NF-κB Binding Sites Research Articles

The Mechanism of Bisphenol A Atherogenicity Involves Apolipoprotein A-I Downregulation through NF-κB Activation.

Apolipoprotein A-I (apoA-I) is the major protein component of high-density lipoproteins (HDL), mediating many of its atheroprotective properties. Increasing data reveal the pro-atherogenic effects of bisphenol A (BPA), one of the most prevalent environmental chemicals. In this study, we investigated the mechanisms by which BPA exerts pro-atherogenic effects. For this, LDLR−/− mice were fed with a high-fat diet and treated with 50 µg BPA/kg body weight by gavage. After two months of treatment, the area of atherosclerotic lesions in the aorta, triglycerides and total cholesterol levels were significantly increased, while HDL-cholesterol was decreased in BPA-treated LDLR−/− mice as compared to control mice. Real-Time PCR data showed that BPA treatment decreased hepatic apoA-I expression. BPA downregulated the activity of the apoA-I promoter in a dose-dependent manner. This inhibitory effect was mediated by MEKK1/NF-κB signaling pathways. Transfection experiments using apoA-I promoter deletion mutants, chromatin immunoprecipitation, and protein-DNA interaction assays demonstrated that treatment of hepatocytes with BPA induced NF-κB signaling and thus the recruitment of p65/50 proteins to the multiple NF-κB binding sites located in the apoA-I promoter. In conclusion, BPA exerts pro-atherogenic effects downregulating apoA-I by MEKK1 signaling and NF-κB activation in hepatocytes.

PNPLA3 I148M mediates the regulatory effect of NF-kB on inflammation in PA-treated HepG2 cells.

Both PNPLA3 I148M and hepatic inflammation are associated with nonalcoholic fatty liver disease (NAFLD) progression. This study aimed to elucidate whether PNPLA3 I148M is involved in NF‐kB‐related inflammation regulation in NAFLD. HepG2 cells homozygous for the PNPLA3 I148M mutation were used. The human PNPLA3 promoter sequence was screened for NF‐kB binding sites using the MATCH and PATCH tools. NF‐kB‐mediated transcriptional regulation of the PNPLA3 gene was assessed by luciferase reporter assay, EMSA and ChIP‐qPCR. Wild‐type (I148I) and mutant (M148M) PNPLA3 were overexpressed using stable lentivirus‐mediated transfection. The pCMV vector and siRNA were transiently transfected into cells to direct NF‐kB overexpression and PNPLA3 silencing, respectively. A putative NF‐kB binding site in the human PNPLA3 promoter was shown to be necessary for basal and NF‐kB‐driven transcriptional activation of PNPLA3 and protein/DNA complex formation. Supershift analysis demonstrated a protein/DNA complex specifically containing the NF‐kB p65 and p50 subunits. ChIP‐qPCR confirmed the endogenous binding of NF‐kB to the human PNPLA3 promoter in response to NF‐kB overexpression and palmitic acid (PA) challenge. The silencing of PNPLA3 blocked the overexpression of NF‐kB or PA‐induced TNF‐α up‐regulation. Moreover, mutant PNPLA3 overexpression prevented NF‐kB inhibitor–induced down‐regulation of TNF‐α expression in PA‐treated HepG2 cells. Finally, the overexpression of mutant but not wild‐type PNPLA3 increased TNF‐α expression and activated the ER stress–mediated and NF‐kB‐independent inflammatory IRE‐1α/JNK/c‐Jun pathway. Human PNPLA3 was shown to be a target of NF‐kB, and PNPLA3 I148M mediated the regulatory effect of NF‐kB on inflammation in PA‐treated HepG2 cells, most likely via the IRE‐1α/JNK/c‐Jun ER stress pathway.

