Notch Intracellular Domain Research Articles

Mucosal Mast Cell-Specific Gene Expression Is Promoted by Interdependent Action of Notch and TGF-β Signaling.

Rodent mast cells are classified into two major subsets, mucosal mast cells (MMCs) and connective tissue mast cells. MMCs arise from mast cell progenitors that are mobilized from the bone marrow to mucosal tissues in response to allergic inflammation or helminth infection. TGF-β is known as an inducer of MMC differentiation in mucosal tissues, but we have previously found that Notch receptor-mediated signaling also leads to the differentiation. Here, we examined the relationship between Notch and TGF-β signaling in MMC differentiation using mouse bone marrow-derived mast cells (BMMCs). We found that the coexistence of Notch and TGF-β signaling markedly upregulates the expression of MMC markers, mouse mast cell protease (mMCP)-1, mMCP-2, and αE integrin/CD103, more than Notch or TGF-β signaling alone, and that their signals act interdependently to induce these marker expressions. Notch and TGF-β-mediated transcription of MMC marker genes were both dependent on the TGF-β signaling transducer SMAD4. In addition, we also found that Notch signaling markedly upregulated mMCP-1 and mMCP-2 expression levels through epigenetic deregulation of the promoter regions of these genes, but did not affect the promoter of the CD103-encoding gene. Moreover, forced expression of the constitutively active Notch2 intracellular domain in BMMCs showed that Notch signaling promotes the nuclear localization of SMADs 3 and 4 and causes SMAD4-dependent gene transcription. These findings indicate that Notch and TGF-β signaling play interdependent roles in inducing the differentiation and maturation of MMCs. These roles may contribute to the rapid expansion of the number of MMCs during allergic mucosal inflammation.

RYBP modulates embryonic neurogenesis involving the Notch signaling pathway in a PRC1-independent pattern.

SummaryRYBP (Ring1 and YY1 binding protein), an essential component of the Polycomb repressive complex 1 (PRC1), plays pivotal roles in development and diseases. However, the roles of Rybp in neuronal development remains completely unknown. In the present study, we have shown that the depletion of Rybp inhibits proliferation and promotes neuronal differentiation of embryonic neural progenitor cells (eNPCs). In addition, Rybp deficiency impairs the morphological development of neurons. Mechanistically, Rybp deficiency does not affect the global level of ubiquitination of H2A, but it inhibits Notch signaling pathway in eNPCs. The direct interaction between RYBP and CIR1 facilitates the binding of RBPJ to Notch intracellular domain (NICD) and consequently activated Notch signaling. Rybp loss promotes CIR1 competing with RBPJ to bind with NICD, and inhibits Notch signaling. Furthermore, ectopic Hes5, Notch signaling downstream target, rescues Rybp-deficiency-induced deficits. Collectively, our findings show that RYBP regulates embryonic neurogenesis and neuronal development through modulating Notch signaling in a PRC1-independent manner.

The NOTCH3 Downstream Target HEYL Is Required for Efficient Human Airway Basal Cell Differentiation.

Basal cells (BCs) are stem/progenitor cells of the mucociliary airway epithelium, and their differentiation is orchestrated by the NOTCH signaling pathway. NOTCH3 receptor signaling regulates BC to club cell differentiation; however, the downstream responses that regulate this process are unknown. Overexpression of the active NOTCH3 intracellular domain (NICD3) in primary human bronchial epithelial cells (HBECs) on in vitro air–liquid interface culture promoted club cell differentiation. Bulk RNA-seq analysis identified 692 NICD3-responsive genes, including the classical NOTCH target HEYL, which increased in response to NICD3 and positively correlated with SCGB1A1 (club cell marker) expression. siRNA knockdown of HEYL decreased tight junction formation and cell proliferation. Further, HEYL knockdown reduced club, goblet and ciliated cell differentiation. In addition, we observed decreased expression of HEYL in HBECs from donors with chronic obstructive pulmonary disease (COPD) vs. normal donors which correlates with the impaired differentiation capacity of COPD cells. Finally, overexpression of HEYL in COPD HBECs promoted differentiation into club, goblet and ciliated cells, suggesting the impaired capacity of COPD cells to generate a normal airway epithelium is a reversible phenotype that can be regulated by HEYL. Overall, our data identify the NOTCH3 downstream target HEYL as a key regulator of airway epithelial differentiation.

