How could the entry of the Notch receptor into endosomes promote efficient signaling? Because signaling depends on intramembrane cleavage of Notch by γ-secretase, we asked whether altered Notch endosomal transport might affect its cleavage. We directly measured the amount of Notch in endocytic mutant tissue that can be cleaved by γ-secretase by using an assay that induces ligand-independent Notch cleavage in tissue extracts (see Materials and methods). Notch cleavage efficiency ex vivo is measured in Western blots by quantifying the amount of the lower band (corresponding to the γ-secretase cleavage product NICD) relative to the upper band (corresponding to its immediate precursor, the membrane-anchored γ-secretase substrate NEXT) of the ∼120-kD doublet recognized by an antibody against an NICD epitope. As expected, generation of free NICD in this assay is completely blocked by treatment with the γ-secretase inhibitor N-(N-[3,5-difluorophenacetyl-l-alanyl])-S-phenylglycine t-butyl ester ...

Notch signalling regulates cell fate in most tissues, promoting precursor cell proliferation in some, but differentiation in others. Accordingly, downregulation or overactivity variously contributes to cancer development. So far, little is known about Notch pathway activity and function in the normal urothelium and in urothelial carcinoma (UC). We have therefore investigated expression of Notch pathway components in UC tissues and cell lines and studied the function of one receptor, NOTCH1, in detail. Expression of canonical Notch pathway components were studied in UC and normal bladder tissues by immunohistochemistry and quantitative RT-PCR and in UC cell lines and normal cultured urothelial cells by qRT-PCR, immunocytochemistry and Western blotting. Pathway activity was measured by reporter gene assays. Its influence on cell proliferation was investigated by γ-secretase inhibition. Effects of NOTCH1 restoration were followed by measuring cell cycle distribution, proliferation, clonogenicity and

Members of the Notch family of transmembrane receptors are found on primitive hematopoietic precursors, and Notch ligand expression has been demonstrated on the surface of stromal cells, suggesting a role for Notch signaling in mammalian blood cell development. The current report examines the expression of Notch receptors and their ligands in murine hematopoietic tissues to determine: A) which blood cell lineages in the adult are influenced by Notch activity, and B) whether fetal hematopoiesis in the embryo involves the Notch pathway. In the adult mouse, a combination of flow cytometry, immunohistochemistry and Northern analysis was used to examine Notch receptor or ligand expression in bone marrow and spleen. In the embryo, Northern analysis and in situ hybridization were used to characterize Notch receptor and ligand expression in fetal liver on embryonic day 12 (E12) through E17, an active period encompassing both erythropoiesis and granulopoeisis. Flow cytometry demonstrated the presence of ...

Notch receptors are important mediators of cell fate during embryogenesis, but their role in adult physiology, particularly in postnatal angiogenesis, remains unknown. Of the Notch receptors, only Notch1 and Notch4 are expressed in vascular endothelial cells. Here we show that blood flow recovery and postnatal neovascularization in response to hindlimb ischemia in haploinsufficient global or endothelial-specific Notch1(+/-) mice, but not Notch4(-/-) mice, were impaired compared with wild-type mice. The expression of vascular endothelial growth factor (VEGF) in response to ischemia was comparable between wild-type and Notch mutant mice, suggesting that Notch1 is downstream of VEGF signaling. Treatment of endothelial cells with VEGF increases presenilin proteolytic processing, gamma-secretase activity, Notch1 cleavage, and Hes-1 (hairy enhancer of split homolog-1) expression, all of which were blocked by treating endothelial cells with inhibitors of phosphatidylinositol 3-kinase/protein

Transfection with siN1 and siN2 selectively suppressed the expression of Notch1 and Notch2 mRNA and protein, respectively. In T-ALL cell lines, NOTCH1 knockdown as well as NOTCH2 knockdown suppressed cell proliferation and induced apoptosis. Immunoblot analysis showed that Myc expression was downregulated in NOTCH1-knockdown cells but not affected in NOTCH2-knockdown cells. In AML cell lines, cell proliferation was not significantly affected by NOTCH siRNAs. NOTCH2 knockdown increased the level of cleaved Notch1 fragment without increasing Notch1 expression. The knockdown of NOTCH1 and NOTCH2 reduced the expression and phosphorylation of mTOR protein in THP-1 cells. To confirm this finding, we examined the effects of activation of Notch by the recombinant Notch ligands, Jagged1 and Delta1, on the expression of mTOR protein. The activation of Notch resulted in an increase in the level of the mTOR protein and its phosphorylation in THP-1 cells. Thus, siRNA-transfection and ligand stimulation of ...

Notch signaling is a ubiquitously used signaling pathway that is highly conserved and used throughout metazoan development. Understanding the regulation of Notch signaling is becoming increasingly important in determining the mechanism and treatment for the myriad of human Notch-related diseases. In Drosophila. melanogaster, the development of external sensory organs provides a context in which Notch can be manipulated and phenotypes can be easily interpreted. Here, we expand upon the growing field of Notch regulation through endocytic trafficking by examining the role of Numb and Sara endosomes. Numb is a potent Notch inhibitor whose function is conserved in higher organisms, but whose mechanism of action has remained elusive. In this study, we dispel a previous hypothesis that Numb promotes Notch internalization and instead demonstrate that Numb is a suppressor of Notch endocytic recycling. In support of this, we show that Numb is necessary and sufficient for Notch trafficking to late endosomes

Notch is a transmembrane receptor for the transmembrane ligands Delta and Jagged (also known as Serrate). Signaling by Notch is important for establishing boundaries during development (see accompanying article by Fortini). Fringe modifies Notch signaling when expressed in Notch-expressing cells. Two groups, Brückner et al. and Moloney et al., show that Fringe is a glycosyltransferase that catalyzes the elongation of O-linked fucose residues on the epidermal growth factor repeats of the Notch receptor. Brückner et al. show that coexpression of Fringe and Notch enhances the interaction between Notch and Delta and that the glycosyltransferase activity of Fringe is essential for this activity. Moloney et al. show that when Notch is modified by Fringe, activation of Notch by Jagged1 is inhibited. Thus, carbohydrate modification of the Notch receptor fine-tunes the cellular responsiveness to Notch ligands, allowing the establishment of discrete boundaries of Notch signaling. Carbohydrate ...

TY - JOUR. T1 - Macrophage Notch Ligand Delta-Like 4 Promotes Vein Graft Lesion Development. T2 - Implications for the Treatment of Vein Graft Failure. AU - Koga, Jun Ichiro. AU - Nakano, Toshiaki. AU - Dahlman, James E.. AU - Figueiredo, Jose Luiz. AU - Zhang, Hengmin. AU - Decano, Julius. AU - Khan, Omar F.. AU - Niida, Tomiharu. AU - Iwata, Hiroshi. AU - Aster, Jon C.. AU - Yagita, Hideo. AU - Anderson, Daniel G.. AU - Keith Ozaki, C.. AU - Aikawa, Masanori. PY - 2015/11/1. Y1 - 2015/11/1. N2 - Objective-Despite its large clinical impact, the underlying mechanisms for vein graft failure remain obscure and no effective therapeutic solutions are available. We tested the hypothesis that Notch signaling promotes vein graft disease. Approach and Results-We used 2 biotherapeutics for Delta-like ligand 4 (Dll4), a Notch ligand: (1) blocking antibody and (2) macrophage-or endothelial cell (EC)-targeted small-interfering RNA. Dll4 antibody administration for 28 days inhibited vein graft lesion ...