Transcriptional activation of USP16 gene expression by NF\u03baB signaling

Ubiquitin Specific Peptidase 16 (USP16) has been reported to contribute to somatic stem-cell defects in Down syndrome. However, how this gene being regulated is largely unknown. To study the mechanism underlying USP16 gene expression, USP16 gene promoter was cloned and analyzed by luciferase assay. We identified that the 5′ flanking region (− 1856 bp ~ + 468 bp) of the human USP16 gene contained the functional promotor to control its transcription. Three bona fide NFκB binding sites were found in USP16 promoter. We showed that p65 overexpression enhanced endogenous USP16 mRNA level. Furthermore, LPS and TNFα, strong activators of the NFκB pathway, upregulated the USP16 transcription. Our data demonstrate that USP16 gene expression is tightly regulated at transcription level. NFκB signaling regulates the human USP16 gene expression through three cis-acting elements. The results provide novel insights into a potential role of dysregulation of USP16 expression in Alzheimer’s dementia in Down Syndrome.

Mutated MYD88 Regulates HCK Pro-Survival Signaling through JunB in MYD88 Mutated B-Lymphoma Cells

Hematopoietic cell kinase (HCK) is a member of the SRC family tyrosine kinases (SFKs) that is down-regulated in later stages of B-cell ontogeny. In MYD88 mutated B-cell lymphomas, HCK is aberrantly over-expressed and is activated and triggers multiple growth and survival pathways including BTK, PI3Kδ/AKT and ERK1/2 which are essential to tumor cell survival. Ibrutinib, a pleiotropic inhibitor of BTK, that produces remarkable responses in MYD88 mutated WM, ABC-DLBCL, and Primary CNS Lymphoma was found also potently inhibits HCK. Mutations that abolish ibrutinib-HCK binding greatly diminish anti-tumor activity in MYD88 mutated lymphoma cells, highlighting the importance of HCK as an essential target in MYD88 driven diseases. To clarify the transcriptional regulation of HCK in MYD88 mutated malignancies, we performed promoter binding TF profiling, PROMO weighted transcription factor (TF) consensus binding analysis, and chromatin immuno-precipitation (ChIP) studies. We identified PAX5, and mutated MYD88 downstream signaling mediators, STAT3, AP-1 and NF-kB as important drivers of HCK transcription. Knockdown of PAX5, a crucial regulatory factor required for B-cell commitment and identity, abrogated HCK transcription in MYD88 mutated lymphoma cells. Deletion of STAT3, AP-1 or NF-kB binding sites greatly reduced corresponding TFs binding and HCK promoter activity, indicating the importance of MYD88 directed signaling in the regulation of HCK transcription. Among AP-1 complex components, JunB but not c-Jun showed greatest relevance to TLR/MYD88 signaling and regulation of HCK transcription. Since STAT3 and NF-kB are known downstream mediators of mutated MYD88 signaling, we focused on the function of JunB. JunB phosphorylation increased following MYD88 pathway activation through TLR4 (with LPS-EB) or TLR9 (with ODN-2006), as well as lentiviral mediated expression of mutated MYD88 (L265P) but not wild type MYD88 (MYD88-WT) at Thr102 and Thr104 in either MYD88 mutated BCWM.1 cells or MYD88-WT Ramos cells. Conversely, c-Jun phosphorylation was not impacted by either intervention. Knockdown of MYD88 in BCWM.1 WM cells also reduced the phosphorylation of JunB, while c-Jun phosphorylation showed modest increase. Moreover, knockdown of JunB reduced HCK protein levels in MYD88 mutated WM and ABC-DLBCL cells. These data demonstrate that JunB is an important mediator of mutated MYD88 signaling among AP1 complex members and is directly involved in the regulation of HCK expression in MYD88 mutated B-cell lymphoma. The findings provide new insights into the transcriptional regulation of HCK by MYD88 driven TFs, and opportunities for further advancing targeted therapeutics in MYD88 driven B-cell malignancies. Disclosures Hunter: Janssen: Consultancy. Castillo:TG Therapeutics: Research Funding; Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Research Funding; BMS: Research Funding; Janssen: Consultancy.

