NF-κB Target Research Articles

An AAV-Based NF-κB-Targeting Gene Therapy (rAAV-DMP-miR533) to Inflammatory Diseases.

BackgroundThe inflammatory diseases pose a great threat to human health. Variant anti-inflammatory agents have been therefore developed. However, the current anti-inflammatory drugs are still challenged by low response and side effects. There remain great unmet treatments to inflammatory diseases.MethodsIn this work, we fabricate a recombinant adeno-associated virus (rAAV), rAAV-DMP-miR533, by packaging a DNA molecule DMP-miR533 into AAV, in which DMP is a NF-κB-activatable promoter composed of a NF-κB decoy and a minimal promoter and miR533 codes an artificial microRNA targeting NF-κB RELA. We evaluate the in vitro and in vivo anti-inflammatory effect of the virus with inflammatory cells and the mice of three typical inflammatory diseases including the dextran sulphate sodium-induced acute colitis, imiquimod-induced psoriasis, and collagen-induced arthritis.ResultsWe found that rAAV-DMP-miR533 had marked anti-inflammatory effect in both cells and mice. In addition, rAAV-DMP-miR533 showed biosafety in mice.ConclusionThis study thus provides a promising gene therapy to variant inflammatory diseases by directly targeting NF-κB, an established hub regulator of inflammation.

Molecular Characterization of Membranous Nephropathy.

Molecular characterization of nephropathies may facilitate pathophysiologic insight, development of targeted therapeutics, and transcriptome-based disease classification. Although membranous nephropathy (MN) is a common cause of adult-onset nephrotic syndrome, the molecular pathways of kidney damage in MN require further definition. We applied a machine-learning framework to predict diagnosis on the basis of gene expression from the microdissected kidney tissue of participants in the Nephrotic Syndrome Study Network (NEPTUNE) cohort. We sought to identify differentially expressed genes between participants with MN versus those of other glomerulonephropathies across the NEPTUNE and European Renal cDNA Bank (ERCB) cohorts, to find MN-specific gene modules in a kidney-specific functional network, and to identify cell-type specificity of MN-specific genes using single-cell sequencing data from reference nephrectomy tissue. Glomerular gene expression alone accurately separated participants with MN from those with other nephrotic syndrome etiologies. The top predictive classifier genes from NEPTUNE participants were also differentially expressed in the ERCB participants with MN. We identified a signature of 158 genes that are significantly differentially expressed in MN across both cohorts, finding 120 of these in a validation cohort. This signature is enriched in targets of transcription factor NF-κB. Clustering these MN-specific genes in a kidney-specific functional network uncovered modules with functional enrichments, including in ion transport, cell projection morphogenesis, regulation of adhesion, and wounding response. Expression data from reference nephrectomy tissue indicated 43% of these genes are most highly expressed by podocytes. These results suggest that, relative to other glomerulonephropathies, MN has a distinctive molecular signature that includes upregulation of many podocyte-expressed genes, provides a molecular snapshot of MN, and facilitates insight into MN's underlying pathophysiology.

NF-κB over-activation portends improved outcomes in HPV-associated head and neck cancer.

Evolving understanding of head and neck squamous cell carcinoma (HNSCC) is leading to more specific diagnostic disease classifications. Among HNSCC caused by the human papilloma virus (HPV), tumors harboring defects in TRAF3 or CYLD are associated with improved clinical outcomes and maintenance of episomal HPV. TRAF3 and CYLD are negative regulators of NF-κB and inactivating mutations of either leads to NF-κB overactivity. Here, we developed and validated a gene expression classifier separating HPV+ HNSCCs based on NF-κB activity. As expected, the novel classifier is strongly enriched in NF-κB targets leading us to name it the NF-κB Activity Classifier (NAC). High NF-κB activity correlated with improved survival in two independent cohorts. Using NAC, tumors with high NF-κB activity but lacking defects in TRAF3 or CYLD were identified; thus, while TRAF3 or CYLD gene defects identify the majority of tumors with NF-κB activation, unknown mechanisms leading to NF-kB activity also exist. The NAC correctly classified the functional consequences of two novel CYLD missense mutations. Using a reporter assay, we tested these CYLD mutations revealing that their activity to inhibit NF-kB was equivalent to the wild-type protein. Future applications of the NF-κB Activity Classifier may be to identify HPV+ HNSCC patients with better or worse survival with implications for treatment strategies.

