ABSTRACT
Immune escape is the main reason that immunotherapy is ineffective in hepatocellular carcinoma (HCC). Here, this study illustrates a pathway mediated by neutrophil extracellular traps (NETs) that can promote immune escape of HCC. Mechanistically, we demonstrated that NETs up-regulated CD73 expression through activating Notch2 mediated nuclear factor kappa B (NF-κB) pathway, promoting regulatory T cells (Tregs) infiltration to mediate immune escape of HCC. In addition, we found the similar results in mouse HCC models by hydrodynamic plasmid transfection. The treatment of deoxyribonuclease I (DNase I) could inhibit the action of NETs and improve the therapeutic effect of anti-programmed cell death protein 1 (PD-1). In summary, our results revealed that targeting of NETs was a promising treatment to improve the therapeutic effect of anti-PD-1.
Subject(s)
5'-Nucleotidase , Carcinoma, Hepatocellular , Extracellular Traps , Liver Neoplasms , Receptor, Notch2 , Tumor Escape , Up-Regulation , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/immunology , Liver Neoplasms/pathology , Animals , Humans , Extracellular Traps/immunology , Extracellular Traps/metabolism , Mice , 5'-Nucleotidase/genetics , 5'-Nucleotidase/metabolism , 5'-Nucleotidase/immunology , Receptor, Notch2/metabolism , Receptor, Notch2/genetics , GPI-Linked Proteins/metabolism , GPI-Linked Proteins/genetics , GPI-Linked Proteins/immunology , Cell Line, Tumor , NF-kappa B/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Signal Transduction , Male , Programmed Cell Death 1 Receptor/metabolismABSTRACT
Memory B cells (MBCs) are essential for humoral immunological memory and can emerge during both the pre-germinal center (GC) and GC phases. However, the transcription regulators governing MBC development remain poorly understood. Here, we report that the transcription regulator Notch2 is highly expressed in MBCs and their precursors at the pre-GC stage and required for MBC development without influencing the fate of GC and plasma cells. Mechanistically, Notch2 signaling promotes the expression of complement receptor CD21 and augments B cell receptor (BCR) signaling. Reciprocally, BCR activation up-regulates Notch2 surface expression in activated B cells via a translation-dependent mechanism. Intriguingly, Notch2 is dispensable for GC-derived MBC formation. In summary, our findings establish Notch2 as a pivotal transcription regulator orchestrating MBC development through the reciprocal enforcement of BCR signaling during the pre-GC phase and suggest that the generation of GC-independent and -dependent MBCs is governed by distinct transcriptional mechanisms.
Subject(s)
Lymphocyte Activation , Memory B Cells , Receptor, Notch2 , Receptors, Antigen, B-Cell , Signal Transduction , Animals , Receptor, Notch2/metabolism , Receptor, Notch2/genetics , Receptors, Antigen, B-Cell/metabolism , Mice , Memory B Cells/metabolism , Memory B Cells/immunology , Lymphocyte Activation/immunology , Mice, Inbred C57BL , Germinal Center/immunology , Germinal Center/metabolism , B-Lymphocytes/metabolism , B-Lymphocytes/immunology , Immunologic Memory , Receptors, Complement 3d/metabolismABSTRACT
Current data on the molecular mechanisms of liver fibrosis and cirrhosis fail to fully explain all stages of their development. Interactions between individual genes and signaling pathways are known to play an important role in their functions. However, data on their relationships are insufficient and often contradictory. For the first time, mRNA expression of Notch1, Notch2, Yap1, Tweak (Tnfsf12), Fn14 (Tnfrsf12a), Ang, Vegfa, Cxcl12 (Sdf), Nos2, and Mmp-9 was studied in detail at several stages of thioacetamide-induced liver fibrosis in Wistar rats. A factor analysis isolated three factors, which combined highly correlated target genes. The first factor included four genes: Cxcl12 (r = 0.829, p < 0.05), Tweak (r = 0.841, p < 0.05), Notch1 (r = 0.848, p < 0.05), and Yap1 (r = 0.921, p < 0.05). The second factor described the correlation between Mmp-9 (r = 0.791, p < 0.05) and Notch2 (r = 0.836, p < 0.05). The third factor included Ang (r = 0.748, p < 0.05) and Vegfa (r = 0.679, p < 0.05). The Nos2 and Fn14 genes were not included in any of the factors. The gene grouping by mRNA expression levels made it possible to assume a pathogenetic relationship between their products in the development of fibrotic changes due to liver toxicity.
