Notch receptors are heterodimeric transmembrane proteins that regulate cell fate, such as differentiation, proliferation, and apoptosis. Dysregulated Notch pathway signaling has been observed in glioblastomas, as well as in other human malignancies. Nerve growth factor (NGF) is essential for cell growth and differentiation in the nervous system. Recent reports suggest that NGF stimulates glioblastoma proliferation. However, the relationship between NGF and Notch1 in glioblastomas remains unknown. Therefore, we investigated expression of Notch1 in a glioblastoma cell line (U87-MG), and examined the relationship between NGF and Notch1 signaling.

Oestrogen receptor β is believed to exert a cardioprotective effect against ischaemic injury. Nonetheless, the mechanism underlying its protective action remains to be fully elucidated. Recently, increased attention has been focused on Notch1 signalling for ameliorating cardiac ischaemic injury. Here, we hypothesised that oestrogen receptor β activation attenuates myocardial infarction (MI)-induced cardiac damage by modulating the Notch1 signalling pathway.

Notch receptors signaling is required for optimal T-cell activation and function. T-cell receptor (TCR) engagement can activate Notch receptors in T-cells in a ligand-independent fashion. In this study, we examined the role of adenosine A2A receptor (A2AR) signaling pathway in regulating the activity of Notch1 induced by TCR stimulation in CD8+T-cells. A selective A2AR agonist decreased Notch1 protein expression and Notch1 cleavage, and reduced transcripts of Notch1-target genes Hes1 and Myc in activated CD8+T-cells. Inhibition of TCR-induced Notch1 expression by an A2AR agonist was accompanied by increased cAMP concentration and mimicked by forskolin. This effect was associated with reduced IFN-γ and granzyme B production. The effect of an A2AR agonist was abrogated by a selective A2AR antagonist and absent in CD8+T-cells harvested from A2AR-/- mice. Stimulation of A2AR reduced Notch1 receptor levels by inhibiting upstream TCR signals, including ZAP70 phosphorylation, in turn impairing the generation of the active Notch1 intracellular domain (N1ICD). Direct activation of PKC with PMA and ionomycin bypassed A2AR-induced Notch1 inhibition. Overexpression of N1ICD in CD8+T-cells prevented the suppressive effects of an A2AR agonist on proliferation and cytokine release during activation. Our results identify the A2AR signaling pathway as an important regulator of TCR-induced Notch1 receptor activation in CD8+T-cells, and Notch as an important target of the immune suppressive effects of A2AR. We propose a mechanism whereby A2AR impairs CD8 T-cells function through inhibition of Notch1 receptor activation.

In vertebrates, embryonic hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of endothelial cells, the hemogenic endothelium (HE), through the endothelial-to-hematopoietic transition (EHT). Notch signaling is essential for HSPC development during embryogenesis across vertebrates. However, whether and how it regulates EHT remains unclear. Here, we show that G protein-coupled receptor 183 (Gpr183) signaling serves as an indispensable switch for HSPC emergence by repressing Notch signaling before the onset of EHT. Inhibition of Gpr183 significantly upregulates Notch signaling and abolishes HSPC emergence. Upon activation by its ligand 7α-25-OHC, Gpr183 recruits β-arrestin1 and the E3 ligase Nedd4 to degrade Notch1 in specified HE cells and then facilitates the subsequent EHT. Importantly, 7α-25-OHC stimulation promotes HSPC emergence in vivo and in vitro, providing an attractive strategy for enhancing the in vitro generation of functional HSPCs.