The regulation of sequence specific NF-κB DNA binding and transcription by IKKβ phosphorylation of NF-κB p50 at serine 80.

Phosphorylation of the NF-κB transcription factor is an important regulatory mechanism for the control of transcription. Here we identify serine 80 (S80) as a phosphorylation site on the p50 subunit of NF-κB, and IKKβ as a p50 kinase. Transcriptomic analysis of cells expressing a p50 S80A mutant reveals a critical role for S80 in selectively regulating the TNFα inducible expression of a subset of NF-κB target genes including pro-inflammatory cytokines and chemokines. S80 phosphorylation regulates the binding of p50 to NF-κB binding (κB) sites in a sequence specific manner. Specifically, phosphorylation of S80 reduces the binding of p50 at κB sites with an adenine at the −1 position. Our analyses demonstrate that p50 S80 phosphorylation predominantly regulates transcription through the p50:p65 heterodimer, where S80 phosphorylation acts in trans to limit the NF-κB mediated transcription of pro-inflammatory genes. The regulation of a functional class of pro-inflammatory genes by the interaction of S80 phosphorylated p50 with a specific κB sequence describes a novel mechanism for the control of cytokine-induced transcriptional responses.

Functional genetic variants can mediate their regulatory effects through alteration of transcription factor binding

Functional variants in the genome are usually identified by their association with local gene expression, DNA methylation or chromatin states. DNA sequence motif analysis and chromatin immunoprecipitation studies have provided indirect support for the hypothesis that functional variants alter transcription factor binding to exert their effects. In this study, we provide direct evidence that functional variants can alter transcription factor binding. We identify a multifunctional variant within the TBC1D4 gene encoding a canonical NFκB binding site, and edited it using CRISPR-Cas9 to remove this site. We show that this editing reduces TBC1D4 expression, local chromatin accessibility and binding of the p65 component of NFκB. We then used CRISPR without genomic editing to guide p65 back to the edited locus, demonstrating that this re-targeting, occurring ~182 kb from the gene promoter, is enough to restore the function of the locus, supporting the central role of transcription factors mediating the effects of functional variants.

The Histone Methyltransferase Setdb2 Modulates Macrophage Phenotype and Uric Acid Production in Diabetic Wound Repair

Macrophage plasticity is critical for normal tissue repair to ensure transition from the inflammatory to the proliferative phase of healing. We examined macrophages isolated from wounds of patients afflicted with diabetes and of healthy controls and found differential expression of the methyltransferase Setdb2. Myeloid-specific deletion of Setdb2 impaired the transition of macrophages from an inflammatory phenotype to a reparative one in normal wound healing. Mechanistically, Setdb2 trimethylated histone 3 at NF-κB binding sites on inflammatory cytokine gene promoters to suppress transcription. Setdb2 expression in wound macrophages was regulated by interferon (IFN) β, and under diabetic conditions, this IFNβ-Setdb2 axis was impaired, leading to a persistent inflammatory macrophage phenotype in diabetic wounds. Setdb2 regulated the expression of xanthine oxidase and thereby the uric acid (UA) pathway of purine catabolism in macrophages, andpharmacologic targeting of Setdb2 or the UApathway improved healing. Thus, Setdb2 regulates macrophage plasticity during normal and pathologic wound repair and is a target for therapeutic manipulation.

Role of NF-κB in cytochrome P450 epoxygenases down-regulation during an inflammatory process in astrocytes