NCOA2 coordinates with the transcriptional KAT2B-NF-κB partner to trigger inflammation response in acute kidney injury

Dysregulation of multiple genes is an important risk factor for acute kidney injury (AKI). Numerous genes, such as proinflammatory cytokines, intracellular cell adhesion molecules (ICAMs), and nitric oxide synthases (NOSs), are implicated in AKI pathogenesis. However, the molecular mechanisms involved in the dysregulation of these genes are still obscure. Herein, we discovered that two subunits of NF-κB (p50 and p65) couple with lysine acetyltransferase 2B (KAT2B) and nuclear receptor coactivator 2 (NOCA2) to assemble a transcriptional complex in a LPS-induced mouse model of AKI. The NCOA2-KAT2B-NF-κB complex bound to the promoters of some NF-κB target genes, such as interleukin 1 beta (IL-1B), IL-6, tumor necrosis factor alpha (TNFA), ICAM1, vascular cell adhesion molecule 1 (VCAM1), cluster of differentiation 38 (CD38), CD40, CD80, and NOS2, and transactivated their expression. In vitro knockdown of components of the NCOA2-KAT2B-NF-κB complex or blockage of KAT2B by its inhibitors (5-chloro-2-(4-nitrophenyl)-3(2H)-isothiazolone [CNIT] and garcinol) significantly decreased the expression of these NF-κB target genes following LPS treatment. The administration of CNIT and garcinol significantly improved the in vivo outcomes of the AKI mice. Our findings reveal the underlying mechanism of NF-κB target upregulation in the pathogenesis of LPS-induced AKI and identify a new therapeutic strategy for AKI that involves targeting the NCOA2-KAT2B-NF-κB complex.

PLGA-Nano-Encapsulated Disulfiram Inhibits Hypoxia-Induced NF-κB, Cancer Stem Cells, and Targets Glioblastoma In Vitro and In Vivo.

Glioblastoma stem cell (GSC) is the major cause of glioblastoma multiforme (GBM) chemotherapy failure. Hypoxia is one of the determinants of GSC. NF-κB plays a pivotal link between hypoxia and cancer stem cells (CSCs). Disulfiram, an antialcoholism drug, has very strong NF-κB-inhibiting and anti-CSC activity. In this study, the in vitro anti-GSC activity of disulfiram and in vivo anti-GBM efficacy of poly lactic-co-glycolic acid nanoparticle-encapsulated disulfiram (DS-PLGA) were examined. We attempt to elucidate the molecular network between hypoxia and GSCs and also examined the anti-GSC activity of disulfiram in vitro and in vivo. The influence of GSCs and hypoxia on GBM chemoresistance and invasiveness was studied in hypoxic and spheroid cultures. The molecular regulatory roles of NF-κB, hypoxia-inducible factor-1α (HIF1α), and HIF2α were investigated using stably transfected U373MG cell lines. The hypoxia in neurospheres determines the cancer stem cell characteristics of the sphere-cultured GBM cell lines (U87MG, U251MG, U373MG). NF-κB is located at a higher hierarchical position than HIF1α/HIF2α in hypoxic regulatory network and plays a key role in hypoxia-induced GSC characters. DS inhibits NF-κB activity and targets hypoxia-induced GSCs. It showed selective toxicity to GBM cells, eradicates GSCs, and blocks migration and invasion at very low concentrations. DS-PLGA efficaciously inhibits orthotopic and subcutaneous U87MG xenograft in mouse models with no toxicity to vital organs.