Drug resistance is one of the major problems in multiple myeloma (MM) clinical treatment. It is reported that Notch pathway was involved in drug resistance. In this study, we demonstrated that Notch activation by Dll1 simulation could induce drug resistance to bortezomib in murine and human MM cells. Blocking Notch pathway by DAPT (Notch inhibitor) could reverse the effect and increased the sensitivity to bortezomib. Notch activation decreased the cell apoptosis which was induced by bortezomib treatment as measured by flow cytometry. Further investigation showed that Dll1 simulation could down-regulate CD138, Blimp-1 and XBP-1 mRNA expression and shifts MM cells to a more resistant CD138- phenotype with a significant increase of CD138- subpopulation as detected by flow cytometry both in murine and human MM cells. Meanwhile, by MACS and FACS sorting of CD138+ subpopulation, we found that Notch activation could up-regulates anti-apoptotic proteins (Bcl-xL, Mcl-1 and Bcl-2) in CD138+ MM ...

TY - JOUR. T1 - The cell giveth and the cell taketh away. T2 - An overview of Notch pathway activation by endocytic trafficking of ligands and receptors. AU - Pratt, Emily B.. AU - Wentzell, Jill S.. AU - Maxson, Julia. AU - Courter, Lauren. AU - Hazelett, Dennis. AU - Christian, Jan L.. PY - 2011/5/1. Y1 - 2011/5/1. N2 - Notch signaling is firmly established as a form of cell-to-cell communication that is critical throughout development. Dysregulation of Notch has been linked to cancer and developmental disorders, making it an important target for therapeutic intervention. One aspect of this pathway that sets it apart from others is its apparent reliance on endocytosis by signal-sending and signal-receiving cells. The subtle details of endocytosis-mediated molecular processing within both ligand- and receptor-presenting cells that are required for the Notch signal to maintain fidelity remain unclear. The endosomal system has long been known to play an important role in terminating signal ...

During regular development heterogeneous expression of Notch ligands can lead to pathway suppression in the signal-sending cell an activity referred to as lateral inhibition. Notch pathway goals in signal-sending cells might occur through the experience of the Notch ligand intracellular domains which Quarfloxin (CX-3543) translocates in to the nucleus. Focusing on how this neoplastic lateral inhibition procedure functions in cancers cells could be essential in concentrating on ligand powered Notch signaling in solid tumors. and households. Within a subset of malignancies including T cell ALL [13] breasts [14] and lung cancers [15] Notch is normally turned on by mutations or translocations that straight alter receptors or various other key pathway associates (analyzed in: [16 17 Generally in most tumors nevertheless Notch signaling is set up when receptors over the tumor bind to ligands portrayed by adjacent cells. In a few tumor microenvironments Notch ligands are extremely portrayed on arteries ...

Variations in the spatial localization of signaling components and crosstalk among signaling cascades are mechanisms through which diversity in signaling networks is generated. The receptor Notch provides an example of regulation by spatial localization: In the canonical Notch signaling pathway, Notch is cleaved to produce the Notch intracellular domain (NICD, also known as NIC), which translocates to the nucleus to regulate gene expression. We describe a T cell receptor-dependent, non-nuclear distribution and function of the processed receptor Notch, which was associated with the improved survival of regulatory T cells (Tregs) in vitro and in vivo and was compromised by T cell-specific deletion of Notch1. Unlike a nuclear-restricted mutant of NICD, mutant NICD that underwent nuclear export or was targeted to the plasma membrane protected Notch1−/− Tregs from apoptosis induced by nutrient deprivation and oxidative stress. Notch signaling integrated with phosphatidylinositol 3-kinase ...

Expression of an activated Notch 1 allele in retrovirally infected cells led to aberrant differentiation of retinal cell types in an otherwise normal environment. Analogously,Notch has been shown to be involved in the differentiation pathway of multiple cell types in Drosophila andXenopus retina. Expression of activated Notch appears to block cell differentiation (Cagan and Ready, 1989; Fortini et al., 1993; Dorsky et al., 1995). This block, however, does not appear to be irreversible in Drosophila. Once Notch activity subsides, cells may recover and differentiate according to the developmental cues present in a later environment (Fortini et al., 1993; Struhl et al., 1993). These studies suggest that Notch activity regulates the ability of cells to respond to differentiation signals. Other studies in mammalian cell cultures and Xenopus embryos also support the model that Notch blocks differentiation of neural and mesodermal cell lineages (Coffman et al., 1993; Kopan and Nye, 1994). Notch ...

Results and Discussion In this paper several approaches have been applied, in order to examine the different properties of the synthetic Notch receptors and to compare them with those of endogenous Notch. They generated libraries of receptors with different EC domains, each coupled with a different IC domain and expressed them in fibroblast. They found that these synthetic receptors are strongly activated by cell-cell contact, if the sender cell expresses cognate ligand on its surface. To determine if the activation occurs by a cleavage mechanism, they used the drug DAPT (N-[N-(3,5-difluorophenacetyl)-lalanyl]-S-phenylglycine t-butyl ester). Treatment with DAPT completely blocks the activation of synNotch. These were eliminatory examinations, which have shown the characteristics of the endogenous Notch necessary to conduct further experiments. Also, they found that the response is reversible upon removal of the ligand - expressing cells. The next phase of the research is dedicated to determining ...

Notch signaling is used for cell fate determination throughout the animal kingdom, and differences in Notch activity between two daughter cells determine their future fates. Thus, Notch signaling promotes progenitor cell identity at the expense of differentiated cell phenotypes (Jadhav et al., 2006; Mizutani et al., 2007). Differences in the Notch activities between two daughter cells can be specified by the asymmetric localization and inheritance of Numb, a negative regulator of the Notch pathway (Guo et al., 1996; Cayouette et al., 2001; Petersen et al., 2002; Shen et al., 2002). In the embryonic lung, Notch signaling controls cell fates in developing airways (Post et al., 2000; Tsao et al., 2008; Tsao et al., 2009), and Notch activation inhibits the differentiation of distal progenitors into alveolar cells (Guseh et al., 2009). Yet the role of asymmetric segregation of cell fate determinant/Notch inhibitor Numb during lung development, and the way the process might be regulated are still ...

In order to achieve a better outcome for pancreatic cancer patients, reliable biomarkers are required which allow for improved diagnosis. These may emanate from a more detailed molecular understanding of the aggressive nature of this disease. Having previously reported that Notch3 activation appeared to be associated with more aggressive disease, we have now examined components of this pathway (Notch1, Notch3, Notch4, HES-1, HEY-1) in more detail in resectable (n = 42) and non-resectable (n = 50) tumours compared to uninvolved pancreas. All three Notch family members were significantly elevated in tumour tissue, compared to uninvolved pancreas, with expression maintained within matched lymph node metastases. Furthermore, significantly higher nuclear expression of Notch1, -3 and -4, HES-1, and HEY-1 (all p ≤ 0.001) was noted in locally advanced and metastatic tumours compared to resectable cancers. In survival analyses, nuclear Notch3 and HEY-1 expression were significantly associated with ...