Abstract 10425: Epigenetic Regulation of Notch Signaling by Prenatal Alcohol Exposure Impacts Cardiac Outflow Tract Defect Development

Introduction: Prenatal alcohol exposure (PAE) leads to congenital heart defects (CHD) with incomplete penetrance and phenotypic heterogeneity. We have demonstrated a gene-environment interaction in this context wherein acute PAE synergizes with second heart field (SHF) haploinsufficiency of Notch signaling, resulting in highly penetrant outflow tract (OFT) defects. Hypothesis: We hypothesize that acute PAE-induced hyperacetylation at histone marker 3 lysine residue 9 (aH3K9) epigenetically inhibits Notch signaling. This potentiates the impact of a pre-existing genetic defect that results in partial loss of Notch signaling leading to loss of viability of SHF progenitors necessary for OFT elongation and alignment. Methods: We studied the effects of acute PAE in vivo in our murine model (two injections of 3g/kg 30% ethanol on E6.5) on acetylation, SHF viability, OFT length, and Notch intracellular domain (NICD) localization. Complimentarily, we evaluated alcohol exposure in vitro using our established thoracic organ culture model. Acetylation was reversed using pan-histone acetyltransferase inhibitor anacardic acid. Results: At E9.5, acute PAE resulted in a significant increase in H3K9 acetylation in SHF associated with restriction of NICD to the cell membrane. This resulted in a 0.28-fold reduction in Notch signaling, as evidenced by reduced Notch reporter expression, and 0.52-fold reduction in SHF cell proliferation (both p<0.001). Loss of SHF progenitors needed for incorporation into the developing OFT reduced OFT length 26% (p<0.001). In vitro thoracic organ culture analyses recapitulated increased acetylation, NICD restriction to cell membrane and reduced SHF proliferation by 60% (p<0.001). Anacardic acid reversed the increase of aH3K9 and allowed NICD nuclear translocation. Notch signaling and SHF proliferation returned to baseline. Conclusions: Acute PAE induces hyperacetylation in the SHF and inhibits Notch signaling by restricting NICD translocation. Lack of Notch signaling reduces SHF viability and prevents elongation of the developing OFT. This hereto unexplored impact of alcohol on the Notch pathway provides a novel area of investigation regarding gene-environment interaction in alcohol teratogenesis.

Phenotype transition of fibroblasts incorporated into patient-derived oral carcinoma organoids.

Cancer-associated fibroblasts (CAFs) are abundantly infiltrated in oral squamous cell carcinoma (OSCC), but the contact-dependent mechanisms that regulate CAFs phenotype in precursor cells, such as paracancerous fibroblasts (PFs), remain unclear. Here, a fibroblast-attached organoid (FAO) model was initiated to determine phenotype transition of fibroblasts triggered by contact with OSCC. Organoids and fibroblasts were generated using OSCC and adjacent tissues. Cell-clusters containing fibroblasts and tumour cells were aggregated to allow for FAOs expansion. Immunoblotting assay was performed to compare expression of Notch intracellular domain (NICD) in CAFs and PFs. Colony formation assay was employed to evaluate morphological activation of fibroblasts. Compared to traditional 3D co-culture, FAOs better modulated the spatial distribution of fibroblasts with tumour nests. The presence of CAFs with multiple branches was stably observed in FAOs during serial passage. Incorporation with organoids promoted the ability of PFs to form multiple branches. Immunoblotting assay confirmed higher NICD level in CAFs than PFs. Treatment with Notch inhibitor, N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (i.e. DAPT) blocked morphological activation of fibroblasts incorporated into FAO. We developed a robust strategy to study contact-dependent mechanisms underlying tumour-stromal interaction, and suggested that Notch activity contributes to biogenesis of OSCC-associated fibroblasts.

The Membrane-Bound Notch Regulator Mnr Supports Notch Cleavage and Signaling Activity in Drosophila melanogaster.