Subject(s)
Chemokine CXCL12 , Cytokine TWEAK , RNA, Messenger , Rats, Wistar , Receptor, Notch1 , YAP-Signaling Proteins , Animals , Rats , YAP-Signaling Proteins/genetics , YAP-Signaling Proteins/metabolism , Male , Receptor, Notch1/genetics , Receptor, Notch1/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Chemokine CXCL12/genetics , Chemokine CXCL12/metabolism , Cytokine TWEAK/genetics , Cytokine TWEAK/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Gene Expression Regulation , Liver Cirrhosis/genetics , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Liver Cirrhosis/chemically induced , Thioacetamide/toxicity , Receptor, Notch2/genetics , Receptor, Notch2/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolismABSTRACT
Development of the mammalian oocyte requires physical contact with the surrounding granulosa cells of the follicle, which provide it with essential nutrients and regulatory signals. This contact is achieved through specialized filopodia, termed transzonal projections (TZPs), that extend from the granulosa cells to the oocyte surface. Transforming growth factor (TGFß) family ligands produced by the oocyte increase the number of TZPs, but how they do so is unknown. Using an inducible Cre recombinase strategy together with expression of green fluorescent protein to verify Cre activity in individual cells, we examined the effect of depleting the canonical TGFß mediator, SMAD4, in mouse granulosa cells. We observed a 20-50% decrease in the total number of TZPs in SMAD4-depleted granulosa cell-oocyte complexes, and a 50% decrease in the number of newly generated TZPs when the granulosa cells were reaggregated with wild-type oocytes. Three-dimensional image analysis revealed that TZPs of SMAD4-depleted cells were longer than controls and more frequently oriented towards the oocyte. Strikingly, the transmembrane proteins, N-cadherin and Notch2, were reduced by 50% in SMAD4-depleted cells. SMAD4 may thus modulate a network of cell adhesion proteins that stabilize the attachment of TZPs to the oocyte, thereby amplifying signalling between the two cell types.
Subject(s)
Granulosa Cells , Oocytes , Smad4 Protein , Animals , Smad4 Protein/metabolism , Smad4 Protein/genetics , Oocytes/metabolism , Oocytes/growth & development , Mice , Female , Granulosa Cells/metabolism , Granulosa Cells/physiology , Receptor, Notch2/metabolism , Receptor, Notch2/genetics , Cadherins/metabolism , Cadherins/genetics , Pseudopodia/metabolism , Pseudopodia/physiologyABSTRACT
Eosinophils not only function as inflammatory effectors in allergic diseases, but also contribute to tissue homeostasis in steady state. Emerging data are revealing tissue eosinophils to be adaptive cells, imprinted by their local tissue microenvironment and exhibiting distinct functional phenotypes that may contribute to their homeostatic vs. inflammatory capacities. However, signaling pathways that regulate eosinophil tissue adaptations remain elusive. Notch signaling is an evolutionarily conserved pathway that mediates differential cell fate programming of both pre- and postmitotic immune cells. This study investigated a role for notch receptor 2 signaling in regulating eosinophil functions and tissue phenotype in both humans and mice. Notch 2 receptors were constitutively expressed and active in human blood eosinophils. Pharmacologic neutralization of notch 2 in ex vivo stimulated human eosinophils altered their activated transcriptome and prevented their cytokine-mediated survival. Genetic ablation of eosinophil-expressed notch 2 in mice diminished steady-state intestine-specific eosinophil adaptations and impaired their tissue retention in a food allergic response. In contrast, notch 2 had no effect on eosinophil phenotype or tissue inflammation within the context of allergic airways inflammation, suggesting that notch 2-dependent regulation of eosinophil phenotype and function is specific to the gut. These data reveal notch 2 signaling as a cell-intrinsic mechanism that contributes to eosinophil survival, function, and intestine-specific adaptations. The notch 2 pathway may represent a viable strategy to reprogram eosinophil functional phenotypes in gastrointestinal eosinophil-associated diseases.
Subject(s)
Allergens , Eosinophils , Receptor, Notch2 , Signal Transduction , Animals , Eosinophils/immunology , Eosinophils/metabolism , Receptor, Notch2/metabolism , Humans , Allergens/immunology , Mice , Intestines/immunology , Intestines/pathology , Mice, Inbred C57BL , Adaptation, Physiological/immunology , Mice, Knockout , Food Hypersensitivity/immunology , Food Hypersensitivity/pathologyABSTRACT
During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1ß, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1ß, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.