The anti-CD20 monoclonal antibodies rituximab and obinutuzumab differ in their mechanisms of action, with obinutuzumab evoking greater direct B cell death. To characterize the signaling processes responsible for improved B cell killing by obinutuzumab, we undertook a phosphoproteomics approach and demonstrate that rituximab and obinutuzumab differentially activate pathways downstream of the B cell receptor. Although both antibodies induce strong ERK and MYC activation sufficient to promote cell-cycle arrest and B cell death, obinutuzumab exceeds rituximab in supporting apoptosis induction by means of aberrant SYK phosphorylation. In contrast, rituximab elicits stronger anti-apoptotic signals by activating AKT, by impairing pro-apoptotic BAD, and by releasing membrane-bound NOTCH1 to up-regulate pro-survival target genes. As a consequence, rituximab appears to reinforce BCL2-mediated apoptosis resistance. The unexpected complexity and differences by which rituximab and obinutuzumab interfere with signaling pathways essential for lymphoma pathogenesis and treatment provide important impetus to optimize and personalize the application of different anti-CD20 treatments.

Intestinal ischemia/reperfusion (I/R) induces disruption of the intestinal barrier function. Aryl hydrocarbon receptor (AhR) has a vital role in maintaining the intestinal barrier function. However, the precise mechanism by which AhR maintains intestinal barrier function remains unclear. Notch1 signaling is downstream of AhR, and has also been reported to have a role in the development of tight junctions (TJs) and maintenance of intestinal homeostasis. Therefore, we hypothesized that AhR activation may attenuate the intestinal barrier dysfunction through increased activation of Notch1 signaling. Adult C57BL/6J mice were divided into three groups: Sham, I/R and I/R + 6-formylindolo(3,2-b)carbazole (Ficz) groups. Mice were sacrificed after I/R for 6 h and the intestine was harvested for histological examination, mRNA and protein content analysis, and mucosal permeability investigation. Additionally, a hypoxic Caco‑2 cell culture model was used to evaluate the role of AhR‑Notch1 signaling in the development of TJs and epithelial permeability in vitro. The AhR‑Notch1 signaling components and TJ proteins were assessed by reverse transcription‑quantitative polymerase chain reaction, western blotting, immunohistochemistry or immunofluorescence staining. Epithelial permeability was detected by transepithelium electrical resistance. The data demonstrated that Ficz significantly attenuated the intestinal tissue damage and the disrupted distribution of TJs, increased the expression of TJ proteins, reversed the decrease in TER and upregulated epithelial Notch1 signaling following intestinal I/R in vivo and hypoxia in vitro. Furthermore, inhibition of Notch1 signaling by N‑[N‑(3,5‑difluorophenacetyl)‑L‑alanyl]‑S‑phenylglycine t‑butyl ester (inhibitor of Notch signaling) counteracted the effects of Ficz on the development of TJs in hypoxic Caco‑2 cells. In conclusion, AhR activation ameliorated epithelial barrier dysfunction following intestinal I/R and hypoxia through upregulation of Notch1 signaling, which suggests that AhR may be a potential pharmaceutical agent to combat this condition.

In the damaged heart, cardiac adaptation relies primarily on cardiomyocyte hypertrophy. The recent discovery of cardiac stem cells in the postnatal heart, however, suggests that these cells could participate in the response to stress via their capacity to regenerate cardiac tissues. Using models of cardiac hypertrophy and failure, we demonstrate that components of the Notch pathway are up-regulated in the hypertrophic heart. The Notch pathway is an evolutionarily conserved cell-to-cell communication system, which is crucial in many developmental processes. Notch also plays key roles in the regenerative capacity of self-renewing organs. In the heart, Notch1 signaling takes place in cardiomyocytes and in mesenchymal cardiac precursors and is activated secondary to stimulated Jagged1 expression on the surface of cardiomyocytes. Using mice lacking Notch1 expression specifically in the heart, we show that the Notch1 pathway controls pathophysiological cardiac remodeling. In the absence of Notch1, cardiac hypertrophy is exacerbated, fibrosis develops, function is altered, and the mortality rate increases. Therefore, in cardiomyocytes, Notch controls maturation, limits the extent of the hypertrophic response, and may thereby contribute to cell survival. In cardiac precursors, Notch prevents cardiogenic differentiation, favors proliferation, and may facilitate the expansion of a transient amplifying cell compartment.