The Notch signaling pathway is pivotal to cellular differentiation. Activation of this pathway involves proteolysis of the Notch receptor and the release of the biologically active Notch intracellular domain, acting as a transcriptional co-activator of Notch target genes. While the regulation of Notch signaling dynamics at the level of ligand–receptor interaction, endocytosis, and transcriptional regulation has been well studied, little is known about factors influencing Notch cleavage. We identified EP555 as a suppressor of the Notch antagonist Hairless (H). EP555 drives expression of CG32521 encoding membrane-bound proteins, which we accordingly rename membrane-bound Notch regulator (mnr). Within the signal-receiving cell, upregulation of Mnr stimulates Notch receptor activation, whereas a knockdown reduces it, without apparent influence on ligand–receptor interaction. We provide evidence that Mnr plays a role in γ-secretase-mediated intramembrane cleavage of the Notch receptor. As revealed by a fly-eye-based reporter system, γ-secretase activity is stimulated by the overexpression of Mnr, and is inhibited by its knockdown. We conclude that Mnr proteins support Notch signaling activity by fostering the cleavage of the Notch receptor. With Mnr, we identified a membrane-bound factor directly augmenting Notch intra-membrane processing, thereby acting as a positive regulator of Notch signaling activity.

The Histone Demethylase KDM6B Is a Genetic Dependency of NOTCH1-Driven T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) arises from the accumulation of genomic abnormalities and the malignant proliferation of immature T-cells. Despite recent advancements in understanding the genetic alterations driving T cell leukemogenesis, patients still suffer from recurrent relapses and treatment-related toxicities. Genome sequencing has revealed significant heterogeneity and important insights into the genetic landscape of T-ALL. Mutations in epigenetic modifiers are frequently observed and serve as an attractive target for novel therapeutic approaches.Histone demethylase enzymes play a critical role in the regulation of gene expression programs in T-ALL. KDM6A (UTX) is known to behave as a tumor suppressor in most T-ALL subtypes. However, it's gene paralog, KDM6B (JMJD3), is never mutated and can be significantly overexpressed in patients. Here, we show that KDM6B is required for T-ALL initiation. Using genetic mouse models, Sca-1 enriched WBM from Vav-Cre: Kdm6b+/+, Vav-Cre: Kdm6bfl/+, and Vav-Cre: Kdm6bfl/fl adult mice was transduced with a retrovirus expressing Notch1 Intracellular Domain (NICD). NOTCH1 gain-of-function mutations are the most frequent driver events in adult T-ALL, and this model recapitulates many of the human pathologies. Transduced cells were transplanted into irradiated mice. While there was robust engraftment in all groups at four weeks post-transplant, T-ALL cells were not sustained in the genetic absence of Kdm6b. Mice receiving control NICD-GFP+ cells succumbed to T-ALL with median survival of 79 days, whereas the only mice receiving Kdm6b-null NICD-GFP+ cells that developed disease were found to retain one copy of the Kdm6b floxed allele (Fig 1). To investigate the translational potential, we targeted KDM6B for genetic inactivation by CRISPR/Cas9 in primary T-ALL patient cells, followed by xenograft into NSG mice. The effect of KDM6B targeting was quantified over time by monitoring the variant allele fraction (VAF) of the transplanted cells. In most patient samples, KDM6B-targeted cells were significantly outcompeted over time, thus further highlighting the requirement of KDM6B in sustaining T-ALL tumorigenesis.To examine the mechanism by which KDM6B sustain T-ALL cells, gene expression profiling was performed by RNA-seq on mouse T-ALL cells of genetic backgrounds Control, Kdm6b-Het, and Kdm6b-KO. Three gene sets were significantly downregulated in Kdm6b-deficient T-ALL cells compared to the Control group, all of which are involved in cell cycle processes. Additional validation of these findings with cell cycle functional studies is currently ongoing. Additionally, while Kdm6b has been described for its H3K27me3 histone demethylase function, recent studies have shown its involvement in regulating various gene expression programs through demethylase-independent mechanisms. To determine if the role of Kdm6b in T-ALL oncogenesis was related to it demethylase activity, we performed the same NICD retroviral transduction experiment with inclusion of a Kdm6b +/H1388A mouse genotype, a point mutation which renders Kdm6b catalytically dead. Our data shows that Kdm6b +/H1388A phenocopies T-ALL with Kdm6b homozygous loss-of-function, showing no evidence of disease in the blood beyond 8-weeks post-transplant. We conclude from this that the leukemogenic role of Kdm6b requires it's H3K27me3 demethylase function. In summary, these data reveal Kdm6b as an oncogenic dependency in Notch1-driven T-ALL in both mouse and human systems, and present Kdm6b as a high value therapeutic target in adult T-ALL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Leukaemia Cells Induced Metabolic Alterations in AML Associated Mesenchymal Stem Cells Via Notch Signalling