Subject(s)
Cytokines , Dendritic Cells , Porphyromonas gingivalis , Th17 Cells , Toll-Like Receptor 2 , Toll-Like Receptor 4 , Porphyromonas gingivalis/immunology , Humans , Dendritic Cells/immunology , Dendritic Cells/metabolism , Dendritic Cells/microbiology , Th17 Cells/immunology , Th17 Cells/metabolism , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 2/genetics , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 4/genetics , Cytokines/metabolism , Cell Differentiation , Th1 Cells/immunology , Interferon Regulatory Factors/metabolism , Interferon Regulatory Factors/genetics , Receptor, Notch2/genetics , Receptor, Notch2/metabolism , Cells, Cultured , Bacterial Capsules/immunology , Bacterial Capsules/metabolism , Bacteroidaceae Infections/immunology , Bacteroidaceae Infections/microbiology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BACKGROUND AND AIM: NOTCH2 is overexpressed in gastric cancer (GC), and its enhanced activity is significantly correlated with worse tumor characteristics. We aim to analyze the clinicopathologic correlation between NOTCH2 and the molecular typing of GC by immunohistochemistry and by transcriptional sequencing. METHODS: In this immunohistochemical study, we detected NOTCH2, EBER, P53, HER2, MLH1, MSH2, PMS2, and MSH6 and evaluated the association of NOTCH2 with clinical and histopathological features in a large single-institutional series of gastric adenocarcinomas (n = 488). The correlation was also investigated between immunohistochemical results and survival outcomes. RESULTS: High NOTCH2 expression (2+/3+) was found in 139/488 (27.5%) samples analyzed. NOTCH2 expression was correlated with early stage T1 (P < 0.0001), GC in the fundus (P = 0.0364), and positive P53 status (P = 0.0019). We did not find an association between NOTCH2 and HER2, microsatellite instability, EBER, and overall survival. Through RNA sequencing, it was revealed that NOTCH2 plays an important biological function in the pathogenesis and development of GC. CONCLUSIONS: Our findings suggested that NOTCH2 may be a potential diagnostic target for GC due to the fact that its high expression is closely associated with the early stages of cancer.
Subject(s)
Adenocarcinoma , Biomarkers, Tumor , Receptor, Notch2 , Stomach Neoplasms , Stomach Neoplasms/pathology , Stomach Neoplasms/genetics , Stomach Neoplasms/diagnosis , Stomach Neoplasms/mortality , Stomach Neoplasms/metabolism , Humans , Receptor, Notch2/genetics , Receptor, Notch2/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adenocarcinoma/metabolism , Adenocarcinoma/diagnosis , Adenocarcinoma/mortality , Female , Male , Middle Aged , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/genetics , Immunohistochemistry , Aged , Neoplasm Staging , Early Detection of Cancer , Gene Expression/genetics , Adult , Microsatellite Instability , Aged, 80 and overABSTRACT
The tumour microenvironment (TME) drives bladder cancer (BLCA) progression. Targeting the TME has emerged as a promising strategy for BLCA treatment in recent years. Furthermore, checkpoint blockade therapies are only beneficial for a minority of patients with BLCA, and drug resistance is a barrier to achieving significant clinical effects of anti-programmed cell death protein-1 (PD-1)/programmed death protein ligand-1 (PD-L1) therapy. In this study, higher low-density lipoprotein receptor-related protein 1 (LRP1) levels were related to a poorer prognosis for patients with various cancers, including those with higher grades and later stages of BLCA. Enrichment analysis demonstrated that LRP1 plays a role in the epithelial-mesenchymal transition (EMT), NOTCH signalling pathway, and ubiquitination. LRP1 knockdown in BLCA cells delayed BLCA progression both in vivo and in vitro. Furthermore, LRP1 knockdown suppressed EMT, reduced DLL4-NOTCH2 signalling activity, and downregulated M2-like macrophage polarisation. Patients with BLCA and higher LRP1 levels responded weakly to anti-PD-1 therapy in the IMvigor210 cohort. Moreover, LRP1 knockdown enhanced the therapeutic effects of anti-PD-1 in mice. Taken together, our findings suggest that LRP1 is a potential target for improving the efficacy of anti-PD-1/PD-L1 therapy by preventing EMT and M2-like macrophage polarisation by blocking the DLL4-NOTCH2 axis.