Cytochrome P450 (CYP) epoxygenases and their metabolic products, epoxyeicosatrienoic acids (EETs), have been proposed as important therapeutic targets in the brain. However, CYP expression can be modified by the presence of diverse pro-inflammatory cytokines and the subsequent activation of the NF-κB pathway. It has been indicated that CYP epoxygenases are down-regulated by inflammation in the heart, kidney and liver. However, up to this point, there has been no evidence regarding regulation of CYP epoxygenases during inflammation in the brain. Therefore, in order to explore the effects of inflammation and NF-κB activation in CYP2J3 and CYP2C11 regulation, rat primary astrocytes cultures were treated with LPS with and without IMD-0354 (selective NF-κB inhibitor). Cyp2j3 and Cyp2c11 mRNA expression was determined by qRT-PCR; protein expression was determined by immunofluorescence and by Western Blot and total epoxygenase activity was determined by the quantification of EETs by ELISA. NF-κB binding sites in Cyp2j3 and Cyp2c11 promoter regions were bioinformatically predicted and Electrophoretic Mobility Shift Assays (EMSA) were performed to determine if each hypothetic response element was able to bind NF-κB complexes. Results shown that LPS treatment is able to down-regulate astrocyte CYP2J3 and CYP2C11 mRNA, protein and activity. Additionally, we have identified NK-κB as the transcription factor involved in this regulation.

Differential Immune Activation in Fetal Macrophage Populations

Distinct macrophage subsets populate the developing embryo and fetus in distinct waves. However little is known about the functional differences between in utero macrophage populations or how they might contribute to fetal and neonatal immunity. Here we tested the innate immune response of mouse macrophages derived from the embryonic yolk sac and from fetal liver. When isolated from liver or lung, CD11bHI fetal liver derived macrophages responded to the TLR4 agonist LPS by expressing and releasing inflammatory cytokines. However F4/80HI macrophages from the yolk sac did not respond to LPS treatment. While differences in TLR4 expression did not appear to explain these data, F4/80HI macrophages had much lower NLRP3 inflammasome expression compared to CD11bHI macrophages. Gene expression profiling also demonstrated LPS-induced expression of inflammatory genes in CD11bHI macrophages, but not in F4/80HI cells. Genes expressed in LPS-treated CD11bHI macrophages were more likely to contain predicted NF-κB binding sites in their promoter regions. Our data show that CD11bHI macrophages derived from fetal liver are the major pro-inflammatory cells in the developing fetus. These findings could have important implications in better understanding the fetal inflammatory response and the unique features of neonatal immunity.

A feedback loop involving FREP and NF-κB regulates the immune response of sea cucumber Apostichopus japonicus

Our previous work indicated that fibrinogen-related protein (AjFREP) from sea cucumber (Apostichopus japonicus) plays important roles in innate immunity. To understand AjFREP expression regulation in A. japonicus, we cloned the AjFREP promoter and characterized the putative transcription-factor binding motifs. The AjFREP promoter region spans 1365bp, containing several transcription-factor binding sites. The full-length sequence and all truncated fragments exhibited high promoter activity in HEK-293 cells. Luciferase activity significantly increased for P1(−1365/+16) after LPS exposure, suggesting that the promoter responded to LPS. We also found that two potential NF-κB binding sites were involved in the promoter region, and co-transfection assay demonstrated that the first binding site was necessary for AjFREP transcription. The expression of AjNF-κB/Rel after AjFREP knock-down followed by LPS injection in vivo was further investigated. We found that AjNF-κB/Rel transcript significantly decreased after silencing AjFREP with LPS challenge compared with the control at 4 and 8h, suggesting that activated AjFREP in turn affected the NF-κB pathway under immune responses. These results provided novel insights into the activation mechanism of AjFREP, i.e., that selectively changing AjFREP expression may prevent pathogen infection in A. japonicus.