Introduction: Acute myeloid leukaemia (AML) is a haematological malignancy with a high relapse rate and poor prognosis. Leukaemia cell proliferation is dependent on its interaction with the bone marrow (BM) microenvironment. AML associated mesenchymal stem cells (AML-MSCs) supported the proliferation of leukaemia cells and contributed to disease progression. Stromal microenvironment promoted a metabolic switch but precise underlying molecular mechanisms are poorly understood. Previous studies have demonstrated transfer of functional mitochondria from AML-MSCs to AML blasts facilitating energy requirements. To further improve our understanding of the crosstalk between leukaemia and AML-MSCs, we sought to determine contribution of AML-MSCs and signalling cascades regulating metabolic processes.Methods: Sorted MSCs from non-leukaemic and MLL-AF9 leukaemic mice were isolated, and gene expression profiling was performed using RNA microarray. Additionally sorted MSCs from long-term cultures were cultured alone or with MLL-AF9 leukaemia cells and analysed by RNA-sequencing. Gene set enrichment analysis (GSEA) was used to identify the hallmark gene sets overrepresented in AML-MSCs. We further cocultured murine wild type BM-MSCs alone or together with murine AML cells (C1498 and MLL-AF9) or the control lineage negative cells (Lin -). Metabolic alterations, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analysed by Agilent Seahorse XFe96 analyser. Additionally, glucose consumption, lactate secretion and mitochondrial DNA copy number were measured.Results: Microarray analysis in sorted MSCs from leukaemic and non-leukaemic mice have identified hallmark oxidative phosphorylation (p<0.01, NES=-1.6) and glycolysis (p<0.01, NES=-1.3) gene sets to be negatively enriched in AML-MSCs. Interestingly, both the gene sets were also negatively enriched in sorted AML-MSCs when cocultured with leukaemia but not control cells. To validate these findings, we analysed OCR and EACR in WT-MSCs in an identical setting. The oxidative phosphorylation was significantly decreased in MSCs cocultured with C1498 (p<0.0001) and MLL-AF9 (p<0.005) but not with Lin - cells. Interestingly, glycolysis rate, glucose consumption, lactate secretion were significantly decreased in MSCs cocultured with leukaemia cells. Mitochondrial DNA copy number were significantly decreased in MSCs cocultured with C1498 (p<0.001) or MLL-AF9 (p<0.005) but not with control cells.Recent evidence from the lab has demonstrated an essential role for Notch signalling in the leukaemia and AML-MSCs interaction. To functionally determine the crosstalk of leukaemia-MSC interaction and subsequent Notch signalling, we ectopically expressed the Notch intracellular domain (Notch-ICN1) to mimic Notch activation in a murine stromal cell line, MS-5. Confirming Notch activation, Hes1 mRNA expression (encoding a transcriptional target of Notch signalling) was significantly increased in these cells. Underscoring a role for Notch signalling and activation, Notch-ICN1 overexpression in MS-5 cells demonstrated less oxidative phosphorylation and glycolysis rates as compared to MS-5 cells transduced with empty vector.Conclusion: In line with our microarray and GSEA analysis, our findings confirmed that leukaemia cells indeed induced metabolic alterations decreasing oxidative phosphorylation and glycolysis, and thereby potentially altering AML-MSCs function. At the molecular level, Notch signalling (via upregulated Notch1 and 2 expressions and Notch-ICN) in AML-MSCs contributed to metabolic alterations. Therefore, therapeutically interfering this pathway could target the bidirectional interaction between leukaemia and AML-MSCs improving therapeutic efficacy of AML. DisclosuresKhandanpour: GSK: Honoraria; Takeda: Honoraria; Janssen: Honoraria; AstraZeneca: Honoraria, Research Funding; Pfizer: Honoraria; Sanofi: Honoraria, Research Funding; BMS/Celgene: Honoraria.