Subject(s)
Drug Resistance, Neoplasm , Epithelial-Mesenchymal Transition , Low Density Lipoprotein Receptor-Related Protein-1 , Receptor, Notch2 , Signal Transduction , Tumor Microenvironment , Urinary Bladder Neoplasms , Humans , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/immunology , Low Density Lipoprotein Receptor-Related Protein-1/metabolism , Low Density Lipoprotein Receptor-Related Protein-1/genetics , Animals , Mice , Epithelial-Mesenchymal Transition/drug effects , Cell Line, Tumor , Receptor, Notch2/metabolism , Receptor, Notch2/genetics , Macrophages/metabolism , Macrophages/immunology , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Programmed Cell Death 1 Receptor/metabolism , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/genetics , Calcium-Binding Proteins/metabolism , Calcium-Binding Proteins/genetics , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Female , Male , Xenograft Model Antitumor Assays , Chemokine CCL2ABSTRACT
The importance of macrophages in adipose tissue (AT) homeostasis and inflammation is well established. However, the potential cues that regulate their function remain incompletely understood. To bridge this important gap, we sought to characterize novel pathways involved using a mouse model of diet-induced obesity. By performing transcriptomics analysis of AT macrophages (ATMs), we found that late-stage ATMs from high-fat diet mice presented with perturbed Notch signaling accompanied by robust proinflammatory and metabolic changes. To explore the hypothesis that the deregulated Notch pathway contributes to the development of AT inflammation and diet-induced obesity, we employed a genetic approach to abrogate myeloid Notch1 and Notch2 receptors. Our results revealed that the combined loss of Notch1 and Notch2 worsened obesity-related metabolic dysregulation. Body and AT weight gain was higher, blood glucose levels increased and metabolic parameters were substantially worsened in deficient mice fed high-fat diet. Moreover, serum insulin and leptin were elevated as were triglycerides. Molecular analysis of ATMs showed that deletion of Notch receptors escalated inflammation through the induction of an M1-like pro-inflammatory phenotype. Our findings thus support a protective role of myeloid Notch signaling in adipose tissue inflammation and metabolic dysregulation.
Subject(s)
Adipose Tissue , Diet, High-Fat , Inflammation , Macrophages , Obesity , Receptor, Notch1 , Receptor, Notch2 , Signal Transduction , Animals , Macrophages/immunology , Macrophages/metabolism , Adipose Tissue/metabolism , Adipose Tissue/immunology , Mice , Diet, High-Fat/adverse effects , Inflammation/immunology , Inflammation/metabolism , Signal Transduction/immunology , Obesity/metabolism , Obesity/immunology , Receptor, Notch1/metabolism , Receptor, Notch1/genetics , Receptor, Notch2/metabolism , Receptor, Notch2/genetics , Mice, Knockout , Mice, Inbred C57BL , MaleABSTRACT
The Notch signaling pathway plays pivotal roles in cell proliferation, stemness and invasion of non-small cell lung cancer (NSCLC). The human Notch family consists of four receptors, namely Notch1, Notch2, Notch3, and Notch4. These receptors are transmembrane proteins that play crucial roles in various cellular processes. Notch1 mostly acts as a pro-carcinogenic factor in NSCLC but sometimes acts as a suppressor. Notch2 has been demonstrated to inhibit the growth and progression of NSCLC, whereas Notch3 facilitates these biological behaviors of NSCLC. The role of Notch4 in NSCLC has not been fully elucidated, but it is evident that Notch4 promotes tumor progression. At present, drugs targeting the Notch pathway are being explored for NSCLC therapy, a majority of which are already in the stage of preclinical research and clinical trials, with bright prospects in the clinical treatment of NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Receptor, Notch1/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Receptors, Notch/metabolism , Receptor, Notch2/metabolism , Receptor, Notch3 , Signal TransductionABSTRACT
NOTCH2 is expressed in pituitary stem cells and is necessary for stem cell maintenance, proliferation, and differentiation. However, the pathways NOTCH2 engages to affect pituitary development remain unclear. In this study, we hypothesized that glycoprotein hormone subunit A2 (GPHA2), a corneal stem cell factor and ligand for the thyroid stimulating hormone receptor (TSHR), is downstream of NOTCH2 signaling. We found Gpha2 is expressed in quiescent pituitary stem cells by RNAscope in situ hybridization and scRNA seq. In Notch2 conditional knockout pituitaries, Gpha2 mRNA is reduced compared with control littermates. We then investigated the possible functions of GPHA2. Pituitaries treated with a GPHA2 peptide do not have a change in proliferation. However, in dissociated adult pituitary cells, GPHA2 increased pCREB expression and this induction was reversed by co-treatment with a TSHR inhibitor. These data suggest GPHA2 is a NOTCH2 related stem cell factor that activates TSHR signaling, potentially impacting pituitary development.