As a previous work has suggested the potential for cross-talk between the Notch and Hedgehog signaling cascades (14), we focused on the upregulation of Hedgehog signaling in response to MRK-003. Increased levels of Notch pathway components are seen in Hedgehog-driven medulloblastoma models (15, 42) and it was initially suggested that these tumors may be dependent on Notch signaling for survival (15). More recent studies, however, indicate that Hedgehog-driven medulloblastomas can grow in the absence of canonical Notch activity (43, 44). Additionally, several groups have demonstrated that Hedgehog pathway components Gli1 and Gli2 are able to positively regulate Hes1 independently of Notch (45, 46). However, when we treated several GBM neurosphere lines with the Hedgehog inhibitor cyclopamine we did not observe decreases in Hes1 or other Notch pathway targets, suggesting that Hedgehog does not play a significant role regulating Notch targets in malignant gliomas (Supplementary Fig. S2A).. We ...

Introduction: Notch ligands of the Delta-like (Dll) family regulate embryonic and postnatal arteriogenesis. However, the role of notch signaling in coronary arteries, especially in the endothelium, is poorly characterized. We have analyzed the role of Notch ligand Dll1 in coronary arteries and myocardial infarction.. Methods and Results: Analysis of Dll1 expression by immunofluorescence and Dll1-lacZ reporter gene studies in adult mouse hearts revealed selective endocardial and coronary endothelial, but not venous or capillary Dll1 expression. Hearts of mice heterozygous for Dll1 (Dll1+/−) were significantly smaller than wildtype (wt) hearts (heart weight (g)/femur length (cm), wt: 0.13± 0.02 vs. het: 0.09± 0.02, n=10, p,0.05) while body weight and survival over 18 months was comparable. Furthermore, the number of medium (,50 -100 um) and large (,100 um) coronary arteries was significantly reduced in Dll1+/− hearts (p,0.01). Reduced coronary numbers were also apparent in neonatal hearts, ...

Delta and Notch function are required for cell fate specification in numerous tissues during embryonic and postembryonic Drosophila development. Delta is expressed by all members of interacting cell populations within which fates are being specified and is subsequently down-regulated as cells stably adopt particular fates. Multiphasic expression in the derivatives of many germ layers implies successive requirements for Delta function in a number of tissues. At the cellular level, Delta and Notch expression are generally coincident within developing tissues. At the subcellular level, Delta and Notch are localized in apparent endocytic vesicles during down-regulation from the surfaces of interacting cells, implying an interaction consistent with their proposed roles as signal and receptor in cellular interactions during development.. ...

Recent data have expanded our understanding of Notch signalling by identifying a C2 domain at the N-terminus of Notch ligands, which has both lipid- and receptor-binding properties. We present novel structures of human ligands Jagged2 and Delta-like4 and human Notch2, together with functional assays, which suggest that ligand-mediated coupling of membrane recognition and Notch binding is likely to be critical in establishing the optimal context for Notch signalling. Comparisons between the Jagged and Delta family show a huge diversity in the structures of the loops at the apex of the C2 domain implicated in membrane recognition and Jagged1 missense mutations, which affect these loops and are associated with extrahepatic biliary atresia, lead to a loss of membrane recognition, but do not alter Notch binding. Taken together, these data suggest that C2 domain binding to membranes is an important element in tuning ligand-dependent Notch signalling in different physiological contexts.

The present study affirms our hypothesis that the Notch pathway plays an important role in macrophages, a key cell type in inflammation and atherosclerosis. Evidence that supports this idea includes the expression of multiple Notch receptors and ligands in human macrophages; markedly increased Dll4 expression in human macrophages stimulated with LPS, mmLDL, or IL-1β, an event that likely involves TLR4 and NF-κB; the ability of Dll4 to bind to macrophages and trigger Notch signaling; the induction of the MAPK, Akt, and NF-κB pathways in macrophages stimulated with Dll4; the Dll4-induced transcription of iNOS, PTX3, Id1, and Dll4 itself; and the presence of Notch pathway components, such as Dll4 and Notch3, in human atherosclerotic plaques rich in macrophages.. Dll4 expression induced in human primary macrophages by proinflammatory stimuli (LPS, IL-1β, and mmLDL) (Figures 2 through 4⇑⇑) and the detection of immunoreactive Dll4 in human atherosclerotic plaques (Figure 8) indicate possible ...

TY - JOUR. T1 - Sox9 mediates Notch1-induced mesenchymal features in lung adenocarcinoma. AU - Capaccione, Kathleen M.. AU - Hong, Xuehui. AU - Morgan, Katherine M.. AU - Liu, Wenyu. AU - Bishop, Michael J.. AU - Liu, Lian Xin. AU - Markert, Elke. AU - Deen, Malik. AU - Minerowicz, Christine. AU - Allen, Thaddeus. AU - Pine, Sharon R.. PY - 2014. Y1 - 2014. N2 - Sox9 has gained increasing importance both functionally and as a prognostic factor in cancer. We demonstrate a functional role for Sox9 in inducing a mesenchymal phenotype in lung ADC. We show that Sox9 mRNA and protein are overexpressed in lung ADC, particularly those with KRAS mutations. Sox9 expression correlated with the Notch target gene Hes1, and numerous other Notch pathway components. We observed that Sox9 is a potent inducer of lung cancer cell motility and invasion, and a negative regulator of E-cadherin, a key protein that is lost during epithelialmesenchymal transition (EMT). Moreover, we show that Notch1 signaling directly ...

Following injury, smooth muscle cells in the wall of the blood vessel can switch from a quiescent, contractile phenotype to a migratory, proliferative phenotype which contributes to lesion formation and vascular occlusive disease. Important regulators of vascular smooth muscle cell phenotype include serum response factor and its cofactor myocardin, growth factors, Krüppel-like factors, microRNA-143/145 and Notch signaling. The Notch signaling pathway is highly conserved and plays a critical role in both vascular development and disease. In mammals, there are five Notch ligands (Jagged1, Jagged2, Delta-like 1, Delta-like 3, and Delta-like 4) and four Notch receptors (Notch1-4). Knockout mouse models of most of the Notch ligand and receptor components display early embryonic lethality due to abnormal vasculogenesis, indicating their essential role in cardiovascular development. Following injury, the vascular remodeling response incorporates unique contributions from several of the Notch ligand and

NOTCH1 is known as an oncogenic or tumor suppressive gene in solid cancer. NOTCH1 mutations in oral squamous cell carcinoma (OSCC) frequently occur near the ligand-binding region. These mutations change the domain structure of this protein and affect the ligand binding activity. When NOTCH1 is activated by ligand binding, NOTCH1 intracellular domain (NICD) is cleaved from the cell membrane. This study investigated the functional change induced by a NOTCH1 mutation detected in OSCC clinical samples using stable transformant analysis. HEK293 cell lines expressing NOTCH1 wild-type (WT cells) or p.A465T NOTCH1 (A465T cells) were established. NOTCH1 expression was analyzed by flow cytometry, western blotting, and immunofluorescence using an anti-human NOTCH1 antibody. mRNA expression levels in WT and A465T cells were determined by quantitative real-time PCR (qPCR). Cell proliferation was analyzed by using cell growth assays and a xenograft tumor assay. Flow cytometry indicated that NOTCH1 expression ...

Here we report that Notch signaling controls gastric epithelial cell homeostasis by regulating antral stem cell function. Our lineage tracing studies in adult NIP1::CreERT2 mice showed that antral stem cells are actively signaling from the Notch1 receptor, thus demonstrating that the Notch pathway directly targets these cells under normal homeostatic conditions. Manipulation of Notch signaling showed that Notch functions to promote overall stem cell proliferation. Blocking Notch by pharmacologic or genetic means reduced stem cell proliferation, while genetic activation of Notch signaling in LGR5+ stem cells increased the number of proliferating stem cells. Expression of the Notch target gene Olfm4 paralleled the changes in stem cell proliferation, suggesting that it may be an antral stem cell marker, similar to what has been reported for LGR5+ stem cells in intestine (van der Flier et al, 2009; VanDussen et al, 2012). Analysis of gastric organoids demonstrated that Notch signaling is intrinsic ...