Cross-regulation of notch/AKT and serum/glucocorticoid regulated kinase 1 (SGK1) in IL-4-stimulated human macrophages

Notch signaling regulates the responses of macrophages to different stimuli in a context-dependent manner. The roles of Notch signaling in proinflammatory macrophages are well characterized, whereas its involvement, if any, in IL-4-stimulated macrophages (M(IL-4)) is still unclear. We observed that Notch signaling is functional in human M(IL-4). We performed transcriptome analysis of the Notch1 intracellular domain (NIC1)-overexpressing human monocytic cell line THP-1 with or without IL-4 stimulation to understand the global impact of Notch signaling in M(IL-4). The results revealed that NIC1-overexpressing THP-1 upregulated proinflammatory-associated genes and target genes of IL-4 signaling. We identified serum/glucocorticoid regulated kinase 1 (SGK1) as one of the genes increased by NIC1 overexpression in M(IL-4). To dissect the signaling pathway leading to SGK1 upregulation, we pretreated THP-1-derived macrophages with specific inhibitors of Notch (DAPT), AKT (LY294002) or ERK (U0126). Among these inhibitors, only LY294002 decreased the SGK1 mRNA levels in M(IL-4), indicating that the AKT pathway plays a key role in SGK1 transcription in M(IL-4). Furthermore, treatment of THP-1-derived macrophages with the SGK1 inhibitor (GSK650394) suppressed AKT phosphorylation, but not STAT6, in response to IL-4, indicating that SGK1 positively regulates AKT pathway in M(IL-4). Finally, GSK650394 treatment of human M(IL-4) increased the levels of PPARG mRNA and its protein, indicating a negative role of SGK1 in M(IL-4) function. Overall, we report that the Notch signaling and AKT pathways cooperatively regulate SGK1 expression in M(IL-4) where SGK1, in turn, plays an important role in suppressing IL-4-induced PPARγ expression.

Effects of Notch-1/Twist-1 axis in the process of epithelial-mesenchymal transition of type II alveolar epithelial cell and its mechanism

Objective: To study the role of Notch-1/Twist-1 axis in the process of epithelial-mesenchymal transition (EMT) of type II alveolar epithelial cells in pulmonary fibrosis (PF) and hope to provide a new theoretical basis for the pathogenesis of PF. Methods: Thirty rats were randomly divided into control group and bleomycin (BLM) group, 15 rats in each group. The PF rat model was induced by intratracheal injection of BLM (7 500 U/kg). Excised inferior lobe of left lung was fixed in 10% formalin for HE staining, Masson staining and transforming growth factor-beta 1 (TGF-β1) immunohistochemistry staining after BLM injection for 28 days. The cultured type II alveolar epithelial cells (RLE-6TN) were divided into 4 groups (Control group, transforming growth factor-beta 1 (TGF-β1) group, Notch-1 negative control siRNA (NC siRNA, 100 pmol/L) group and Notch-1 siRNA (100 pmol/L) group), each group was established nine holes. The cells were treated with TGF-β1 (5.0 ng/ml) for 24 h following NC siRNA or Notch-1 siRNA for 48 h. The mRNA and (or) proteins levels of TGF-β1, collagen I, collagen III, E-Cadherin, zonula occludens-1 (ZO-1), Vimentin, E-Cadherin, Notch-1, Notch-1 intracellular domain (NICD), Hes-1 and Twist-1 were detected in lung tissue and type II alveolar epithelial cells. Results: In vivo, compared with the control group, the alveolar atrophy, collapse and fusion occurred, alveolar septum widened significantly, and a large number of inflammatory cells infiltrated in the pulmonary interstitial of the rats in the BLM group. And compared with control group, BLM obviously increased collagen deposition and collagen I and collagen III expressions, while the expressions of ZO-1 and E-cadherin were decreased, and the expressions of Vimentin and N-cadherin were increased, and concomitantly with increasing Notch-1, NICD, Hes-1 and Twist-1 expression in lung tissues of rats (P＜0.01). In vitro, compared with control group, TGF-β1 treatment obviously induced collagen I, collagen III, Notch-1, NICD, Hes-1 and Twist-1 expressions, and the expressions of E-cadherin and ZO-1 were decreased and the expressions of Vimentin and N-cadherin were increased(P＜0.01). Compared with TGF-β1 group, Notch-1 siRNA treatment significantly inhibited the expressions of Notch-1, NICD, Hes-1 and Twist-1, and the expressions of E-cadherin and ZO-1 were increased and the expressions of Vimentin and N-cadherin were decreased, and also obviously reduced the expressions of collagen I and collagen III induced by TGF-β1 (P＜0.05 or P＜0.01). Conclusion: Notch-1/Twist-1 axis is involved in the EMT process of type II alveolar epithelial cells, suggesting that Notch-1/Twist-1 signaling may be involved in the development of pulmonary fibrosis.