Subject(s)
Pituitary Gland , Stem Cell Factor , Adult , Humans , Pituitary Gland/metabolism , Receptor, Notch2/genetics , Receptor, Notch2/metabolism , Receptors, Thyrotropin , Stem Cell Factor/metabolism , Stem Cells/metabolismABSTRACT
Atherosclerosis (AS) is a major chronic non-communicable disease and a primary cause of cardiovascular disease. Recent studies have shown that circRNAs are potential epigenetic factors that regulate vascular endothelial inflammatory responses and AS progression. Therefore, identification of the circRNAs that regulate ox-LDL levels is a critical step to understanding the pathology of AS. Our study is aim to investigate how circLZIC regulates atherosclerosis (AS) via the Micro-330-5p/NOTCH2 regulatory axis. The results showed that CircLZIC and NOTCH2 are highly expressed in human AS clinical samples, while Micro-330-5p is expressed locally. The CCK-8 experiment results showed that circLZIC promotes the proliferation of HUVECS cells. Flow cytometry analysis showed that circLZIC act as an inhibitor of HUVEC cell apoptosis. The expression level of Micro-330-5p can be up-regulated by transfection of small interfering RNA against circLZIC. Further, Starbase predicted that Micro-330-5p could target and regulate NOTCH2. Next, we confirmed that overexpression of Micro-330-5p could significantly reduce the expression of fluorescein using the double Luciferase reporter assay. RIP-qRT-PCR experiment showed that Micro-330-5p and NOTCH2 mRNAs are effectively enriched by ago2 protein. Further, we found that knocking down circLZIC increases the expression of Micro-330-5p and promotes cell apoptosis, while inhibiting the expression of NOTCH2 and cell activity. On the other hand, co-transfection of Micro-330-5p inhibitor decreases Micro-330-5p expression and inhibit cell apoptosis, while increasing NOTCH2 expression and cell activity. In conclusion, CircLZIC regulates HUVEC cell activity by the Micro-330-5p/NOTCH2 signaling pathway, suggesting that circLZIC plays a key role in atherosclerosis development.
Subject(s)
Apoptosis , Atherosclerosis , Cell Proliferation , Human Umbilical Vein Endothelial Cells , Lipoproteins, LDL , RNA, Circular , Receptor, Notch2 , Humans , Apoptosis/drug effects , Atherosclerosis/metabolism , Atherosclerosis/pathology , Cell Proliferation/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Lipoproteins, LDL/pharmacology , MicroRNAs/metabolism , MicroRNAs/genetics , Receptor, Notch2/metabolism , Receptor, Notch2/genetics , RNA, Circular/genetics , Signal Transduction/drug effectsABSTRACT
Notch signaling plays a key regulatory role in bone remodeling and NOTCH2 enhances osteoclastogenesis, an effect that is mostly mediated by its target gene Hes1. In the present study, we explored mechanisms responsible for the enhanced osteoclastogenesis in bone marrow-derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice. Notch2tm1.1Ecan mice are osteopenic and have enhanced osteoclastogenesis. Bulk RNA-Seq and gene set enrichment analysis of Notch2tm1.1Ecan BMMs cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand revealed enrichment of genes associated with enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis. These pathways were not enhanced in the context of a Hes1 inactivation. Analysis of single cell RNA-Seq data of pooled control and Notch2tm1.1Ecan BMMs treated with M-CSF or M-CSF and receptor activator of NF-κB ligand for 3 days identified 11 well-defined cellular clusters. Pseudotime trajectory analysis indicated a trajectory of clusters expressing genes associated with osteoclast progenitors, osteoclast precursors, and mature cells. There were an increased number of cells expressing gene markers associated with the osteoclast and with an unknown, albeit related, cluster in Notch2tm1.1Ecan than in control BMMs as well as enhanced expression of genes associated with osteoclast progenitors and precursors in Notch2tm1.1Ecan cells. In conclusion, BMM cultures display cellular heterogeneity, and NOTCH2 enhances osteoclastogenesis, increases mitochondrial and metabolic activity of osteoclasts, and affects cell cluster allocation in BMMs.
