activation. A major challenge for the application of this cell of origin (COO) classification in routine clinical practice is to establish a robust clinical assay amenable to routine formalin-fixed paraffin-embedded (FFPE) diagnostic biopsies. In this study, we investigated the possibility of COO-classification using FFPE tissue RNA samples by massive parallel quantitative reverse transcription PCR (qRT-PCR). We established a protocol for parallel qRT-PCR using FFPE RNA samples with the Fluidigm BioMark HD system, and quantified the expression of the COO classifier genes and the NF-κB targeted-genes that characterize ABC-DLBCL in 143 cases of DLBCL. We also trained and validated a series of basic machine-learning classifiers and their derived meta classifiers, and identified SimpleLogistic as the top classifier that gave excellent performance across various GEP data sets derived from fresh-frozen or FFPE tissues by different microarray platforms. Finally, we applied SimpleLogistic to our data set generated by qRT-PCR, and the ABC and GCB-DLBCL assigned showed the respective characteristics in their clinical outcome and NF-κB target gene expression. The methodology established in this study provides a robust approach for DLBCL sub-classification using routine FFPE diagnostic biopsies in a routine clinical setting.

Elevated free cholesterol in a p62 overexpression model of non-alcoholic steatohepatitis.

To characterize how insulin-like growth factor 2 (IGF2) mRNA binding protein p62/IMP2-2 promotes steatohepatitis in the absence of dietary cholesterol. Non-alcoholic steatohepatitis (NASH) was induced in wild-type mice and in mice overexpressing p62 specifically in the liver by feeding the mice a methionine and choline deficient (MCD) diet for either two or four weeks. As a control, animals were fed a methionine and choline supplemented diet. Serum triglycerides, cholesterol, glucose, aspartate aminotransferase and alanine transaminase were determined by standard analytical techniques. Hepatic gene expression was determined by real-time reverse transcription-polymerase chain reaction. Generation of reactive oxygen species in liver tissue was quantified as thiobarbituric acid reactive substances using a photometric assay and malondialdehyde as a standard. Tissue fatty acid profiles and cholesterol levels were analyzed by gas chromatography-mass spectrometry after hydrolysis. Hepatocellular iron accumulation was determined by Prussian blue staining in paraffin-embedded formalin-fixed tissue. Filipin staining on frozen liver tissue was used to quantify hepatic free cholesterol levels. Additionally, nuclear localization of the nuclear factor kappa B (NF-κB) subunit p65 was examined in frozen tissues. Liver-specific overexpression of the insulin-like growth factor 2 mRNA binding protein 2-2 (IGF2BP2-2/IMP2-2/p62) induces steatosis with regular chow and amplifies NASH-induced fibrosis in the MCD mouse model. Activation of NF-κB and expression of NF-κB target genes suggested an increased inflammatory response in p62 transgenic animals. Analysis of hepatic lipid composition revealed an elevation of monounsaturated fatty acids as well as increased hepatic cholesterol. Moreover, serum cholesterol was significantly elevated in p62 transgenic mice. Dietary cholesterol represents a critical factor for the development of NASH from hepatic steatosis. Filipin staining revealed increased free cholesterol in p62 transgenic livers, which were not diet-derived. The mRNA levels of the rate-limiting enzyme for cholesterol synthesis 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase or HMGCR) were not significantly upregulated, potentially due to increased cholesterol biosynthesis via elevated sterol regulatory element binding transcription factor 2 (SREBF2) gene expression and increased iron deposition in transgenic animals. This study provides evidence that p62/IGF2BP2-2 drives the progression of NASH through elevation of hepatic iron deposition and increased production of hepatic free cholesterol.

Neurotropin suppresses inflammatory cytokine expression and cell death through suppression of NF-κB and JNK in hepatocytes.