Nup54-induced CARM1 nuclear importation promotes gastric cancer cell proliferation and tumorigenesis through transcriptional activation and methylation of Notch2.

Gastric cancer (GC) has the fifth highest incidence globally, but its molecular mechanisms are not well understood. Here, we report that coactivator-associated arginine methyltransferase 1 (CARM1) is specifically highly expressed in gastric cancer and that its overexpression correlates with poor prognosis in patients with gastric cancer. Nucleoporin 54 (Nup54) was identified as a CARM1-interacting protein that promoted CARM1 nuclear importation. In the nucleus, CARM1 cooperates with transcriptional factor EB (TFEB) to activate Notch2 transcription by inducing H3R17me2 of the Notch2 promoter but not H3R26me2. Additionally, the Notch2 intracellular domain (N2ICD) was identified as a CARM1 substrate. Methylation of N2ICD at R1786, R1838, and R2047 by CARM1 enhanced the binding between N2ICD and mastermind-like protein 1 (MAML1) and increased gastric cancer cell proliferation in vitro and tumor formation in vivo. Our findings reveal a molecular mechanism linking CARM1-mediated transcriptional activation of the Notch2 signaling pathway to Notch2 methylation in gastric cancer progression.

Proprotein convertase furina is required for heart development in zebrafish.

Cardiac looping and trabeculation are key processes during cardiac chamber maturation. However, the underlying mechanisms remain incompletely understood. Here, we report the isolation, cloning and characterization of the proprotein convertase furina from the cardiovascular mutant loft in zebrafish. loft is an ethylnitrosourea-induced mutant and has evident defects in the cardiac outflow tract, heart looping and trabeculation, the craniofacial region and pharyngeal arch arteries. Positional cloning revealed that furina mRNA was barely detectable in loft mutants, and loft failed to complement the TALEN-induced furina mutant pku338, confirming that furina is responsible for the loft mutant phenotypes. Mechanistic studies demonstrated that Notch reporter Tg(tp1:mCherry) signals were largely eliminated in mutant hearts, and overexpression of the Notch intracellular domain partially rescued the mutant phenotypes, probably due to the lack of Furina-mediated cleavage processing of Notch1b proteins, the only Notch receptor expressed in the heart. Together, our data suggest a potential post-translational modification of Notch1b proteins via the proprotein convertase Furina in the heart, and unveil the function of the Furina-Notch1b axis in cardiac looping and trabeculation in zebrafish, and possibly in other organisms.

GAS1 is required for NOTCH-dependent facilitation of SHH signaling in the ventral forebrain neuroepithelium.

ABSTRACTGrowth arrest-specific 1 (GAS1) acts as a co-receptor to patched 1, promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in induced pluripotent stem cell-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating NOTCH signaling, which is essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives NOTCH pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating NOTCH and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations.