Subject(s)
Osteoclasts , Osteogenesis , Receptor, Notch2 , Transcriptome , Animals , Mice , Bone Marrow Cells/metabolism , Cell Differentiation/genetics , Gene Expression Regulation, Developmental/genetics , Macrophage Colony-Stimulating Factor/metabolism , Mice, Inbred C57BL , Mutation , Osteoclasts/cytology , Osteoclasts/metabolism , Osteogenesis/genetics , Receptor Activator of Nuclear Factor-kappa B/metabolism , Receptor, Notch2/genetics , Receptor, Notch2/metabolism , Transcription Factor HES-1/metabolism , Transcriptome/geneticsABSTRACT
Glioma cell cultures are used in basic researches of tumor processes, personalized medicine for selecting treatment regimens depending on individual characteristics of patients and pharmacology for assessing the effectiveness of chemotherapy. Suppression of glioma culture growth without reduction of malignancy grade is common. Drug cancellation may be followed by substitution of precursor cells by more malignant clones. Therefore, analysis of culture cell malignancy grade is important. In the future, intraoperative analysis of glioma cell malignancy grade can be used to select individual therapy. OBJECTIVE: We analyzed the relationship between expression of marker genes TUBB3, CD133, CDK4, CDK6, CIRBP, DR4, DR5, EGFR, FGFR, FSHR, GDNF, GFAP, L1CAM, LEF1, MAP2, MDM2, MELK, NANOG, NOTCH2, OCT4, OLIG2, PDGFRA, PDGFA, PDGFB and SOX2 and glioma cell malignancy grade, as well as created appropriate prognostic model. MATERIAL AND METHODS: We analyzed expression of 25 marker genes in 22 samples of human glioma cultures using quantitative real-time PCR. Statistical analysis was performed using the IBM SPSS Statistics 26.0 software. We used the Kolmogorov-Smirnov and Shapiro-Wilk tests to assess distribution normality. Nonparametric Jonckheere-Terpstra and Spearman tests were applied. RESULTS: We obtained a prognostic model for assessing the grade III and IV glioma cell malignancy based on expression of marker genes MDM2, MELK, SOX2, CDK4, DR5 and OCT4. Predictive accuracy was 83% (Akaike information criterion -55.125).
Subject(s)
Glioma , Humans , Prognosis , Glioma/genetics , Receptor, Notch2/genetics , Receptor, Notch2/metabolism , Gene Expression , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Proto-Oncogene Proteins c-mdm2/therapeutic use , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/therapeutic use , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 4/therapeutic use , RNA-Binding Proteins/genetics , RNA-Binding Proteins/therapeutic use , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolismABSTRACT
Transfer RNA-derived fragments (tRFs) are a class of small non-coding regulatory RNAs that are involved in the pathophysiology of many diseases. However, the role of tRFs in cancer progression remains largely elusive. Here, we demonstrate that a pan-cancer 3'-tRF, CAT1 (cancer associated tRF 1), is ubiquitously upregulated in tumors and associated with poor prognosis of a variety of cancers, including lung cancer. The upregulated CAT1 in cancer cells binds to RNA-binding protein with multiple splicing (RBPMS) and displaces NOTCH2 association from RBPMS, thereby inhibiting the subsequent CCR4-NOT deadenylation-complex-mediated NOTCH2 mRNA decay. The CAT1-enhanced NOTCH2 expression promotes lung cancer cell proliferation and metastasis in vitro and in vivo. In addition, plasma CAT1 levels are substantially increased in patients with lung cancer compared to non-cancer control subjects. Our findings reveal an intrinsic connection between cancer-specific upregulation of CAT1 and cancer progression, show the regulation of NOTCH signaling in cancer by a 3'-tRF, and highlight its great clinical potential.