Maladaptation after a virus host switch leads to increased activation of the pro-inflammatory NF-κB pathway

Myxoma virus (MYXV) causes localized cutaneous fibromas in its natural hosts, tapeti and brush rabbits; however, in the European rabbit, MYXV causes the lethal disease myxomatosis. Currently, the molecular mechanisms underlying this increased virulence after cross-species transmission are poorly understood. In this study, we investigated the interaction between MYXV M156 and the host protein kinase R (PKR) to determine their crosstalk with the proinflammatory nuclear factor kappa B (NF-κB) pathway. Our results demonstrated that MYXV M156 inhibits brush rabbit PKR (bPKR) more strongly than European rabbit PKR (ePKR). This moderate ePKR inhibition could be improved by hyperactive M156 mutants. We hypothesized that the moderate inhibition of ePKR by M156 might incompletely suppress the signal transduction pathways modulated by PKR, such as the NF-κB pathway. Therefore, we analyzed NF-κB pathway activation with a luciferase-based promoter assay. The moderate inhibition of ePKR resulted in significantly higher NF-κB–dependent reporter activity than complete inhibition of bPKR. We also found a stronger induction of the NF-κB target genes TNFα and IL-6 in ePKR-expressing cells than in bPKR-expressing cells in response to M156 in both transfection and infections assays. Furthermore, a hyperactive M156 mutant did not cause ePKR-dependent NF-κB activation. These observations indicate that M156 is maladapted for ePKR inhibition, only incompletely blocking translation in these hosts, resulting in preferential depletion of short–half-life proteins, such as the NF-κB inhibitor IκBα. We speculate that this functional activation of NF-κB induced by the intermediate inhibition of ePKR by M156 may contribute to the increased virulence of MYXV in European rabbits.

ST08 Altered NF-κB Pathway in Breast Cancer Cells In Vitro as Revealed by miRNA-mRNA Analysis and Enhanced the Effect of Cisplatin on Tumour Reduction in EAC Mouse Model

ST08 is a novel curcumin derivative that exhibited apoptotic and anti-migratory activity in MDA-MB-231, triple-negative breast cancer cells reported earlier. In this study, we further explored the anticancer properties of ST08. ST08 reduced tumor burden in vivo and induced apoptosis through the mitochondrial pathway both in vitro and in vivo. ST08 potentiated the effect of cisplatin in vitro and in vivo in mouse EAC breast cancer models with minimal toxicity. ST08 induced alterations in the gene expression were studied by parallel analysis of miRNA and mRNA. 74 differentially expressed miRNA regulated 114 mRNA in triple-negative (MDA-MB-231) cancer cells. Pathway related to the ECM was altered in mesenchymal MDA-MB-231 cells. We constructed a unique miRNA-mRNA interaction network, and one of the pathways regulated by miRNA was NF-κB. Targets of NF-κB like MMP1, PTX3, and MMP2 were downregulated in MDA-MB-231 in response to ST08 treatment. PMA induced cell proliferation was abrogated by ST08 treatment, and no additional cell cytotoxicity was observed when used in combination with IKK-16 indicating ST08 regulation of NF-κB pathway in MDA-MB-231 cells.

Allergic inflammation in lungs and nasal epithelium of rat model is regulated by tissue-specific miRNA expression

IntroductionAtopic asthma and allergic rhinitis are common chronic inflammatory diseases affecting lower airways and nasal mucosa, respectively. Several reports demonstrated frequent co-occurrence of these two diseases, however, the exact molecular mechanism has not been described. The present study aimed to investigate if small non-coding RNA might be responsible for the co-occurrence of asthma and allergic rhinitis in an animal model of allergic airway inflammation. Materials and methodsAs an in vivo model of allergic airway inflammation, we used Brown Norway rats exposed intranasally to house dust mite (HDM). Histological analysis, total IgE concentration, eosinophil counts and iNOS gene expression were determined to confirm inflammatory changes. Small RNA sequencing in the lung tissue and nasal epithelium was performed with TruSeq Small RNA Library Preparation Kit and analyzed using the BaseSpace tool. Validation of sequencing results was performed using qPCR. To assess the functional role of hsa-miR-223–3p, we transfected normal human bronchial epithelial (NHBE) cells with specific LNA-inhibitor and measured phosphorylated protein level of NF-kB with ELISA. Expression analysis of NF-kB pathway-related genes was performed using qPCR with SYBR Green and analyzed in DataAssist v3.01. Statistical analysis were done with STATISTICA version 13. ResultsWe found 9 miRNA genes differentially expressed in the lungs of allergic rats. In nasal epithelium, only rno-miR-184 was upregulated in animals exposed to HDM. Validation with qPCR confirmed increased expression only for rno-miR-223–3p in the lungs from allergic rats. The expression of this miRNA was also increased in normal bronchial epithelial ALI cell culture stimulated with IL-13, but not in cells cultured in monolayer due to the low mRNA level of IL13RA1 and IL13RA2. Transfecting NHBE cells with hsa-miR-223–3p inhibitor increased the amount of phosphorylated NF-kB protein level and expression of MUC5AC, CCL24 and TSLP genes. ConclusionsThese findings suggest that miRNAs that regulate allergic inflammation in the lungs and nasal epithelium are specific for upper and lower airways. Furthermore, our study provides new insight on the role of hsa-miR-223–3p, that via targeting NF-kB signaling pathway, regulates the expression of MUC5AC, CCL24 and TSLP. Taken together, our study suggests that miR-223–3p is a regulator of allergic inflammation and could potentially be used to develop novel and targeted therapy for asthma.