High expression of Notch-1 and/or Jagged-1 has negative prognostic significance in breast cancer ( 14, 15), and Notch-1 can transform HMECs ( 16). Our data confirm that Notch-1 and Notch-4 are commonly coexpressed in infiltrating breast cancers of ductal and lobular histologies, which also express Notch ligands Jagged-1 and Delta-1. However, our observations on breast cancer cell lines suggest that there may not be a simple correlation between protein levels of Notch receptor and ligands and Notch pathway activity level. Our data suggest that estrogen inhibits Notch signaling through an ERα-dependent effect, which is at least in part mediated by inhibition of Notch cleavage by γ-secretase. Inhibition of Notch activation by estrogen is observed under physiologic, ligand-induced Notch activation conditions, but it is independent of Notch ligands. Estradiol did not affect expression of Jagged-1, the most abundant Notch ligand in these cells (data not shown). The membrane accumulation of uncleaved ...

Specific functional consequences of mutated Notch receptors and ligands are largely not yet experimentally defined, but the clustering of mutations in known functional elements invites speculation with regard to whether the specific Notch receptor likely functions as a tumor suppressor or oncogene in a specific cancer type (Fig. 2).. More NOTCH1 gene mutations were observed than mutations in the other NOTCH receptor genes. This was in part, but not entirely, due to the greater number of tumors with NOTCH1 sequencing data. For HNSCC, lung SCC, and breast, NOTCH1 mutations were relatively frequent, with 5% to 15% of tumors harboring protein coding changes. Many of these missense mutations occurred at or near identified important domains such as the ligand-binding domain (EGF repeats 11 and 12) or the ankyrin domains (Fig. 2). Nonsense mutations observed in HNSCC would be predicted to result in truncated Notch1 proteins lacking domains important for transcription activation. The data suggest that ...

TY - JOUR. T1 - Identification of Deleterious NOTCH Mutation as Novel Predictor to Efficacious Immunotherapy in NSCLC. AU - Zhang, Kai. AU - Hong, Xiaohua. AU - Song, Zhengbo. AU - Xu, Yu. AU - Li, Chengcheng. AU - Wang, Guoqiang. AU - Zhang, Yuzi. AU - Zhao, Xiaochen. AU - Zhao, Zhengyi. AU - Zhao, Jing. AU - Huang, Mengli. AU - Huang, Depei. AU - Qi, Chuang. AU - Gao, Chan. AU - Cai, Shangli. AU - Gu, Feifei. AU - Hu, Yue. AU - Xu, Chunwei. AU - Wang, Wenxian. AU - Lou, Zhenkun. AU - Zhang, Yong. AU - Liu, Li. PY - 2020/7/15. Y1 - 2020/7/15. N2 - PURPOSE: NOTCH signaling is associated with tumorigenesis, mutagenesis, and immune tolerance in non-small cell lung cancer (NSCLC), indicating its association with the clinical benefit of immune checkpoint inhibitors (ICI). We hypothesized that NOTCH mutation in NSCLC might be a robust predictor of immunotherapeutic efficacy. EXPERIMENTAL DESIGN: Multiple-dimensional data including genomic, transcriptomic, and clinical data from cohorts of NSCLC ...

Clone REA1204 recognizes human Notch2, a member of the Notch receptor family. Upon binding its membrane-bound ligand of the Delta-like or Jagged family members (Jagged1, Jagged2, and Delta1), the Notch receptor undergoes two proteolytic cleavage steps eventually releasing the Notch intracellular domain (NICD), which subsequently translocates into the nucleus, where it activates transcription within a multiprotein complex. The Notch signaling is essential for the physiologic development of natural killer (NK) cells. Notch2 is expressed on various hematopetic cells, including T cell precursors in the thymus and activated peripheral T cells. In addition, the altered expression of Notch2 in various leukemias, lymphomas, and cancers has been reported.Additional information: Clone REA1204 displays negligible binding to Fc receptors. | Principat dAndorra

Clone REA1204 recognizes human Notch2, a member of the Notch receptor family. Upon binding its membrane-bound ligand of the Delta-like or Jagged family members (Jagged1, Jagged2, and Delta1), the Notch receptor undergoes two proteolytic cleavage steps eventually releasing the Notch intracellular domain (NICD), which subsequently translocates into the nucleus, where it activates transcription within a multiprotein complex. The Notch signaling is essential for the physiologic development of natural killer (NK) cells. Notch2 is expressed on various hematopetic cells, including T cell precursors in the thymus and activated peripheral T cells. In addition, the altered expression of Notch2 in various leukemias, lymphomas, and cancers has been reported.Additional information: Clone REA1204 displays negligible binding to Fc receptors. | Canada

Differentiation of arteries and veins is essential for the development of a functional circulatory system. In vertebrate embryos, genetic manipulation of Notch signaling has demonstrated the importance of this pathway in driving artery endothelial cell differentiation. However, when and where Notch activation occurs to affect endothelial cell fate is less clear. Using transgenic zebrafish bearing a Notch-responsive reporter, we demonstrate that Notch is activated in endothelial progenitors during vasculogenesis prior to blood vessel morphogenesis and is maintained in arterial endothelial cells throughout larval stages. Furthermore, we find that endothelial progenitors in which Notch is activated are committed to a dorsal aorta fate. Interestingly, some arterial endothelial cells subsequently downregulate Notch signaling and then contribute to veins during vascular remodeling. Lineage analysis, together with perturbation of both Notch receptor and ligand function, further suggests several distinct

Notch1 has been regarded as a fundamental regulator in tissue differentiation and stem cell properties. Recently, Notch1 mutations have been reported intensively both in solid tumors and in hematopoietic malignancies. However, little is known about the biological effect and the clinical implication of these reported mutations. Previously, we discovered several missense mutations in the Notch1 receptor in a Chinese population with oral squamous cell carcinoma (OSCC). We selected a hotspot mutation in the Abruptex domain (C1133Y). The expression of Notch1 was determined by western blot and real-time qPCR in OSCC cell lines transfected with pcDNA3.1-Notch1WT, pcDNA3.1-Notch1C1133Y, or pcDNA3.1 empty vector. CCK-8 assays were used to assess cell proliferation. Flow cytometry and western blot were used to confirm the alteration of cell cycle after transfection. Transwell assays and the detection of Epithelial-to-mesenchymal transition (EMT) markers were used to determine the invasive ability. The effects

TY - JOUR. T1 - Notch and NOXA-related pathways in melanoma cells.. AU - Nickoloff, Brian J.. AU - Hendrix, Mary J.C.. AU - Pollock, Pamela M.. AU - Trent, Jeffrey M.. AU - Miele, Lucio. AU - Qin, Jian Zhong. PY - 2005/11. Y1 - 2005/11. N2 - Notch receptor-mediated intracellular events represent an ancient cell signaling system, and alterations in Notch expression are associated with various malignancies in which Notch may function as an oncogene or less commonly as a tumor suppressor. Notch signaling regulates cell fate decisions in the epidermis, including influencing stem cell dynamics and growth/differentiation control of cells in skin. Because of increasing evidence that the Notch signaling network is deregulated in human malignancies, Notch receptors have become attractive targets for selective killing of malignant cells. Compared with proliferating normal human melanocytes, melanoma cell lines are characterized by markedly enhanced levels of activated Notch-1 receptor. By using a small ...