Subject(s)
Lung Neoplasms , RNA, Transfer , Humans , RNA, Messenger/genetics , RNA, Transfer/metabolism , Cell Transformation, Neoplastic , Lung Neoplasms/genetics , RNA-Binding Proteins , Receptor, Notch2/genetics , Receptor, Notch2/metabolismABSTRACT
Notch regulates the immune and inflammatory response and has been associated with the pathogenesis of osteoarthritis in humans and preclinical models of the disease. Notch2tm1.1Ecan mice harbor a NOTCH2 gain-of-function and are sensitized to osteoarthritis, but the mechanisms have not been explored. We examined the effects of tumor necrosis factor α (TNFα) in chondrocytes from Notch2tm1.1Ecan mice and found that NOTCH2 enhanced the effect of TNFα on Il6 and Il1b expression. Similar results were obtained in cells from a conditional model of NOTCH2 gain-of-function, Notch22.1Ecan mice, and following the expression of the NOTCH2 intracellular domain in vitro. Recombination signal-binding protein for immunoglobulin Kappa J region partners with the NOTCH2 intracellular domain to activate transcription; in the absence of Notch signaling it inhibits transcription, and Rbpj inactivation in chondrocytes resulted in Il6 induction. Although TNFα induced IL6 to a greater extent in the context of NOTCH2 activation, there was a concomitant inhibition of Notch target genes Hes1, Hey1, Hey2, and Heyl. Electrophoretic mobility shift assay demonstrated displacement of recombination signal-binding protein for immunoglobulin Kappa J region from DNA binding sites by TNFα explaining the increased Il6 expression and the concomitant decrease in Notch target genes. NOTCH2 enhanced the effect of TNFα on NF-κB signaling, and RNA-Seq revealed increased expression of pathways associated with inflammation and the phagosome in NOTCH2 overexpressing cells in the absence and presence of TNFα. Collectively, NOTCH2 has important interactions with TNFα resulting in the enhanced expression of Il6 and inflammatory pathways in chondrocytes.
Subject(s)
Chondrocytes , Osteoarthritis , Receptor, Notch2 , Tumor Necrosis Factor-alpha , Animals , Humans , Mice , Chondrocytes/cytology , Chondrocytes/drug effects , Chondrocytes/metabolism , Immunoglobulins , Interleukin-6/genetics , Interleukin-6/metabolism , Osteoarthritis/genetics , Osteoarthritis/metabolism , Receptor, Notch2/genetics , Receptor, Notch2/metabolism , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Inflammation , Disease Models, Animal , Chondrogenesis , Signal Transduction/drug effects , Protein Domains/immunology , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Gene Deletion , Gene Expression Regulation/drug effectsABSTRACT
OBJECTIVE: Trastuzumab is the preferred drug for the treatment of breast cancer. However, research on the cellular mechanisms of trastuzumab's potential cardiotoxicity is insufficient. The purpose of this study was to explore the toxic effects and potential mechanism of action of trastuzumab on cardiomyocytes. METHOD: Human Cardiomyocyte (HCM) viability was assessed using the MTT method. HCM apoptosis was detected using the Hoechst33342/PI Fluorescent staining. The LDH and CK activities of the cell were measured using commercially available LDH and CK assay kits. The expression levels of Notch2, JAK2, STAT3, cleaved caspase 3, bax, and bcl 2 in HCMs were detected using western blotting. RESULTS: The results showed that 250 mg/L trastuzumab induced cardiomyocyte injury and apoptosis, inhibited viability, activated the Notch2 receptor, and inhibited JAK2/STAT3 expression in HCM. Inhibition of Notch2 expression in HCM by targeted siNotch2 transfection reversed the trastuzumab-induced injury and apoptosis, and the expression of JAK2/STAT3 returned to normal levels. CONCLUSIONS: Trastuzumab induces Notch2 expression by inhibiting the JAK2/STAT3 pathway of HCMs, promotes cell apoptosis, and causes cardiomyocyteinjury. Notch2 may be a potential target of trastuzumab-inducedmyocardial injury. This experiment reveals the mechanism of trastuzumab-induced cardiotoxicity, providing a theoretical basis for the application of trastuzumab.
Subject(s)
Cardiotoxicity , Myocytes, Cardiac , Humans , Myocytes, Cardiac/metabolism , Trastuzumab/adverse effects , Trastuzumab/metabolism , Cardiotoxicity/etiology , Cardiotoxicity/metabolism , Receptor, Notch2/metabolism , Apoptosis , Janus Kinase 2/metabolism , Janus Kinase 2/pharmacology , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/pharmacologyABSTRACT
Notch signaling regulates cell fate in multiple tissues including the skeleton. Hajdu-Cheney-Syndrome (HCS), caused by gain-of-function mutations in the Notch2 gene, is a rare inherited disease featuring early-onset osteoporosis and increased risk for fractures and non-union. As the impact of Notch2 overactivation on fracture healing is unknown, we studied bone regeneration in mice harboring a human HCS mutation. HCS mice, displaying high turnover osteopenia in the non-fractured skeleton, exhibited only minor morphologic alterations in the progression of bone regeneration, evidenced by static radiological and histological outcome measurements. Histomorphometry showed increased osteoclast parameters in the callus of HCS mice, which was accompanied by an increased expression of osteoclast and osteoblast markers. These observations were accompanied by inferior biomechanical stability of healed femora in HCS mice. Together, our data demonstrate that structural indices of bone regeneration are normal in HCS mice, which, however, exhibit signs of increased callus turnover and display impaired biomechanical stability of healed fractures.