Fanconi Anemia (FA) is an inherited bone marrow failure syndrome caused by mutation in FA pathway proteins, involved in Interstrand Cross Link (ICL) repair. FA cells exhibit in vitro proliferation arrest due to accumulated DNA damage, hence understanding the rescue mechanism that renders proliferation advantage is required. Gene expression profiling performed in FA patients Peripheral Blood Mononuclear Cells (PBMCs) revealed a wide array of dysregulated biological processes. Functional enrichment and gene clustering analysis showed crippled autophagy process and escalated Notch signalling pathway in FA clinical samples and cell lines. Notch pathway mediators overexpression were reverted in FANCA mutant cells when treated with Rapamycin, an autophagy inducer. Additionally, Rapamycin stabilized cell viability after treatment with the DNA damaging agent, MitomycinC (MMC) and enhanced cell proliferation genes expression in FANCA mutant cells. Inherently FANCA mutant cells express impaired autophagy; thus activation of autophagy channelizes Notch signalling cascade and sustains cell viability.

Methamphetamine (METH), a widely abused stimulant drug, induces psychosis in approximately half of abusers; this effect is becoming a major concern for society. Although the Notch1 signalling pathway has been shown to play a part in the pathogenesis of some psychiatric disorders, its role in METH-induced psychosis (MIP) is still unknown. Here, the METH-induced locomotor sensitization model in rodents is considered to represent the underlying neurochemical changes driving psychoses. We found that the Notch1 signalling was downregulated in the medial prefrontal cortex (mPFC) in sensitized mice. Direct genetic and pharmacological manipulations of Notch1 signalling bidirectionally altered METH-induced locomotor sensitization and other MIP-related behaviours through governing neuronal activity in the mPFC. Moreover, Notch1 signalling negatively regulated GABAB1 receptor expression in the mPFC of METH-sensitized mice through Hes1, a transcriptional repressor in Notch1 signalling. Further, we show that Hes1 can directly bind to the GABAB1 receptor promoter. Notably, pharmacological regulation of the GABAB receptor in the mPFC reversed the changes in METH-induced locomotor sensitization caused by the dysfunction of Notch1 signalling. Together, our findings uncover a previously unrecognised Notch1-Hes1-GABAB1 receptor-dependent mechanism involved in regulating mPFC neuronal activity and behavioural phenotypes in MIP. Our work provides mechanistic insight into the aetiology and pathophysiology of MIP.

The Notch signaling pathway is a crucial regulator of cell differentiation as well as tissue organization, whose deregulation is linked to the pathogenesis of different diseases. NOTCH1 plays a key role in breast cancer progression by increasing proliferation, maintenance of cancer stem cells, and impairment of cell death. NOTCH1 is a mechanosensitive receptor, where mechanical force is required to activate the proteolytic cleavage and release of the Notch intracellular domain (NICD). We circumvent this limitation by regulating Notch activity by light. To achieve this, we have engineered an optogenetic NOTCH1 receptor (optoNotch) to control the activation of NOTCH1 intracellular domain (N1ICD) and its downstream transcriptional activities. Using optoNotch we confirm that NOTCH1 activation increases cell proliferation in MCF7 and MDA-MB-468 breast cancer cells in 2D and spheroid 3D cultures, although causing distinct cell-type specific migratory phenotypes. Additionally, optoNotch activation induced chemoresistance on the same cell lines. OptoNotch allows the fine-tuning, ligand-independent, regulation of N1ICD activity and thus a better understanding of the spatiotemporal complexity of Notch signaling. Video Abstract.

Prostate cancer (PCa) is the second leading cause of mortality and a leading cause of malignant tumors in males. Prostate cancer stem cells (PCSCs) are likely the responsible cell types for cancer initiation, clinical treatment failure, tumor relapse, and metastasis. Estrogen receptor alpha (ERα) is mainly expressed in the basal layer cells of the normal prostate gland and has key roles in coordinating stem cells to control prostate organ development. Here, we investigated the roles of the estrogen-ERα signaling pathway in regulating PCSCs.