Transcriptional landscape of pulmonary lymphatic endothelial cells during fetal gestation

The genetic programs responsible for pulmonary lymphatic maturation prior to birth are not known. To address this gap in knowledge, we developed a novel cell sorting strategy to collect fetal pulmonary lymphatic endothelial cells (PLECs) for global transcriptional profiling. We identified PLECs based on their unique cell surface immunophenotype (CD31+/Vegfr3+/Lyve1+/Pdpn+) and isolated them from murine lungs during late gestation (E16.5, E17.5, E18.5). Gene expression profiling was performed using whole-genome microarrays, and 1,281 genes were significantly differentially expressed with respect to time (FDR q < 0.05) and grouped into six clusters. Two clusters containing a total of 493 genes strongly upregulated at E18.5 were significantly enriched in genes with functional annotations corresponding to innate immune response, positive regulation of angiogenesis, complement & coagulation cascade, ECM/cell-adhesion, and lipid metabolism. Gene Set Enrichment Analysis identified several pathways coordinately upregulated during late gestation, the strongest of which was the type-I IFN-α/β signaling pathway. Upregulation of canonical interferon target genes was confirmed by qRT-PCR and in situ hybridization in E18.5 PLECs. We also identified transcriptional events consistent with a prenatal PLEC maturation program. This PLEC-specific program included individual genes (Ch25h, Itpkc, Pcdhac2 and S1pr3) as well as a set of chemokines and genes containing an NF-κB binding site in their promoter. Overall, this work reveals transcriptional insights into the genes, signaling pathways and biological processes associated with pulmonary lymphatic maturation in the fetal lung.

Aberrant histone deacetylase 1 expression upregulates vimentin expression via an NF‑κB‑dependent pathway in hepatocellular carcinoma

Aberrantly elevated expression levels of histone deacetylase 1 (HDAC1) and vimentin are closely associated with disease progression in hepatocellular carcinoma (HCC). It was previously demonstrated that knocking down expression of HDAC1 resulted in a concurrent decrease in the expression levels of vimentin. However, a causal link between these two proteins has not yet been demonstrated, to the best of our knowledge. In the present study, the association between HDAC1 and vimentin was investigated using an HDAC1 overexpression platform. HDAC1 and vimentin were significantly increased in HCC cells, and HDAC1 overexpression enhanced vimentin mRNA and protein expression levels in an HDAC1 dose-dependent manner. Subsequently, truncation and mutation of a vimentin promoter demonstrated that HDAC1-induced vimentin expression was dependent on a nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) binding site in the vimentin promoter sequence. Furthermore, HDAC1 induced vimentin expression by promoting NF-κB translocation between the cytoplasm and the nucleus, as opposed to modulating the total expression level of vimentin directly. The data in the present study demonstrated that HDAC1 is overexpressed in HCC and that HDAC1 may upregulate vimentin expression through the NF-κB signaling pathway, thus demonstrating a causal link between HDAC1 and vimentin in HCC, and may provide valuable information in understanding the pathogenesis of HCC.

Considering Abundance, Affinity, and Binding Site Availability in the NF-κB Target Selection Puzzle.

The NF-κB transcription regulation system governs a diverse set of responses to various cytokine stimuli. With tools from in vitro biochemical characterizations, to omics-based whole genome investigations, great strides have been made in understanding how NF-κB transcription factors control the expression of specific sets of genes. Nonetheless, these efforts have also revealed a very large number of potential binding sites for NF-κB in the human genome, and a puzzle emerges when trying to explain how NF-κB selects from these many binding sites to direct cell-type- and stimulus-specific gene expression patterns. In this review, we surmise that target gene transcription can broadly be thought of as a function of the nuclear abundance of the various NF-κB dimers, the affinity of NF-κB dimers for the regulatory sequence and the availability of this regulatory site. We use this framework to place quantitative information that has been gathered about the NF-κB transcription regulation system into context and thus consider questions it answers, and questions it raises. We end with a brief discussion of some of the future prospects that new approaches could bring to our understanding of how NF-κB transcription factors orchestrate diverse responses in different biological contexts.