Subject(s)
Bone Diseases, Metabolic , Hajdu-Cheney Syndrome , Osteoporosis , Humans , Mice , Animals , Fracture Healing , Hajdu-Cheney Syndrome/genetics , Hajdu-Cheney Syndrome/metabolism , Hajdu-Cheney Syndrome/pathology , Bone Diseases, Metabolic/pathology , Osteoporosis/pathology , Osteoclasts/metabolism , Receptor, Notch2/metabolismABSTRACT
OBJECTIVE: To investigate the effect of Notch2 gene knockout in Treg cells on head and neck squamous cell carcinoma (HNSCC) in mice. METHODS: A mouse model of HNSCC was constructed. Flow cytometry and immunofluorescence were used to examine the numbers of related immune cells and programmed cell death in tumor cells in the spleen and tumor microenvironment of mice. Western blotting was used to measure the expression of related proteins in tumor tissues. RESULTS: The tumor volume of regulatory T (Treg) cell-specific Notch2-knockout mice (experimental group) was significantly smaller than that of control mice (control group) (P < 0.05). Compared with those in the control group, the number of Treg cells and the expression of Ki67 in Treg cells in the spleen and tumor tissue were significantly decreased in the experimental group, while the numbers of CD45+ hematopoietic cells, CD4+ T cells, CD8+ T cells, T helper 1 (Th1) cells, CD11b+ cells (macrophages), and CD11b+CD11c+ cells (dendritic cells) and the expression of Ki67 in CD4+ T cells and CD8+ T cells were significantly increased (P < 0.05). There was no significant difference in the number of Th2 cells between the two groups (P > 0.05). Immunofluorescence analysis showed that the numbers of CD4+ T cells and CD8+ T cells in the tumor tissue in the experimental group were significantly higher than those in the control group (P < 0.05). Compared with that in the control group, programmed cell death in the experimental group was significantly increased (P < 0.05). Moreover, the expression levels of NLRP3, Caspase-1 and GSDMD in the tumor tissues of the experimental group were higher than those in the control group (P < 0.01), while the expression levels of BCL2, Bax, ATG5, LC3 and p62 were not significantly different (P > 0.05). CONCLUSIONS: Specific knockout of the Notch2 gene in Treg cells significantly decreases the function of Treg cells, inhibits the growth of HNSCC and improves the immune microenvironment in mice, thus effectively treating HNSCC.
Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Receptor, Notch2 , Animals , Mice , Carcinoma, Squamous Cell/metabolism , Cell Proliferation , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Ki-67 Antigen/metabolism , Mice, Knockout , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/metabolism , T-Lymphocytes, Regulatory , Tumor Microenvironment , Receptor, Notch2/genetics , Receptor, Notch2/metabolismABSTRACT
Female reproductive lifespan is largely determined by the size of the primordial follicle pool, which is established in early life. Dibutyl phthalate (DBP), a popular plasticiser, is a known environmental endocrine disruptor that poses a potential threat to reproductive health. However, DBP impact on early oogenesis has been rarely reported. In this study, maternal exposure to DBP in gestation disrupted germ-cell cyst breakdown and primordial follicle assembly in foetal ovary, impairing female fertility in adulthood. Subsequently, altered autophagic flux with autophagosome accumulation was observed in DBP-exposed ovaries carrying CAG-RFP-EGFP-LC3 reporter genes, whereas autophagy inhibition by 3-methyladenine attenuated the impact of DBP on primordial folliculogenesis. Moreover, DBP exposure reduced the expression of NOTCH2 intracellular domain (NICD2) and decreased interactions between NICD2 and Beclin-l. NICD2 was observed within the autophagosomes in DBP-exposed ovaries. Furthermore, NICD2 overexpression partially restored primordial folliculogenesis. Furthermore, melatonin significantly relieved oxidative stress, decreased autophagy, and restored NOTCH2 signalling, consequently reversing the effect on folliculogenesis. Therefore, this study demonstrated that gestational DBP exposure disrupts primordial folliculogenesis by inducing autophagy, which targets NOTCH2 signalling, and this impact has long-term consequences on fertility in adulthood, strengthening the potential contribution of environmental chemicals to the development of ovarian dysfunctional diseases.