The spatiotemporal localization and expression of Dll4 are critical for sprouting angiogenesis. However, the related mechanisms are poorly understood. Here, we show that G-protein-coupled receptor-kinase interacting protein-1 (GIT1) is a robust endogenous inhibitor of Dll4-Notch1 signaling that specifically controls stalk cell fate. GIT1 is highly expressed in stalk cells but not in tip cells. GIT1 deficiency remarkably enhances Dll4 expression and Notch1 signaling, resulting in impaired retinal sprouting angiogenesis, which can be rescued by treatment with the Notch inhibitor or Dll4 neutralizing antibody. Notch1 regulates Dll4 expression by binding to recombining binding protein suppressor of hairless (RBP-J, a transcriptional regulator of Notch) via a highly conserved ankyrin (ANK) repeat domain. We show that GIT1, which also contains an ANK domain, inhibits the Notch1-Dll4 signaling pathway by competing with Notch1 ANK domain for binding to RBP-J in stalk cells.

Canonical NOTCH signaling, known to be essential for tissue development, requires the Delta-Serrate-LAG2 (DSL) domain for NOTCH to interact with its ligand. However, despite lacking DSL, Delta-like 1 homolog (DLK1), a protein that plays a significant role in mammalian development, has been suggested to interact with NOTCH1 and act as an antagonist. This non-canonical interaction is, however controversial, and evidence for a direct interaction, still lacking in mammals. In this study, we elucidated the putative DLK1-NOTCH1 interaction in a mammalian context. Taking a global approach and using Dlk1(+/+) and Dlk1(-/-) mouse tissues at E16.5, we demonstrated that several NOTCH signaling pathways indeed are affected by DLK1 during tissue development, and this was supported by a lower activation of NOTCH1 protein in Dlk1(+/+) embryos. Likewise, but using a distinct Dlk1-manipulated (siRNA) setup in a mammalian cell line, NOTCH signaling was substantially inhibited by DLK1. Using a mammalian two-hybrid system, we firmly established that the effect of DLK1 on NOTCH signaling was due to a direct interaction between DLK1 and NOTCH1. By careful dissection of this mechanism, we found this interaction to occur between EGF domains 5 and 6 of DLK1 and EGF domains 10-15 of NOTCH1. Thus, our data provide the first evidence for a direct interaction between DLK1 and NOTCH1 in mammals, and substantiate that non-canonical NOTCH ligands exist, adding to the complexity of NOTCH signaling.

Mechanosensory hair cells (HCs) and surrounding supporting cells (SCs) in the mouse cochlea are important for hearing and are derived from the same prosensory progenitors. Notch1 signaling plays dual but contrasting and age-dependent roles in mouse cochlear development: early lateral induction and subsequent lateral inhibition. However, it has been difficult to directly visualize mouse cochlear cells experiencing various levels of Notch1 activity at single cell resolution. Here, we characterized two knock-in mouse lines, Notch1(Cre (Low)/+) and Notch1(Cre (High)/+) , with different Cre recombinase activities, that can detect Notch1 receptor proteolysis or Notch1 activity at high and low thresholds, respectively. Using both lines together with a highly sensitive Cre reporter line, we showed that Notch1 activity is nearly undetectable during lateral induction but increases to medium and high levels during lateral inhibition. Furthermore, we found that within the neonatal organ of Corti, the vast majority of cells that experience Notch1 activity were SCs not HCs, suggesting that HCs kept undetectable Notch1 activity during the entire lineage development. Furthermore, among SC subtypes, ∼85-99% of Deiters' and outer pillar cells but only ∼19-38% of inner pillar cells experience medium and high levels of Notch1 activity. Our results demonstrate that Notch1 activity is highly heterogeneous: 1) between lateral induction and inhibition; 2) between HC and SC lineages; 3) among different SC subtypes; 4) among different cells within each SC subtype. Such heterogeneity should elucidate how the development of the cochclear sensory epithelium is precisely controlled and how HC regeneration can be best achieved in postnatal cochleae.