Loss of Numb promotes hepatic progenitor expansion and intrahepatic cholangiocarcinoma by enhancing Notch signaling

Numb, a stem cell fate determinant, acts as a tumor suppressor and is closely related to a wide variety of malignancies. Intrahepatic cholangiocarcinoma (iCCA) originates from hepatic progenitors (HPCs); however, the role of Numb in HPC malignant transformation and iCCA development is still unclear. A retrospective cohort study indicated that Numb was frequently decreased in tumor tissues and suggests poor prognosis in iCCA patients. Consistently, in a chemically induced iCCA mouse model, Numb was downregulated in tumor cells compared to normal cholangiocytes. In diet-induced chronic liver injury mouse models, Numb ablation significantly promoted histological impairment, HPC expansion, and tumorigenesis. Similarly, Numb silencing in cultured iCCA cells enhanced cell spheroid growth, invasion, metastasis, and the expression of stem cell markers. Mechanistically, Numb was found to bind to the Notch intracellular domain (NICD), and Numb ablation promoted Notch signaling; this effect was reversed when Notch signaling was blocked by γ-secretase inhibitor treatment. Our results suggested that loss of Numb plays an important role in promoting HPC expansion, HPC malignant transformation, and, ultimately, iCCA development in chronically injured livers. Therapies targeting suppressed Numb are promising for the treatment of iCCA.

Notch1 Signaling Contributes to Mechanical Allodynia Associated with Cyclophosphamide-Induced Cystitis by Promoting Microglia Activation and Neuroinflammation.

Aims Notch1 signaling regulates microglia activation, which promotes neuroinflammation. Neuroinflammation plays an essential role in various kinds of pain sensation, including bladder-related pain in bladder pain syndrome/interstitial cystitis (BPS/IC). However, the impact of Notch1 signaling on mechanical allodynia in cyclophosphamide- (CYP-) induced cystitis is unclear. This study is aimed at determining whether and how Notch1 signaling modulates mechanical allodynia of CYP-induced cystitis. Methods CYP was peritoneally injected to establish a bladder pain syndrome/interstitial cystitis (BPS/IC) rat model. A γ-secretase inhibitor, DAPT, was intrathecally injected to modulate Notch1 signaling indirectly. Mechanical withdrawal threshold in the lower abdomen was measured with von Frey filaments using the up-down method. The expression of Notch1 signaling, Iba-1, OX-42, TNF-α, and IL-1β in the L6-S1 spinal dorsal horn (SDH) was measured with Western blotting analysis and immunofluorescence staining. Results Notch1 and Notch intracellular domain (NICD) were both upregulated in the SDH of the cystitis group. Moreover, the expression of Notch1 and NICD was negatively correlated with the mechanical withdrawal threshold of the cystitis rats. Furthermore, treatment with DAPT attenuated mechanical allodynia in CYP-induced cystitis and inhibited microglia activation, leading to decreased production of TNF-α and IL-1β. Conclusion Notch1 signaling contributes to mechanical allodynia associated with CYP-induced cystitis by promoting microglia activation and neuroinflammation. Our study showed that inhibition of Notch1 signaling might have therapeutic value for treating pain symptoms in BPS/IC.

Cleaved Delta like 1 intracellular domain regulates neural development via Notch signal-dependent and -independent pathways

ABSTRACTNotch-Delta signaling regulates many developmental processes, including tissue homeostasis and maintenance of stem cells. Upon interaction of juxtaposed cells via Notch and Delta proteins, intracellular domains of both transmembrane proteins are cleaved and translocate to the nucleus. Notch intracellular domain activates target gene expression; however, the role of the Delta intracellular domain remains elusive. Here, we show the biological function of Delta like 1 intracellular domain (D1ICD) by modulating its production. We find that the sustained production of D1ICD abrogates cell proliferation but enhances neurogenesis in the developing dorsal root ganglia (DRG), whereas inhibition of D1ICD production promotes cell proliferation and gliogenesis. D1ICD acts as an integral component of lateral inhibition mechanism by inhibiting Notch activity. In addition, D1ICD promotes neurogenesis in a Notch signaling-independent manner. We show that D1ICD binds to Erk1/2 in neural crest stem cells and inhibits the phosphorylation of Erk1/2. In summary, our results indicate that D1ICD regulates DRG development by modulating not only Notch signaling but also the MAP kinase pathway.

Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor.

Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.

Neonatal Anesthesia by Ketamine in Neonatal Rats Inhibits the Proliferation and Differentiation of Hippocampal Neural Stem Cells and Decreases Neurocognitive Function in Adulthood via Inhibition of the Notch1 Signaling Pathway.