Design and analysis of stably integrated reporters for inducible transgene expression in human T cells and CAR NK-cell lines

BackgroundCytotoxic activity of T- and NK-cells can be efficiently retargeted against cancer cells using chimeric antigen receptors (CARs) and rTCRs. In the context of solid cancers, use of armored CAR T- and NK cells secreting additional anti-cancer molecules such as cytokines, chemokines, antibodies, BiTEs, inverted cytokine receptors, and checkpoint inhibitors, appears particularly promising, as this may help overcome immunosuppressive tumor microenvironment, attract bystander immune cells, and boost CAR T/NK-cell persistence. Placing the expression of such molecules under the transcriptional control downstream of CAR-mediated T/NK-cell activation offers the advantage of targeted delivery, high local concentration, and reduced toxicity. Several canonic DNA sequences that are known to function as activation-inducible promoters in human T and B cells have been described to date and typically encompass the multimers of NFkB and NFAT binding sites. However, relatively little is known about the DNA sequences that may function as activation-driven switches in the context of NK cells. We set out to compare the functionality of several activation-inducible promoters in primary human T cells, as well as in NK cell lines NK-92 and YT.MethodsLentiviral constructs were engineered to express two fluorescent reporters: mCherry under 4xNFAT, 2xNFkB, 5xNFkB, 10xNFkB, 30xNFkB promoters, as well as two variants of the CD69 promoter, and copGFP under the strong constitutive promoter of the human EF1a gene. Pseudotyped lentiviral particles obtained using these constructs were transduced into primary human T cells and NK-92 and YT cell lines expressing a CAR specific for PSMA. The transgenic cells obtained were activated by CD3/CD28 beads (T cells) or via a CAR (CAR-NK cell lines). Promoter activity before and after activation was assayed using FACS analysis.ResultsIn T cells, the CD69 promoter encompassing CNS1 and CNS2 regions displayed the highest signal/noise ratio. Intriguingly, in the context of CAR-YT cell line neither of the seven promoters tested displayed acceptable activation profile. In CAR-NK-92 cells, the largest fold activation (which was modest) was achieved with the 10xNFkB and 30xNFkB promoters, however its expression was clearly leaky in “resting” non-activated cells.ConclusionsUnlike in T cells, the robust activation-driven inducible expression of genetic cassettes in NK cells requires unbiased genome-wide identification of promoter sequences.

Abstract P5-05-03: Estrogen receptor beta elicits anti-cancer effects in triple negative breast cancer through suppression of NFκB signaling

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, Estrogen Receptor Beta (ERβ) protein has been shown to be expressed in approximately 25% of TNBCs and is associated with improved patient outcomes. Using multiple ERβ +/- TNBC cell lines and PDX models, we have demonstrated that ligand-mediated activation of ERβ by estradiol (E2) decreases cell proliferation, invasion, and migration in vitro, as well as primary tumor growth and metastatic spread in vivo. Methods: To determine the mechanisms by which ERβ elicits these anti-cancer effects, we elucidated the ERβ transcriptome and cistrome via Microarray and ChIPseq, respectively, in TNBC cells stably expressing ERβ in a doxycycline-inducible manner. We also performed gene expression and luciferase assays to assess the impact of ERβ on NFκB signaling, followed by ChIP-PCR and ChIPseq to assess how ERβ modifies chromatin architecture near NFκB target genes. Results: Pathway analysis of ERβ-regulated genes identified NFκB signaling as one of the most suppressed pathways in response to E2 treatment. Indeed, numerous NFκB target genes were among the most down-regulated genes following E2 treatment but only in the presence of ERβ expression. Chromatin Immunoprecipitation followed by sequencing (ChIPseq) revealed that ERβ primarily associated with estrogen response elements (EREs), but was also enriched around NFκB binding sites following E2 treatment. In fact, 12% of all ERβ binding sites were enriched for NFκB response elements and ERβ 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 200 genes to be significantly regulated by TNFα in TNBC cells, of which 81 were significantly altered in the presence of E2+TNFα. ChIPseq 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 are also associated with recruitment of the histone methyltransferase, EZH2, to enhancer elements of these NFκB target genes. Drug-mediated blockade of HDAC and EZH2 activity reversed suppression of NFκB target gene expression by ERβ. Conclusions: Our data suggest that ERβ may elicit 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 role of estradiol in ERβ expressing TNBC and to further evaluate the cross-talk between ERβ and NFκB signaling in TNBC. Citation Format: Aspros K, Nelson A, Ye Z, Sun Z, Chernukhin I, Carroll J, Ingle J, Goetz M, Hawse J. Estrogen receptor beta elicits anti-cancer effects in triple negative breast cancer through suppression of NFκB signaling [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-03.