Extracellular vesicle-derived miR-1249–5p regulates influenza A virus-induced acute lung injury in RAW246.7 cells through targeting SLC4A1

Acute lung injury (ALI) is characterized by tissue damage that leads to pulmonary epithelial membrane dysfunction and macrophage activation. Currently however, the exact mechanism by which the initial mediators of mouse lung epithelial (MLE-12) cells induce inflammation remines unclear. We constructed co-culture systems of MLE-12 cells with mouse macrophage cells RAW246.7 which were realized by the supernatant and Transwell chamber. In previous study, we successfully constructed an influenza A virus-induced MLE-12 cells model. Extracellular Vesicles (EVs) from cells supernatant were isolated by differential ultracentrifugation and confirmed by transmission electron microscopy. High-throughput sequencing results showed that MLE-12 cells stimulated by influenza A virus had higher level of miR-1249–5p. The results were validated by RT-qPCR analysis. The research aimed to investigate the roles and mechanisms of miR-1249–5p in ALI. RAW246.7 cells were transfected with miR-1249–5p mimic/inhibitor. The concentrations of TNF-α, IL-6 were determined by ELISA and the uptake of EVs was monitored by confocal laser scanning microscope. Western blotting detected changes in the SLC4A1 and NF-κB signaling pathway. The results indicated that miR-1249–5p played an important role in ALI, and further investigation of its target gene SLC4A1 and NF-κB signaling pathway provides ideas for new therapeutic targets and strategies for ALI.

Transcriptomics-Based Investigation of Molecular Mechanisms Underlying Apoptosis Induced by ZnO Nanoparticles in Human Diffuse Large B-Cell Lymphoma.

IntroductionZinc oxide nanoparticles (ZnO NPs) show anti-cancer activity. Diffuse Large B-cell Lymphoma (DLBCL) is a type of B-cell malignancies with unsatisfying treatment outcomes. This study was set to assess the potential of ZnO NPs to selectively induce apoptosis in human DLBCL cells (OCI-LY3), and to describe possible molecular mechanisms of action.MethodsThe impact of ZnO NPs on DLBCL cells and normal peripheral blood mononuclear cells (PBMCs) was studied using cytotoxicity assay and flow-cytometry. Transcriptomics analysis was conducted to identify ZnO NPs-dependent changes in the transcriptomic profiles of DLBCL cells.ResultsZnO NPs selectively induced apoptosis in DLBCL cells, and caused changes in their transcriptomes. Deferential gene expression (DGE) with fold change (FC) ≥3 and p ≤ 0.008 with corrected p ≤ 0.05 was identified for 528 genes; 125 genes were over-expressed and 403 genes were under-expressed in ZnO NPs-treated DLBCL cells. The over-expressed genes involved in biological processes and pathways like stress response to metal ion, cellular response to zinc ion, metallothioneins bind metals, oxidative stress, and negative regulation of growth. In contrast, the under-expressed genes were implicated in DNA packaging complex, signaling by NOTCH, negative regulation of gene expression by epigenetic, signaling by WNT, M phase of cell cycle, and telomere maintenance. Setting the FC to ≥1.5 with p ≤ 0.05 and corrected p ≤ 0.1 showed ZnO NPs to induce over-expression of anti-oxidant genes and under-expression of oncogenes; target B-cell receptor (BCR) signaling pathway and NF-κB pathway; and promote apoptosis by intrinsic and extrinsic pathways.DiscussionOverall, ZnO NPs selectively induced apoptosis in DLBCL cells, and possible molecular mechanisms of action were described.