Inflammatory response and cell death in hepatocytes are hallmarks of chronic liver disease, and, therefore, can be effective therapeutic targets. Neurotropin® (NTP) is a drug widely used in Japan and China to treat chronic pain. Although NTP has been demonstrated to suppress chronic pain through the descending pain inhibitory system, the action mechanism of NTP remains elusive. We hypothesize that NTP functions to suppress inflammatory pathways, thereby attenuating disease progression. In the present study, we investigated whether NTP suppresses inflammatory signaling and cell death pathways induced by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) in hepatocytes. NTP suppressed nuclear factor-κB (NF-κB) activation induced by IL-1β and TNFα assessed by using hepatocytes isolated from NF-κB-green fluorescent protein (GFP) reporter mice and an NF-κB-luciferase reporter system. The expression of NF-κB target genes, Il6, Nos2, Cxcl1, ccl5 and Cxcl2 induced by IL-1β and TNFα was suppressed after NTP treatment. We also found that NTP suppressed the JNK phosphorylation induced by IL-1β and TNFα. Because JNK activation contributes to hepatocyte death, we determined that NTP treatment suppressed hepatocyte death induced by IL-1β and TNFα in combination with actinomycin D. Taken together, our data demonstrate that NTP attenuates IL-1β and TNFα-mediated inflammatory cytokine expression and cell death in hepatocytes through the suppression of NF-κB and JNK. The results from the present study suggest that NTP may become a preventive or therapeutic strategy for alcoholic and non-alcoholic fatty liver disease in which NF-κB and JNK are thought to take part.

Bmi1 regulates self-renewal and epithelial to mesenchymal transition in breast cancer cells through Nanog.

BackgroundThe Bmi1 polycomb ring finger oncogene, a transcriptional repressor belonging to the Polycomb group of proteins plays an important role in the regulation of stem cell self-renewal and is elevated in several cancers. In the current study, we have explored the role of Bmi1 in regulating the stemness and drug resistance of breast cancer cells.MethodsUsing real time PCR and immunohistochemistry primary breast tissues were analyzed. Retro- and lentiviruses were utilized to overexpress and knockdown Bmi1, RT-PCR and Western blot was performed to evaluate mRNA and protein expression. Stemness properties were analyzed by flow cytometry and sphere-formation and tumor formation was determined by mouse xenograft experiments. Dual luciferase assay was employed to assess promoter activity and MTT assay was used to analyze drug response.ResultsWe found Bmi1 overexpression in 64% of grade III invasive ductal breast adenocarcinomas compared to normal breast tissues. Bmi1 overexpression in immortalized and transformed breast epithelial cells increased their sphere-forming efficiency, induced epithelial to mesenchymal transition (EMT) with an increase in the expression of stemness-related genes. Knockdown of Bmi1 in tumorigenic breast cells induced epithelial morphology, reduced expression of stemness-related genes, decreased the IC50 values of doxorubicin and abrogated tumor-formation. Bmi1-high tumors showed elevated Nanog expression whereas the tumors with lower Bmi1 showed reduced Nanog levels. Overexpression of Bmi1 increased Nanog levels whereas knockdown of Bmi1 reduced its expression. Dual luciferase promoter-reporter assay revealed Bmi1 positively regulated the Nanog and NFκB promoter activity. RT-PCR analysis showed that Bmi1 overexpression activated the NFκB pathway whereas Bmi1 knockdown reduced the expression of NFκB target genes, suggesting that Bmi1 might regulate Nanog expression through the NFκB pathway.ConclusionsOur study showed that Bmi1 is overexpressed in several high-grade, invasive ductal breast adenocarcinomas, thus supporting its role as a prognostic marker. While Bmi1 overexpression increased self-renewal and promoted EMT, its knockdown reversed EMT, reduced stemness, and rendered cells drug sensitive, thus highlighting a crucial role for Bmi1 in regulating the stemness and drug response of breast cancer cells. Bmi1 may control self-renewal through the regulation of Nanog expression via the NFκB pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-785) contains supplementary material, which is available to authorized users.