Notch intercellular communication instructs tissue-specific T-cell development and function. In this study, we explored the roles of dendritic cell (DC)-expressed Notch ligands in the regulation of T-cell effector function. We generated mice with CD11c lineage-specific deletion of Notch Delta-like ligand (Dll)1 and Jagged (Jag)2. Using these genetically-ablated mice and engineered pharmacological Notch ligand constructs, the roles of various Delta-like and Jagged ligands in the regulation of T-cell-mediated immunity were investigated. We assessed tumor growth, mouse survival, cytokine production, immunophenotyping of myeloid and lymphoid populations infiltrating the tumors, expression of checkpoint molecules and T-cell function in the experimental settings of murine lung and pancreatic tumors and cardiac allograft rejection. Correlative studies were also performed for the expression of NOTCH ligands, NOTCH receptors and PD-1 on various subsets of myeloid and lymphoid cells in tumor-infiltrating immune

ViraQuest Inc. , Uncategorized , MiR-34a targeting of Notch ligand delta-like 1 impairs CD15+/CD133+ tumor-propagating cells and supports neural differentiation in medulloblastoma ...

A recent study has shown that Cr-1 controls processing of the Nodal proprotein by recruiting proprotein convertases such as furin or PACE4 (Blanchet et al., 2008). Because processing by furin-like convertases (S1 cleavage) is also a prerequisite to generate mature heterodimerized Notch receptors (Logeat et al., 1998), we hypothesized that CR-1 may affect this processing step. Similar to the sequestration of the Nodal precursor protein into lipid rafts (Blanchet et al., 2008), forced expression of CR-1 in CR-1-deficient CHO cells enhanced the localization of the FL Notch1 protein in the lipid raft fraction in which glycosylphosphatidylinositol-anchored proteins such as CR-1 are enriched (Fig. 4 B). Furthermore, we assessed the effect of CR-1 expression on S1 cleavage of Notch1 in the presence of the γ-secretase inhibitor DAPT to exclude the effect on ligand-induced S3 cleavage (Fig. 4, C and D). CR-1 expression caused a dose-dependent increase in enhancement of the cleaved form of Notch1. This ...

Purpose: : To explore the role of Notch signaling in corneal endothelial-mesenchymal transformation (EnMT). Methods: : EnMT was induced in rat corneal endothelial cells (RCECs) by serial passages or TGF-β treatment, with or without 10µM DAPT. Cell phenotype and transformation were evaluated by EnMT markers, growth curve, scratch test, immunostaining and RT-qPCR. An in vivo EnMT rat model was induced by transcorneal freezing, with or without topical treatment of 50µM DAPT for 14 days. The wound endothelium was evaluated by slit lamp, stereomicroscope, immunostaining and RT-qPCR. Results: : Corneal EnMT in vitro was evidenced by changed morphology, downregulated tight junctions (ZO-1, Cx43 and N-cadherin), increased α-SMA and Notch signaling (Notch1, Notch2, Jag1 and Hes1). DAPT blocked EnMT induction, also reversed the phenotype, morphology and hyperplasia of the transformed RCECs. In rat, DAPT treatment blocked the EnMT process, with normal tight junction and suppressed α-SMA and Notch ...

Wnt signaling is important for T-cell differentiation at the early CD4(-)CD8(-) stage and is subsequently downregulated with maturation. To assess the importance of this downregulation, we generated a mouse line (R26-βcat) in which high levels of active β-catenin are maintained throughout T-cell development. Young R26-βcat mice show a differentiation block at the CD4(+)CD8(+) double-positive (DP) stage. These DP cells exhibit impaired apoptosis upon irradiation or dexamethasone treatment. All R26-βcat mice develop T-cell leukemias at 5 to 6 months of age. R26-βcat leukemias remain dependent on β-catenin function but lack Notch pathway activation. They exhibit recurrent secondary genomic rearrangements that lead to Myc overexpression and loss of Pten activity. Because β-catenin activation and Myc translocations were previously found in murine T-cell acute lymphoblastic leukemias (T-ALLs) deficient for Pten, our results suggest that activation of the canonical Wnt pathway is associated with a

The Notch signaling pathway is an evolutionarily conserved, intercellular signaling mechanism essential for proper embryonic development in all metazoan organisms in the Animal kingdom. The Notch proteins (Notch1-Notch4 in vertebrates) are single-pass receptors that are activated by the Delta (or Delta-like) and Jagged/Serrate families of membrane-bound ligands. They are transported to the plasma membrane as cleaved, but otherwise intact polypeptides. Interaction with ligand leads to two additional proteolytic cleavages that liberate the Notch intracellular domain (NICD) from the plasma membrane. The NICD translocates to the nucleus, where it forms a complex with the DNA binding protein CSL, displacing a histone deacetylase (HDAc)-co-repressor (CoR) complex from CSL. Components of an activation complex, such as MAML1 and histone acetyltransferases (HATs), are recruited to the NICD-CSL complex, leading to the transcriptional activation of Notch target genes ...

Notch and Angiopoietin-1 (Ang1)/Tie2 pathways are crucial for vascular maturation and stability. Here we identify the transcription factor ERG as a key regulator of endothelial Notch signalling. We show that ERG controls the balance between Notch ligands by driving Delta-like ligand 4 (Dll4) while repressing Jagged1 (Jag1) expression. In vivo, this regulation occurs selectively in the maturing plexus of the mouse developing retina, where Ang1/Tie2 signalling is active. We find that ERG mediates Ang1-dependent regulation of Notch ligands and is required for the stabilizing effects of Ang1 in vivo. We show that Ang1 induces ERG phosphorylation in a phosphoinositide 3-kinase (PI3K)/Akt-dependent manner, resulting in ERG enrichment at Dll4 promoter and multiple enhancers. Finally, we demonstrate that ERG directly interacts with Notch intracellular domain (NICD) and {beta}-catenin and is required for Ang1-dependent {beta}-catenin recruitment at the Dll4 locus. We propose that ERG coordinates Ang1, ...

One of the principal signaling mechanisms that control development of multicellular organisms is the Notch signaling pathway. The transmembrane Notch receptor, presented at the cell surface, interacts with ligand on neighbouring cells. Ligand interaction is followed by proteolytic processing, where cleavage by the gamma-secretase complex releases the intracellular (IC) part of the receptor. The Notch IC translocates to the nucleus, where it displaces corepressors from the DNA-binding protein CSL. Notch IC in complex with CSL, mastermind, p300, PCAF and other associated proteins activate transcription of target genes, including genes of the HES and HEY families. This thesis work is focused on two specific aspects of Notch signaling: i) processing of the receptor by the Á-secretase complex and ii) the mechanisms by which the IC functions as a transcriptional activator. The gamma-secretase complex, consisting of presenilin (PS), nicastrin, Aph-1 and Pen-2 proteins, processes not only the Notch ...