The Notch signaling pathway plays an important role in the regulation of neurogenesis. The objective of this study was to investigate whether the Notch signaling pathway was involved in the neurogenesis impairment and long-term neurocognitive dysfunction caused by neonatal exposure to ketamine. On postnatal day 7 (PND-7), male Sprague-Dawley (SD) rats were intraperitoneally injected with 40mg/kg ketamine four consecutive times (40mg/kg × 4) at 1-h intervals. Notch ligand Jagged1 (0.5mg/kg) and lentivirus overexpressing the Notch1 intracellular domain (LV-NICD1) were microinjected into the hippocampal dentate gyrus (DG) 1h or 4days before ketamine administration, respectively. The expression of Notch1 signaling pathway-related proteins was detected by Western blotting 24h after ketamine administration. The proliferation and differentiation of the neural stem cells (NSCs) in the hippocampal DG were evaluated by double immunofluorescence staining 24h after treatment. Moreover, changes in hippocampus-dependent spatial memory of 2-month-old rats were investigated with the Morris water maze test. Ketamine anesthesia in neonatal rats decreased the expression levels of Jagged1, Notch1, NICD1, and hairy enhancer of split 1 (Hes1); inhibited the proliferation and astrocytic differentiation of NSCs; and promoted the differentiation of neurons. Neonatal exposure to ketamine caused deficits in hippocampus-dependent spatial reference memory tasks in 2-month-old rats. Microinjection of Jagged1 or LV-NICD1 reversed the inhibitory effect of ketamine on the expression of Notch1-related proteins in the hippocampal DG, attenuated the ketamine-mediated decrease in NSC proliferation and differentiation, and improved the cognitive function of 2-month-old rats after neonatal exposure to ketamine. These results suggest that neonatal exposure to ketamine in rats inhibits the proliferation and differentiation of hippocampal NSCs and impairs neurocognitive function in adulthood. The Notch1 signaling pathway may be involved in the impairment of hippocampus-dependent learning and memory during adulthood caused by neonatal exposure to ketamine. These findings contribute to further understanding the neurotoxicity induced by neonatal exposure to ketamine and the underlying mechanisms.

Noncanonical Notch signals have opposing roles during cardiac development

The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stage-specific roles during cardiac development.

Notch‐1 and Notch‐3 Mediate Hypoxia‐Induced Activation of Synovial Fibroblasts in Rheumatoid Arthritis

To fully understand the molecular mechanism of hypoxia-induced rheumatoid arthritis synovial fibroblast cell (RASFC) activation via Notch-1 and Notch-3 signalling, and to evaluate its potential as a therapeutic target. Notch-1 and Notch-3 intracellular domain (N1ICD), Notch-3 intracellular domain (N3ICD), and hypoxia-inducible factor-1α (HIF-1α) were detected in RA synovial tissues via immunohistology. RASFC were cultured under hypoxic and normoxic conditions with or without small interfering RNAs, and N1ICD and N3ICD were overexpressed under normoxic conditions. Collagen-induced arthritis (CIA) rats were administered with LY411575 (inhibition of N1ICD and N3ICD) for 15 and 28 days, and its therapeutic efficacy was assessed by histology, radilology and inflammatory cytokine detection. In the study, we found that N1ICD, N3ICD and HIF-1α were abundantly expressed in RA patient synovial tissues. Meanwhile, HIF-1α was found to directly regulate the expression of Notch-1 and Notch-3 genes under hypoxic conditions. Moreover, hypoxia induced N1ICD and N3ICD expression in RASFC was blocked by HIF-1α small interfering RNA (siHIF-1α).Notch-1 small interfering RNA (siNotch-1) and Notch-3 small interfering RNA (siNotch-3) inhibited hypoxia-induced RASFC invasion and angiogenesis in vitro, whereas N1ICD and N3ICD overexpression promoted these processes. In addition, it was revealed that Notch-1 regulates RASFC migration and epithelial-mesenchymal transition (EMT) under hypoxia, whereas Notch-3 regulates anti-apoptosis and autophagy. Further, in vivo studies showed that N1ICD and N3ICD inhibitor LY411575 had a therapeutic effect on CIA rats. Collectively, this study has identified a functional link between HIF-1α, Notch-1, and Notch-3 signalling in regulating RASFC activation and rheumatoid arthritis.