Intracerebral hemorrhage (ICH) is a leading medical problem and has no effective treatment approach up until now. The transcription factor androgen receptor (AR) has been indicated in the cerebrovascular function recently. However, its participation in ICH remains unclear. The present study aims to expound the regulation of AR in microglia/macrophage phenotypes and the secondary brain injury in a rat model with ICH, and to discuss the involved pathway. Following the induction of ICH in rats, we found that ICH led to increased mNSS score, enhanced microglial activity, and promoted levels of inflammatory factors and apoptosis of brain cells. Using microarray analysis, AR was found to be significantly overexpressed in ICH rat brain tissues. AR repressed the transcription of Jumonji d3 (JMJD3, histone 3 demethylase). JMJD3 inhibited the methylation of Botch and promoted the activity of Notch1. JMJD3 hampered microglial activity and ameliorated secondary brain injury in rats, whereas upregulation of AR or downregulation of Botch reversed the protective effects of JMJD3. In conclusion, we found that AR promoted microglial activation and secondary brain injury via transcriptionally repressing JMJD3 and mediating the subsequent Botch/Notch1 pathway, which may provide novel insights into therapeutic options for the treatment of ICH.

Notch signaling induced interleukin (IL)-22 secretion by CD4+ T cells via retinoid-related orphan nuclear receptor γt (RORγt) or aryl hydrocarbon receptor (AhR). Previous studies have demonstrated that Notch-AhR-IL-22 axis took part in the pathogenesis of chronic viral infection, however, its role in cancer has not been fully elucidated. Thus, the aim of current study was to investigate the involvement of Notch-AhR-IL-22 axis in the pathogenesis of lung adenocarcinoma. A total of 37 late-stage lung adenocarcinoma patients and 17 healthy individuals were enrolled. CD4+ T cells were purified from peripheral bloods and bronchoalveolar lavage fluids (BALF), and were stimulated with γ-secretase inhibitor (GSI). mRNA corresponding to Notch receptors and transcriptional factors were measured by real-time PCR. IL-22 concentration was investigated by ELISA. The bioactivity (including cellular proliferation, cell cycle, apoptosis, and invasion) of lung adenocarcinoma cell line A549 was also assessed in response to recombinant IL-22 stimulation in vitro. Notch1 mRNA expression was significantly elevated in CD4+ T cells purified from peripheral bloods and tumor site BALF in lung adenocarcinoma patients. IL-22 expression and RORγt/AhR mRNA in BALF was also remarkably increased in tumor site. Inhibition of Notch signaling by GSI did not affect cellular proliferation, but reduced IL-22 production in CD4+ T cells from BALF, along with down-regulation of AhR, but not RORγt. Moreover, IL-22 stimulation promoted A549 cells invasion. The current data indicated that elevated Notch1 induced higher IL-22 secretion by CD4+ T cells in lung adenocarcinoma patients, and Notch-AhR-IL-22 axis took part in the pathogenesis of lung adenocarcinoma.

Calcific aortic valve disease (CAVD) is a deadly disease that is rising in prevalence due to population aging. While the disease is complex and poorly understood, one well-documented driver of valvulopathy is serotonin agonism. Both serotonin overexpression, as seen with carcinoid tumors and drug-related agonism, such as with Fenfluramine use, are linked with various diseases of the valves. Thus, the objective of this study was to determine if genetic ablation or pharmacological antagonism of the 5-HT2B serotonin receptor (gene: Htr2b) could improve the hemodynamic and histological progression of calcific aortic valve disease. Htr2b mutant mice were crossed with Notch1+/- mice, an established small animal model of CAVD, to determine if genetic ablation affects CAVD progression. To assess the effect of pharmacological inhibition on CAVD progression, Notch1+/- mice were treated with the 5-HT2B receptor antagonist SB204741. Mice were analyzed using echocardiography, histology, immunofluorescence, and real-time quantitative polymerase chain reaction. Htr2b mutant mice showed lower aortic valve peak velocity and mean pressure gradient-classical hemodynamic indicators of aortic valve stenosis-without concurrent left ventricle change. 5-HT2B receptor antagonism, however, did not affect hemodynamic progression. Leaflet thickness, collagen density, and CAVD-associated transcriptional markers were not significantly different in any group. This study reveals that genetic ablation of Htr2b attenuates hemodynamic development of CAVD in the Notch1+/- mice, but pharmacological antagonism may require high doses or long-term treatment to slow progression.