Maintaining the homeostasis of germinal zones in adult organs is a fundamental but mechanistically poorly understood process. In particular, what controls stem cell activation remains unclear. We have previously shown that Notch signaling limits neural stem cell (NSC) proliferation in the adult zebrafish pallium. Combining pharmacological and genetic manipulations, we demonstrate here that long-term Notch invalidation primarily induces NSC amplification through their activation from quiescence and increased occurrence of symmetric divisions. Expression analyses, morpholino-mediated invalidation and the generation of a notch3-null mutant directly implicate Notch3 in these effects. By contrast, abrogation of notch1b function results in the generation of neurons at the expense of the activated NSC state. Together, our results support a differential involvement of Notch receptors along the successive steps of NSC recruitment. They implicate Notch3 at the top of this hierarchy to gate NSC activation and

Signaling through the Notch1 receptor is essential for T cell development in the thymus. Stromal OP9 cells ectopically expressing the Notch ligand Delta-like1 mimic the thymic environment by inducing hemopoietic stem cells to undergo in vitro T cell development. Notch1 is also expressed on Pax5-/- pro-B cells, which are clonable lymphoid progenitors with a latent myeloid potential. In this study, we demonstrate that Pax5-/- progenitors efficiently differentiate in vitro into CD4+CD8+ alphabeta and gammadelta T cells upon coculture with OP9-Delta-like1 cells. In vitro T cell development of Pax5-/- progenitors strictly depends on Notch1 function and progresses through normal developmental stages by expressing T cell markers and rearranging TCRbeta, gamma, and delta loci in the correct temporal sequence. Notch-stimulated Pax5-/- progenitors efficiently down-regulate the expression of B cell-specific genes, consistent with a role of Notch1 in preventing B lymphopoiesis in the thymus. At the same ...

TY - JOUR. T1 - Structure and stability of the ankyrin domain of the Drosophila Notch receptor. AU - Zweifel, Mark E.. AU - Leahy, Daniel J.. AU - Hughson, Frederick M.. AU - Barrick, Doug. PY - 2003/11. Y1 - 2003/11. N2 - The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 Å resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue ...

TY - JOUR. T1 - Association of Transcription Factor YY1 with the High Molecular Weight Notch Complex Suppresses the Transactivation Activity of Notch. AU - Tsai, Tian-Shun. AU - Lin, Yu Min. AU - Hsieh, Rong-Hong. AU - Tseng, Min Jen. PY - 2003/10/24. Y1 - 2003/10/24. N2 - Notch receptors are evolutionarily conserved from Drosophila to human and play important roles in cell late decisions. After ligand binding, Notch receptors are cleaved to release their intracellular domains. The intracellular domains, the activated form of Notch receptors, are then translocated into the nucleus where they interact with other transcriptional machinery to regulate the expression of cellular genes. To dissect the molecular mechanisms of Notch signaling, the cellular targets that interact with Notch1 receptor intracellular domain (N1IC) were screened. In this study, we found that endogenous transcription factor Ying Yang 1 (YY1) was associated with exogenous N1IC in human K562 erythroleukemic cells. The ankyrin ...

The Notch signaling pathway plays a substantial role on human NK cell development, however the role of Notch on KIR upregulation and acquisition of effector function has not been explored. To evaluate how Notch influences terminal differentiation, cord blood derived NK cells or sorted KIR- peripheral blood NK cells were cultured with IL-15 for 7 days in the presence or absence of gamma-secretase inhibitor, known to inhibit Notch signaling. Inhibition of Notch signaling significantly decreased KIR expression. Conversely, co-culturing the same cells with OP9 cells bearing Notch ligands enhanced KIR expression. Overexpression of the active portion of Notch on cord blood derived NK cells after 28 days of culture resulted in a 2-fold increase in KIR expression indicating that Notch signaling plays a direct, cell intrinsic, role in KIR regulation. By knocking down delta-like 1 (DLL1) on NK cells and co-culturing them with OP9 cells expressing Notch ligands we show that DLL1 mediated cis-inhibition ...

Electroacupuncture (EA) pretreatment can induce the tolerance against focal cerebral ischemia. However, the underlying mechanisms have not been fully understood. Emerging evidences suggest that canonical Notch signaling may be involved in ischemic brain injury. In the present study, we tested the hypothesis that EA pretreatment-induced tolerance against focal cerebral ischemia is mediated by Notch signaling. EA pretreatment significantly enhanced Notch1, Notch4 and Jag1 gene transcriptions in the striatum, except Notch1 intracellular domain level, which could be increased evidently by ischemia. After ischemia and reperfusion, Hes1 mRNA and Notch1 intracellular domain level in ischemic striatum in EA pretreatment group were increased and reached the peak at 2 h and 24 h, respectively, which were both earlier than the peak achieved in control group. Intraventricular injection with the γ-secretase inhibitor MW167 attenuated the neuroprotective effect of EA pretreatment. EA pretreatment induces the

The homeobox gene TLX1 (for T-cell leukemia homeobox 1, previously known as HOX11) is inappropriately expressed in a major subgroup of T cell acute lymphoblastic leukemia (T-ALL) where it is strongly associated with activating NOTCH1 mutations. Despite the recognition that these genetic lesions cooperate in leukemogenesis, there have been no mechanistic studies addressing how TLX1 and NOTCH1 functionally interact to promote the leukemic phenotype. Global gene expression profiling after downregulation of TLX1 and inhibition of the NOTCH pathway in ALL-SIL cells revealed that TLX1 synergistically regulated more than 60% of the NOTCH-responsive genes. Structure-function analysis demonstrated that TLX1 binding to Groucho-related TLE corepressors was necessary for maximal transcriptional regulation of the NOTCH-responsive genes tested, implicating TLX1 modulation of the NOTCH-TLE regulatory network. Comparison of the dataset to publicly available biological databases indicated that the TLX1/NOTCH-coregulated

Thymus function depends on the epithelial compartment of the thymic stroma. Cortical thymic epithelial cells (cTECs) regulate T cell lineage commitment and positive selection, while medullary (m) TECs impose central tolerance on the T cell repertoire. During thymus organogenesis, these functionally distinct sub-lineages are thought to arise from a common thymic epithelial progenitor cell (TEPC). The mechanisms controlling cTEC and mTEC production from the common TEPC are not however understood. Here, we show that emergence of the earliest mTEC lineage-restricted progenitors requires active NOTCH signaling in progenitor TEC and that, once specified, further mTEC development is NOTCH-independent. In addition, we demonstrate that persistent NOTCH activity favors maintenance of undifferentiated TEPC at the expense of cTEC differentiation. Finally, we uncover a cross-regulatory relationship between NOTCH and FOXN1, a master regulator of TEC differentiation. These data establish NOTCH as a potent ...

Several studies have revealed that endosomal sorting controls the steady-state levels of Notch at the cell surface in normal cells and prevents its inappropriate activation in the absence of ligands. However, whether this highly dynamic physiologic process can be exploited to counteract dysregulated Notch signaling in cancer cells remains unknown. T-ALL is a malignancy characterized by aberrant Notch signaling, sustained by activating mutations in Notch1 as well as overexpression of Notch3, a Notch paralog physiologically subjected to lysosome-dependent degradation in human cancer cells. Here we show that treatment with the pan-HDAC inhibitor Trichostatin A (TSA) strongly decreases Notch3 full-length protein levels in T-ALL cell lines and primary human T-ALL cells xenografted in mice without substantially reducing NOTCH3 mRNA levels. Moreover, TSA markedly reduced the levels of Notch target genes, including pT alpha, CR2, and DTX-1, and induced apoptosis of T-ALL cells. We further observed that ...