The role of NF-κB and Elk-1 in the regulation of mouse ADAM17 expression.

ABSTRACTADAM17 is a cell membrane metalloproteinase responsible for the release of ectodomains of numerous proteins from the cell surface. Although ADAM17 is often overexpressed in tumours and at sites of inflammation, little is known about the regulation of its expression. Here we investigate the role of NF-κB and Elk-1 transcription factors and upstream signalling pathways, NF-κB and ERK1/2 in ADAM17 expression in mouse brain endothelial cells stimulated with pro-inflammatory factors (TNF, IL-1β, LPS) or a phorbol ester (PMA), a well-known stimulator of ADAM17 activity. Notably, NF-κB inhibitor, IKK VII, interfered with the IL-1β- and LPS-mediated stimulation of ADAM17 expression. Furthermore, Adam17 promoter contains an NF-κB binding site occupied by p65 subunit of NF-κB. The transient increase in Adam17 mRNA in response to PMA was strongly reduced by an inhibitor of ERK1/2 phosphorylation, U0126. Luciferase reporter assay with vectors encoding the ERK1/2 substrate, Elk-1, fused with constitutively activating or repressing domains, indicated Elk-1 involvement in Adam17 expression. The site-directed mutagenesis of potential Elk-1 binding sites pointed to four functional Elk-1 binding sites in Adam17 promoter. All in all, our results indicate that NF-κB and Elk-1 transcription factors via NF-κB and ERK1/2 signalling pathways contribute to the regulation of mouse Adam17 expression.

Involvement of JNK signaling pathway in lipopolysaccharide-induced complement C3 transcriptional activation from amphioxus Branchiostoma belcheri

Complement C3 is a pivotal component of three cascades of complement activation. C3 in circulation is mainly provided by the hepatic cecum. The expression and secretion of C3 by hepatocytes is increased during acute inflammation. The detailed information on the regulationary mechanism underlying C3 transcriptional activation is limited. Here, we characterized the 5′-flanking region of the amphioxus C3 gene. To functionally analyze the upstream regulatory region of the C3 gene, a series of luciferase reporter gene constructs containing deleted or mutant regulatory elements were prepared. Using luciferase assay, we revealed that a potential C-JUN-1 binding sites within the proximal promoter region were necessary for full activation of the C3 promoter, whereas NF-κB, AP-1, C-JUN-2 and NFAT transcription factor binding sites played roles in governing the promoter activity at a homeostatic level. Our data also indicated that sp600125, a c-Jun N-terminal kinase (JNK) inhibitor, decreased lipopolysaccharide (LPS)-stimulated C3 promoter activity, mRNA expression and protein secretion using western blotting and quantitative real-time PCR analysis. These findings demonstrated that JNK signaling pathway is involved in the regulation of C3 gene transcription by targeting C-JUN transcription factor binding sites in the 5′-flanking promoter region, leading to LPS-induced C3 activation and therefore providing a potential target for regulating C3 expression.

Lack of Vitamin D Receptor Leads to Hyperfunction of Claudin-2 in Intestinal Inflammatory Responses.