Interferon‐γ Induces Bcl3 Expression in Ovarian Cancer Cells by JAK1/STAT1 Signaling

The proto‐oncogene Bcl3 is an atypical member of IkB family, which regulates expression of a number of NFkB target genes, including anti‐apoptotic genes, cytokines, and immune checkpoints. Bcl3 expression is increased in many types of human cancers and is associated with increased tumor growth and malignant potential. Interferon‐γ (IFNγ) is a pleiotropic cytokine that has been used in cancer treatment; in addition, its expression is induced in response to radiation therapy and immune checkpoint blockade during cancer treatment. We have recently shown that IFNγ induces Bcl3 expression in ovarian cancer cells, resulting in their increased proliferation, migration, and invasion. In this study, we have investigated the mechanisms of how IFNγ induces the Bcl3 expression in ovarian cancer cells. Our results demonstrate that the IFNγ‐induced Bcl3 expression is dependent on JAK1 and STAT1 signaling, as well as on p65 NFkB. In addition, we have identified a potential interferon regulatory factor 1 (IRF1) binding site in human Bcl3 promoter and found that the IFNγ‐induced Bcl3 expression in ovarian cancer cells is associated with an increased occupancy of IRF1, Ser‐727 phosphorylated STAT1 and acetylated histone H3 at the IRF1 binding site in Bcl3 promoter. These findings identify Bcl3 as a novel target of IFNγ/JAK1/STAT1 signaling and suggest that targeting the JAK1/STAT1 pathway may suppress the IFNγ‐induced Bcl3 expression in ovarian cancer.

Novel Nexus with NFκB, β-catenin, and RB1 empowers PSMD10/Gankyrin to counteract TNF-α induced apoptosis establishing its oncogenic role.

NFκB is a critical rapid-acting transcription factor that protects cancer cells from programmed cell death induced by stress or therapy. While NFκB works in nexus with non-classical oncoproteins such as STAT3 and AKT under a variety of conditions, it is a major antiapoptotic factor activated by TNF-α of the tumor microenvironment. Therefore, it is surprising that PSMD10, an oncoprotein overexpressed in several cancers and a marker of poor prognosis, is reported to inhibit the NFκB pathway. In this study, we explore the role of PSMD10 in cancer cells exposed to TNF-α. We screen several breast and colon cancer cell lines and select SW480, a colon cancer cell line highly resistant to TNF-α, and demonstrate that PSMD10 knockdown sensitizes these cells to TNF-α induced cell death. One of the mechanisms involves transcriptional regulation of β-catenin and RB1, two key colon cancer cell specific anti-apoptotic factors. Surprisingly, we find that PSMD10 is required for optimal phosphorylation and transcriptional activation of NFκB (RELA). Thus, upon PSMD10 knockdown, there is significant downregulation of anti-apoptotic NFκB target genes TNFAIP3 (A20), BIRC2 (cIAP1), BIRC3 (cIAP2), and XIAP. Our study, for the first time, shows that PSMD10 is required for the activation of the pro-survival arm via NFκB transcriptional activation to prevent cancer cells from succumbing to TNF-induced cell death. In addition by transcriptional regulation of two major antiapoptotic players RB1 and β-catenin, PSMD10 proves to be a coveted oncoprotein with a key role in tumorigenesis.

Toll Like Receptor 4 Inhibition Improves Antitumor Responses to MBQ‐167 in Triple Negative Breast Cancer Cells in vitro