The current study identifies a potential interaction between the FA pathway and Notch signaling in HSC differentiation and establishes a role of FA proteins in the control of balance between renewal and lineage commitment. There are several findings that highlight the significance of our study: 1) loss of murine FA proteins results in enhanced Notch signaling in MPPs, which is correlated with decreased phenotypic and functional LT-HSCs and increased formation of MPP1 progenitors; 2) deletion of the Fanca or Fancc gene deregulates genes in the Notch signaling and the NF-κB pathway; 3) TNF-α stimulation enhances Notch signaling in Fanca−/− and Fancc−/− LSK cells, leading to decreased HSC quiescence and compromised HSC self-renewal; 4) inflammation-activated Notch target gene expression in Fanca−/− and Fancc−/− MPP cells requires NF-κB; 5) genetic ablation or pharmacologic inhibition of NF-κB reduces Notch signaling in FA MPPs to near WT levels and significantly increases ...

Osteoclastogenesis plays an important role in the bone erosion of rheumatoid arthritis (RA). Recently, Notch receptors have been implicated in the development of osteoclasts. However, the responsible Notch ligands have not been identified yet. This study was undertaken to determine the role of individual Notch receptors and ligands in osteoclastogenesis. Mouse bone marrow-derived macrophages or human peripheral blood monocytes were used as osteoclast precursors and cultured with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) to induce osteoclasts. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining. K/BxN serum-induced arthritic mice and ovariectomized mice were treated with anti-mouse Delta-like 1 (Dll1) blocking monoclonal antibody (mAb). Blockade of a Notch ligand Dll1 with mAb inhibited osteoclastogenesis and, conversely, immobilized Dll1-Fc fusion protein enhanced it in both mice and humans. In contrast,

Abstract Background Hand, foot and mouth disease (HFMD), a virus-induced infectious disease that usually affects infants and children, has an increased incidence in China in recent years. This study attempted to investigate the role of the Notch signaling pathway in the pathogenesis of HFMD. Methods Eighty-two children diagnosed with HFMD were enrolled into this study. The HFMD group was further divided into the uncomplicated HFMD and HFMD with encephalitis groups. The control group included 40 children who underwent elective surgery for treatment of inguinal hernias. Results Children with HFMD displayed significantly reduced CD3+, CD3+CD4+ and CD3+CD8+ cell subsets, but substantially enhanced CD3−CD19+ cell subset (p , 0.05 versus control subjects). The expression levels of Notch ligands Dll1 and Dll4 in the peripheral blood of the HFMD group were significantly higher than those in the control group (p , 0.05). There were statistically significant differences in CD3+, CD3+CD4+ and CD3−CD19+ ...

The Notch signaling pathway is an evolutionarily conserved, intercellular signaling mechanism essential for proper embryonic development in all metazoan organisms in the Animal kingdom. The Notch proteins (Notch1-Notch4 in vertebrates) are single-pass receptors that are activated by the Delta (or Delta-like) and Jagged/Serrate families of membrane-bound ligands. They are transported to the plasma membrane as cleaved, but otherwise intact polypeptides. Interaction with ligand leads to two additional proteolytic cleavages that liberate the Notch intracellular domain (NICD) from the plasma membrane. The NICD translocates to the nucleus, where it forms a complex with the DNA binding protein CSL, displacing a histone deacetylase (HDAc)-co-repressor (CoR) complex from CSL. Components of an activation complex, such as MAML1 and histone acetyltransferases (HATs), are recruited to the NICD-CSL complex, leading to the transcriptional activation of Notch target genes ...

Next generation sequencing was used to identify Notch mutations in a large collection of diverse solid tumors. NOTCH1 and NOTCH2 rearrangements leading to constitutive receptor activation were confined to triple negative breast cancers (TNBC, 6 of 66 tumors). TNBC cell lines with NOTCH1 rearrangements associated with high levels of activated NOTCH1 (N1-ICD) were sensitive to the gamma-secretase inhibitor (GSI) MRK-003, both alone and in combination with pacitaxel, in vitro and in vivo, whereas cell lines with NOTCH2 rearrangements were resistant to GSI. Immunohistochemical staining of N1-ICD in TNBC xenografts correlated with responsiveness, and expression levels of the direct Notch target gene HES4 correlated with outcome in TNBC patients. Activating NOTCH1 point mutations were also identified in other solid tumors, including adenoid cystic carcinoma (ACC). Notably, ACC primary tumor xenografts with activating NOTCH1 mutations and high N1-ICD levels were sensitive to GSI, whereas N1-ICD-low ...

The Notch signalling pathway is important in development and differentiation of a diverse range of both embryonic and adult tissues. There is now strong evidence implicating aberrant Notch signalling in the pathogenesis of T-cell acute lymphoblastic leukaemia (T-ALL), with over 50% of paediatric patients having activating mutations in NOTCH-1. This thesis aims to explore several aspects of the Notch pathway in both T-ALL and acute myeloid leukaemia (AML). Chapter one summarises the published data on the Notch signalling pathway itself, addressing the basic understanding of Notch activation through cell-to-cell interaction, as well as the mechanisms through which it is regulated. The role that Notch signaling plays in normal haematopoiesis is also discussed. Chapter three addresses the incidence and characteristics of NOTCH-1 mutations in a cohort of adult patients with T-ALL in comparison to the published study of paediatric T-ALL, as well as in a cohort of patients with infantile leukaemia and ...

NOTCH1 is frequently mutated in adenoid cystic carcinoma (ACC). To test the idea that immunohistochemical (IHC) staining can identify ACCs with NOTCH1 mutations, we performed IHC for activated NOTCH1 (NICD1) in 197 cases diagnosed as ACC from 173 patients. NICD1 staining was positive in 194 cases (98%) in 2 major patterns: subset positivity, which correlated with tubular/cribriform histology; and diffuse positivity, which correlated with a solid histology. To determine the relationship between NICD1 staining and NOTCH1 mutational status, targeted exome sequencing data were obtained on 14 diffusely NICD1-positive ACC specimens from 11 patients and 15 subset NICD1-positive ACC specimens from 15 patients. This revealed NOTCH1 gain-of-function mutations in 11 of 14 diffusely NICD1-positive ACC specimens, whereas all subset-positive tumors had wild-type NOTCH1 alleles. Notably, tumors with diffuse NICD1 positivity were associated with significantly worse outcomes (P=0.003). To determine whether ...

TY - JOUR. T1 - The Notch inhibitor cowanin accelerates nicastrin degradation. AU - Arai, Midori A.. AU - Akamine, Ryuta. AU - Tsuchiya, Anna. AU - Yoneyama, Tatsuro. AU - Koyano, Takashi. AU - Kowithayakorn, Thaworn. AU - Ishibashi, Masami. N1 - Funding Information: This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), Strategic Priority Research Promotion Program, Chiba University, Phytochemical Plant Molecular Sciences, and by a Workshop on Chirality at Chiba University (WCCU). This work was inspired by the international and interdisciplinary environments of the JSPS Core-to-Core Program, Asian Chemical Biology Initiative. Publisher Copyright: © 2018 The Author(s).. PY - 2018/12/1. Y1 - 2018/12/1. N2 - Aberrant activation of Notch signaling contributes to the pathogenesis of several different types of cancer, and Notch pathway inhibitors may have significant therapeutic potential. Using a unique cell-based assay ...