Vitamin D3 and vitamin D receptor (VDR) are involved in the pathogenesis of inflammatory bowel disease (IBD) and bacterial infection. Claudin-2 is a junction protein that mediates paracellular water transport in epithelia. Elevation of Claudin-2 is associated with active IBD. However, VDR involved in epithelial junctions under inflammation and infection remains largely unknown. We investigated the mechanisms on how VDR and Claudin-2 are related in inflamed states. Using cultured VDR-/- enteroids, human intestinal epithelial cells, VDR-/- mice with Salmonella- or DSS-colitis, and human IBD samples, we investigated the mechanisms how VDR and Claudin-2 are related in inflamed states. After Salmonella infection had taken place, we observed significantly enhanced Claudin-2 and an increased bacterial invasion and translocation. A lack of VDR regulation led to a robust increase of Claudin-2 at the mRNA and protein levels post-infection. In DSS-treated VDR-/- mice, Claudin-2 was significantly increased. Location and quantification of Claudin-2 protein in the mouse colons treated with DSS also confirmed these results. Inflammatory cytokines were significantly higher in the serum and mRNA levels in intestine, which are known to increase Claudin-2. Furthermore, in inflamed intestine of ulcerative colitis patients, VDR expression was low and Claudin-2 was enhanced. Mechanistically, we identified the enhanced Claudin-2 promoter activity through the binding sites of NF-κB and STAT in inflamed VDR-/- cells. Our studies have identified a new role for intestinal epithelial VDR in regulating barrier functions in the context of infection and inflammation.

Practical exercises with the EFSA WEB app based on PROAST: Presentation of case studies and modelling of provided datasets

Aristolochic acid I (AAI) is a phytocompound that is linked to the progressive renal disease and development of human urothelial carcinoma. The bladder cancer-associated protein (BLCAP) gene exhibits a tumor suppressor function in various tumors, including bladder carcinoma. This study evaluated the effect of AAI on BLCAP expression and its associated mechanism in human cells. Administering AAI to human embryonic kidney cells (HEK293), human proximal tubule epithelial cells (HK-2) and urinary bladder cancer cells (HT-1376) significantly reduced the expression of BLCAP mRNA and protein. AAI also effectively suppressed the luciferase activities driven by BLCAP promoters of various lengths in HEK293 cells. AAI significantly reduced both activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) activities in reporter assays, but further point mutations revealed that Ap-1 and NF-κB binding sites on the BLCAP promoter were not AAI-responsive elements. Application of the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC), reversed the decline of BLCAP expression that had been induced by AAI. However, AAI exposure did not alter hypermethylation of the BLCAP promoter, determined by methyl-specific polymerase chain reaction (PCR) and bisulfate sequencing. Knocking down BLCAP in HEK293 cell line enhanced the potential for cellular migration, invasion, and proliferation, along with the induction of a capacity for anchorage-independent growth. In conclusion, AAI down-regulated the expression of BLCAP gene and the deficiency in BLCAP expression contributed to the malignant transformation of human cells, implying that BLCAP may have a role in mediating AAI-associated carcinogenesis.

Triptolide attenuates proteinuria and podocyte apoptosis via inhibition of NF-\u03baB/GADD45B

Podocyte injury is a primary contributor to proteinuria. Triptolide is a major active component of Tripterygium wilfordii Hook F that exhibits potent antiproteinuric effects. We used our previously developed in vivo zebrafish model of inducible podocyte-target injury and found that triptolide treatment effectively alleviated oedema, proteinuria and foot process effacement. Triptolide also inhibited podocyte apoptosis in our zebrafish model and in vitro. We also examined the mechanism of triptolide protection of podocyte. Whole-genome expression profiles of cultured podocytes demonstrated that triptolide treatment downregulated apoptosis pathway-related GADD45B expression. Specific overexpression of gadd45b in zebrafish podocytes abolished the protective effects of triptolide. GADD45B is a mediator of podocyte apoptosis that contains typical NF-κB binding sites in the promoter region, and NF-κB p65 primarily transactivates this gene. Triptolide inhibited NF-κB signalling activation and binding of NF-κB to the GADD45B promoter. Taken together, our findings demonstrated that triptolide attenuated proteinuria and podocyte apoptosis via inhibition of NF-κB/GADD45B signalling, which provides a new understanding of the antiproteinuric effects of triptolide in glomerular diseases.