Triple negative breast cancer (TNBC) is an aggressive variant of breast cancer that lacks the expression of estrogen, progesterone, and human epithelial growth factor receptors. Due to the scarce availability of receptor‐targeted or hormonal treatments, TNBC patients are submitted to taxane‐based chemotherapies like paclitaxel. Patients initially respond to paclitaxel, but 4 out of 10 patients may develop tumor recurrence along with high metastasis and mortality rates. Several studies demonstrate that paclitaxel may induce chemoresistance and metastasis through the activation of Toll‐Like Receptor 4 (TLR4)/NFKB signaling pathway, a known cancer prognostic marker in breast cancer. Hence, new treatments are needed to improve TNBC prognosis in patients. Recently, our laboratory and collaborators developed MBQ‐167, a potent Rac and Cdc42 inhibitor that decreases tumor burden, cell proliferation, and metastasis in TNBC mouse models. In preliminary studies with TNBC mice, we observed that MBQ‐167/Paclitaxel combination treatment prevented metastasis progression compared to paclitaxel treatment alone. This led us to hypothesize that MBQ‐167 chemosensitizes aggressive breast cancer cells and reduces metastasis by blocking Rac/Cdc4, hence decreasing their downstream target NfkB activity. This research project aims to evaluate cellular and molecular events that explain how MBQ‐167 prevents paclitaxel‐induced metastasis. Here, we generated TLR4‐knockdowns (TLR4‐KD) in MDA‐MB‐231 TNBC cell lines using lentiviral particles with shRNAs against the TLR4 mRNA. After knockdown validation with western blot analysis (WB), Wildtype (MDA‐MB‐231), Scramble control knockdowns (Sc‐KD), and TLR4‐KD cells were treated with different concentrations of MBQ‐167, paclitaxel, doxorubicin, or combinations. After treatments, we evaluated the effect on cell viability from MTT assays at 96 and 120 hours, cell apoptosis through Caspase‐Glo 3/7 Assays at 48 hours, and cellular migration through scratch wound healing assays. Our results demonstrate that partial inhibition of TLR4 decreases the cell viability of cells, increases apoptosis, and decreases cell migration after treatment with MBQ‐167 (250 nM and 500 nM), Paclitaxel (5 nM and 10 nM), or Doxorubicin (250 nM and 500 nM). We also observed an additive response upon combinatorial treatments with MBQ‐167/Paclitaxel and MBQ‐167/Doxorubicin when TLR4‐KD cells were compared to wild‐type and Sc‐KD cells. Altogether, these results suggest that TLR4‐KD chemosensitizes cells to Paclitaxel and Doxorubicin chemotherapies. In addition, TLR4 inhibition improves cellular response to Rac/Cdc42 inhibitor, MBQ‐167. Therefore, TLR4‐KD may reduce Rac and NfkB signaling, improving treatment response to MBQ‐167, MBQ‐167/Paclitaxel, and MBQ‐167/Doxorubicin. Other cellular studies, including Rac and NfkB activation assays, are being evaluated in our laboratory.

Factors Regulating or Regulated by Myogenic Regulatory Factors in Skeletal Muscle Stem Cells.

MyoD, Myf5, myogenin, and MRF4 (also known as Myf6 or herculin) are myogenic regulatory factors (MRFs). MRFs are regarded as master transcription factors that are upregulated during myogenesis and influence stem cells to differentiate into myogenic lineage cells. In this review, we summarize MRFs, their regulatory factors, such as TLE3, NF-κB, and MRF target genes, including non-myogenic genes such as taste receptors. Understanding the function of MRFs and the physiology or pathology of satellite cells will contribute to the development of cell therapy and drug discovery for muscle-related diseases.

Aortic Fibrosis in Insulin-Sensitive Mice with Endothelial Cell-Specific Deletion of Ceacam1 Gene.