Angiogenesis, a marker of cancer development, affects response to radiotherapy sensibility. This preclinical study aims to understand the receptor tyrosine kinase-mediated angiogenesis in head neck squamous cell carcinoma (HNSCC). The receptor tyrosine kinase activity in a transgenic mouse model of HNSCC was assessed. The anti-tumorigenetic and anti-angiogenetic effects of cetuximab-induced epidermal growth factor receptor (EGFR) inhibition were investigated in xenograft and transgenic mouse models of HNSCC. The signaling transduction of Notch1 and hypoxia-inducible factor-1α (HIF-1α) was also analyzed. EGFR was overexpressed and activated in the Tgfbr1/Pten deletion (2cKO) mouse model of HNSCC. Cetuximab significantly delayed tumor onset by reducing tumor angiogenesis. This drug exerted similar effects on heterotopic xenograft tumors. In the human HNSCC tissue array, increased EGFR expression correlated with increased HIF-1α and micro vessel density. Cetuximab inhibited tumor-induced angiogenesis in vitro and in vivo by significantly downregulating HIF-1α and Notch1. EGFR is involved in the tumor angiogenesis of HNSCC via the HIF-1α and Notch1 pathways. Therefore, targeting EGFR by suppressing hypoxia- and Notch-induced angiogenesis may benefit HNSCC therapy.

The Notch1 signaling pathway contributes to tumorigenesis by influencing differentiation, proliferation and apoptosis. Here, we demonstrate that inhibition of the Notch1 signaling pathway sensitizes glioblastoma cell lines and glioblastoma initiating cells to apoptosis induced by the death ligand TRAIL. This sensitization occurs through transcriptional upregulation of the death receptor 5 (DR5, TRAIL-R2). The increase in DR5 expression is abrogated by concomitant repression of the transcription factor Sp1, which directly binds to the DR5 promoter in the absence of Notch1 as revealed by chromatin immunoprecipitation. Consistent with these findings, Notch1 inhibition resulted in increased DR5 promoter activity, which was impaired by mutation of one out of two Sp1-binding sites within the proximal DR5 promoter. Moreover, we demonstrate that JNK signaling contributes to the regulation of DR5 expression by Notch1. Taken together, our results identify Notch1 as key driver for TRAIL resistance and suggest Notch1 as a promising target for anti-glioblastoma therapy.

Endothelial cells transduce mechanical forces from blood flow into intracellular signals required for vascular homeostasis. Here we show that endothelial NOTCH1 is responsive to shear stress, and is necessary for the maintenance of junctional integrity, cell elongation, and suppression of proliferation, phenotypes induced by laminar shear stress. NOTCH1 receptor localizes downstream of flow and canonical NOTCH signaling scales with the magnitude of fluid shear stress. Reduction of NOTCH1 destabilizes cellular junctions and triggers endothelial proliferation. NOTCH1 suppression results in changes in expression of genes involved in the regulation of intracellular calcium and proliferation, and preventing the increase of calcium signaling rescues the cell-cell junctional defects. Furthermore, loss of Notch1 in adult endothelium increases hypercholesterolemia-induced atherosclerosis in the descending aorta. We propose that NOTCH1 is atheroprotective and acts as a mechanosensor in adult arteries, where it integrates responses to laminar shear stress and regulates junctional integrity through modulation of calcium signaling.