(1) Background: Mice with global Ceacam1 deletion developed plaque-like aortic lesions even on C57BL/6J background in the presence of increased endothelial cell permeability and insulin resistance. Loss of endothelial Ceacam1 gene caused endothelial dysfunction and reduced vascular integrity without affecting systemic insulin sensitivity. Because endothelial cell injury precedes atherosclerosis, we herein investigated whether the loss of endothelial Ceacam1 initiates atheroma formation in the absence of insulin resistance. (2) Methods: Endothelial cell-specific Ceacam1 null mice on C57BL/6J.Ldlr−/− background (Ldlr−/−VECadCre+Cc1fl/fl) were fed an atherogenic diet for 3–5 months before metabolic, histopathological, and en-face analysis of aortae were compared to their control littermates. Sirius Red stain was also performed on liver sections to analyze hepatic fibrosis. (3) Results: These mice displayed insulin sensitivity without significant fat deposition on aortic walls despite hypercholesterolemia. They also displayed increased inflammation and fibrosis. Deleting Ceacam1 in endothelial cells caused hyperactivation of VEGFR2/Shc/NF-κB pathway with resultant transcriptional induction of NF-κB targets. These include IL-6 that activates STAT3 inflammatory pathways, in addition to endothelin-1 and PDGF-B profibrogenic effectors. It also induced the association between SHP2 phosphatase and VEGFR2, downregulating the Akt/eNOS pathway and reducing nitric oxide production, a characteristic feature of endothelial dysfunction. Similarly, hepatic inflammation and fibrosis developed in Ldlr−/−VECadCre+Cc1fl/fl mice without an increase in hepatic steatosis. (4) Conclusions: Deleting endothelial cell Ceacam1 caused hepatic and aortic inflammation and fibrosis with increased endothelial dysfunction and oxidative stress in the presence of hypercholesterolemia. However, this did not progress into frank atheroma formation. Because these mice remained insulin sensitive, the study provides an in vivo demonstration that insulin resistance plays a critical role in the pathogenesis of frank atherosclerosis.

Molecular dynamics simulation and docking analysis of NF-κB protein binding with sulindac acid.

It is of interest to document the Molecular Dynamics Simulation and docking analysis of NF-κB target with sulindac sodium in combating COVID-19 for further consideration. Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) of the arylalkanoic acid class that is marketed by Merck under the brand name Clinoril. We show the binding features of sulindac sodium with NF-κB that can be useful in drug repurposing in COVID-19 therapy.

The Effect of the NFκB-USP9X-Cx43 Axis on the Dynamic Balance of Bone Formation/Degradation during Ossification of the Posterior Longitudinal Ligament of the Cervical Spine

Connexin 43- (Cx43-) mediated nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling has been found involved in the ossification of the posterior longitudinal ligament (OPLL). However, the underlying mechanism how OPLL is regulated has not been elucidated. In the present study, primary ligament fibroblast were isolated; immunoprecipitation (IP) and liquid chromatography-mass spectrometry (LC-MS) assays were applied to identify potential binding proteins of Cx43. Protein interaction was then confirmed by co-IP assay. Alkaline phosphatase (ALP) activity and alizarin red staining were used to evaluate ossification. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were employed to assess the binding between NF-κB p65 and target gene. Lipoxygenase inhibitor (5,8,11-eicosatriynoic acid, EPA) was applied to induce endoplasmic reticulum (ER) stress, and 4-phenylbutyrate (4-PBA) was used as an ER-stress inhibitor. Expression of USP9X, Cx43, and nuclei p65 in ligaments from patients and controls was detected by Western blotting. The results showed that ubiquitin-specific protease 9 X-chromosome (USP9X), a deubiquitylating enzyme, was a candidate of Cx43 binding proteins, and USP9X inhibited Cx43 ubiquitination. In vitro experiments showed that USP9X promoted ossification of primary ligament fibroblasts and nuclear translocation of NF-κB p65 by regulating Cx43 expression. Moreover, NF-κB can bind to the USP9X promoter to promote its transcription. When ER stress was inhibited by 4-PBA, USP9X levels, NF-κB nuclei translocation, and ALP activity were decreased. Reverse results were obtained when ER stress was induced by EPA. PDTC, an NF-κB inhibitor, could abolish the effects of EPA. Furthermore, USP9X, Cx43, and nuclei p65 were significantly upregulated in ligaments from OPLL patients than non-OPLL controls. USP9X was positively correlated with CX43 and nuclei p65 in OPLL samples. Overall, the findings suggest that the ER stress–NFκB-USP9X-Cx43 signaling pathway plays an important role in OPLL progression.

Investigation on the mechanism of 2,3,4′,5-Tetrahydroxystilbene 2-o-D-glucoside in the treatment of inflammation based on network pharmacology

BackgroundInflammation is the pathogenesis of various chronic diseases plaguing clinic for years.Fallopia multiflora (Thunb.) is a traditional Chinese herbal medicine with a long history of application in detoxification and anti-inflammation. 2,3,4′,5-Tetrahydroxystilbene 2-o-D-glucoside (TSG) is a main active compound of F. multiflora. However, the mechanism of TSG in the treatment of inflammation remains unknown. MethodsNetwork pharmacology and molecular docking were employed to explore the mechanism of anti-inflammatory effect of TSG. Potential targets of TSG and inflammation were obtained from Swiss Target Prediction, Pharm Mapper, and GeneCards database. Protein-protein interaction (PPI) networks, GO and KEGG pathway enrichment analysis were performed to elucidate the interaction of targets. Moreover, the anti-inflammatory effect of TSG was validated by in vitro experiments using flow cytometry, RT-qPCR, Western blot, and immunocytochemistry assays. ResultsPPI network and gene enrichment analysis showed that TSG may exert a protein kinase binding activity, and IKBKB, MAPK1, NFKBIA, and RELA were predicted as the targets of anti-inflammation. Verified by molecular docking and Western blot, TSG may target NF-κB and ERK2 related signals to alleviate inflammatory damage. Furthermore, TSG effectively downregulated the expression of inflammatory cytokine, the nuclear translocation of NF-κB p65, and the production of reactive oxygen species (ROS). ConclusionTSG possesses significant anti-inflammatory effect. TSG may display a protein kinase binding activity and target NF-κB and ERK2 related signals to treat the inflammation. This work may enlighten the potential application of TSG in anti-inflammation and indicate network pharmacology was an effective tool for the further study of TCM.

Effects of Human RelA Transgene on Murine Macrophage Inflammatory Responses.

The NFκB transcription factors are major regulators of innate immune responses, and NFκB signal pathway dysregulation is linked to inflammatory disease. Here, we utilised bone marrow-derived macrophages from the p65-DsRedxp/IκBα-eGFP transgenic strain to study the functional implication of xenogeneic (human) RelA(p65) protein introduced into the mouse genome. Confocal imaging showed that human RelA is expressed in the cells and can translocate to the nucleus following activation of Toll-like receptor 4. RNA sequencing of lipid A-stimulated macrophages, revealed that human RelA impacts on murine gene transcription, affecting both non-NFκB and NFκB target genes, including immediate-early and late response genes, e.g., Fos and Cxcl10. Validation experiments on NFκB targets revealed markedly reduced mRNA levels, but similar kinetic profiles in transgenic cells compared to wild-type. Enrichment pathway analysis of differentially expressed genes revealed interferon and cytokine signaling were affected. These immune response pathways were also affected in macrophages treated with tumor necrosis factor. Data suggests that the presence of xenogeneic RelA protein likely has inhibitory activity, altering specific transcriptional profiles of key molecules involved in immune responses. It is therefore essential that this information be taken into consideration when designing and interpreting future experiments using this transgenic strain.

The NCOA1-CBP-NF-κB transcriptional complex induces inflammation response and triggers endotoxin-induced myocardial dysfunction

Inflammatory pathways represented by TLR4/NF-κB (Toll-like receptor 4/Nuclear factor-κB) axis signaling are activated in the pathogenesis of endotoxin-induced myocardial dysfunction (EIMD). However, the underlying mechanism by which NF-κB coordinates with other transcriptional coactivators/corepressors to regulate the expression of proinflammatory cytokine genes remains unclear. We established an EIMD-mouse model by intraperitoneal injection of lipopolysaccharides (LPS), and we discovered that NCOA1 (nuclear receptor coactivator 1) assembled with CBP (CREB binding protein) and NF-κB subunits to form a transcriptional complex that specifically bound to promoters of proinflammatory cytokine genes to activate their expression. LPS treatment also inhibited DNMT1 (DNA methyltransferase 1) expression, thereby decreasing DNA methylation of a CpG island located on the promoter of NCOA1 and causing NCOA1 overexpression. Screening small molecules that abolished NCOA1-CBP interaction in a yeast system identified a compound PSSM2126 that effectively blocked the NCOA1-CBP interaction in vitro and in vivo. Administration of PSSM2126 to EIMD mice significantly alleviated the inflammation response and improved cardiac function. Collectively, our results reveal that an NCOA1-dependent transactivation mechanism can regulate proinflammatory cytokine expression, thereby improving our understanding of the activation of NF-κB targets. The promising inhibition of the NCOA1-CBP interaction by PSSM2126 may provide a new therapeutic option for EIMD.