BACKGROUND: The epidermal growth factor receptor (EGFR) and Notch signaling pathways have been implicated in self-renewal of normal breast stem cells. We investigated the involvement of these signaling pathways in ductal carcinoma in situ (DCIS) of the breast. METHODS: Samples of normal breast tissue (n = 15), pure DCIS tissue of varying grades (n = 35), and DCIS tissue surrounding an invasive cancer (n = 7) were used for nonadherent (i.e., mammosphere) culture. Mammosphere cultures were treated at day 0 with gefitinib (an EGFR inhibitor), DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) (a gamma-secretase inhibitor), or Notch 4-neutralizing antibody. Mammosphere-forming efficiency (MFE) was calculated by dividing the number of mammospheres of 60 microm or more formed by the number of single cells seeded and is expressed as a percentage. The Notch 1 intracellular domain (NICD) was detected immunohistochemically in paraffin-embedded DCIS tissue from 50 patients with at least

We show here that the Notch3-RBPJκ signaling pathway is necessary to maintain both the differentiated state of VSMCs and the integrity of the coronary vasculature via the regulation of the VEGFA/VEGFR2/DLL4 pathway, in response to pressure overload. These results are paramount because this is the demonstration of a direct association between a primary alteration of the coronary vasculature and the development of chronic and acute decompensated HF, besides myocardial infarction.. Notch3 loss-of-function was previously shown to be protective in a monocrotaline-induced pulmonary hypertension.16 In this model, proliferation of VSMCs in small pulmonary arteries leads to a detrimental increase in pulmonary artery resistance, which is dependent on Notch3 signaling. Because Notch3−/− mice subjected to angII-induced arterial hypertension exhibited a lower increase in blood pressure than their WT counterparts,12 one may anticipate a similar benefit of the loss of Notch3 signaling in hypertension. To ...

TY - JOUR. T1 - Notch signaling mediates hypoxia-induced tumor cell migration and invasion. AU - Sahlgren, Cecilia. AU - Gustafsson, Maria V.. AU - Jin, Shaobo. AU - Poellinger, Lorenz. AU - Lendahl, Urban. N1 - Copyright: Copyright 2008 Elsevier B.V., All rights reserved.. PY - 2008/4/29. Y1 - 2008/4/29. N2 - Tumor hypoxia is linked to increased metastatic potential, but the molecular mechanisms coupling hypoxia to metastasis are poorly understood. Here, we show that Notch signaling is required to convert the hypoxic stimulus into epithelial-mesenchymal transition (EMT), increased motility, and invasiveness. Inhibition of Notch signaling abrogated hypoxia-induced EMT and invasion, and, conversely, an activated form of Notch could substitute for hypoxia to induce these processes. Notch signaling deploys two distinct mechanisms that act in synergy to control the expression of Snail-1, a critical regulator of EMT. First, Notch directly upregulated Snail-1 expression by recruitment of the Notch ...

The Notch pathway plays diverse and complex roles in cell signaling during development. In the mammalian ovary, Notch is important for the initial formation and growth of follicles, and for regulating the proliferation and differentiation of follicular granulosa cells during the periovulatory period. This study seeks to determine the contribution of female germ cells toward the initial activation and subsequent maintenance of Notch signaling within somatic granulosa cells of the ovary. To address this issue, transgenic Notch reporter (TNR) mice were crossed with Sohlh1-mCherry (S1CF) transgenic mice to visualize Notch-active cells (EGFP) and germ cells (mCherry) simultaneously in the neonatal ovary. To test the involvement of oocytes in activation of Notch signaling in ovarian somatic cells, we ablated germ cells using busulfan, a chemotherapeutic alkylating agent, or investigated KitWv/Wv (viable dominant white-spotting) mice that lack most germ cells. The data reveal that Notch pathway ...

Organ morphogenesis depends on the precise orchestration of cell migration, cell fate specification and cell shape chages. Results in this thesis demonstrate that Notch signaling is an integral part of the feedback loop between Wnt and Fgf signaling that underlies the self‐organization of rosette‐shaped sensory organs in the zebrafish lateral line system. Notch cell autonomously induces apical constriction and cell adhesion downstream of Fgf signaling and organizes lateral line organs into rosettes independent of patterning cues normally provided by a Wnt/Fgf signaling system. We also show that the ectopic Notch signaling induces larger organs independently of proliferation and the Hippo pathway. Transplantation and RNASeq analyses revealed that Notch signaling induces cell adhesion and tight junction proteins that interact with cytoskeleton causing cells to self‐organize into fewer larger organs rather than several smaller ones. Thus, Notch plays an essential role in coordinating ...

Glioblastomas co-opt stem cell regulatory pathways to maintain brain tumor-initiating cells (BTICs), also known as cancer stem cells. NOTCH signaling has been a molecular target in BTICs, but NOTCH antagonists have demonstrated limited efficacy in clinical trials. Recombining binding protein suppressor of hairless (RBPJ) is considered a central transcriptional mediator of NOTCH activity. Here, we report that pharmacologic NOTCH inhibitors were less effective than targeting RBPJ in suppressing tumor growth. While NOTCH inhibitors decreased canonical NOTCH gene expression, RBPJ regulated a distinct profile of genes critical to BTIC stemness and cell cycle progression. RBPJ was preferentially expressed by BTICs and required for BTIC self-renewal and tumor growth. MYC, a key BTIC regulator, bound the ...

Vascular development and angiogenesis initially depend about endothelial tip cell invasion, which is definitely followed by a series of maturation steps, including lumen formation and recruitment of perivascular cells. secreted healthy proteins, and growth factors coordinately regulate angiogenesis and boat stabilization, the Notch signaling pathway is definitely unique in that it is definitely involved at multiple phases of angiogenesis, from initial vascular plexus formation and artery/vein patterning, to vascular clean muscle mass cell (VSMC) recruitment and vascular redesigning. Notch signaling, which is definitely characterized by heterotypic cell-cell relationships between Notch-ligand and -receptor articulating cells, represents an evolutionarily conserved mechanism that is definitely known to become important for cell-fate decisions during embryogenesis and, more recently, angiogenesis. Mammals communicate 4 Notch receptors, Notch1-4, and 5 Notch ligands, Delta-like ligand (Dll) 1, Dll3, ...

Lysophosphatidic acid (LPA) is a potent lipid mediator with various biological functions mediated through six G protein-coupled receptors (GPCRs), LPA1-LPA6. Previous studies have demonstrated that LPA-Gα12/Gα13 signaling plays an important role in embryonic vascular development. However, the responsible LPA receptors and underlying mechanisms are poorly understood. Here, we show a critical role of LPA4 and LPA6 in developmental angiogenesis. In mice, Lpa4;Lpa6 double-knockout (DKO) embryos were lethal due to global vascular deficiencies, and endothelial cell-specific (EC-specific) Lpa4;Lpa6-DKO retinas had impaired sprouting angiogenesis. Mechanistically, LPA activated the transcriptional regulators YAP and TAZ through LPA4/LPA6-mediated Gα12/Gα13-Rho-ROCK signaling in ECs. YAP/TAZ knockdown increased endothelial expression of the Notch ligand delta-like ligand 4 (DLL4) that was mediated by β-catenin and Notch intracellular domain (NICD). Fibrin gel sprouting assay revealed that LPA4/LPA6, ...

A Phase 2, Open-Label, Multi-centre Study of AL101 in Patients With Adenoid Cystic Carcinoma (ACC) Bearing Activating Notch Mutations

Intracranial aneurysm (IA) incidence is about 1~2%. However, the specific mechanisms of IA onset and development need further study. Our objective was to discover novel IA-related genes to determine possible etiologies further. We performed next-generation sequencing on nineteen Chinese patients with familial IA and one patient with sporadic IA. We obtained mRNA expression data of 129 samples from Gene Expression Omnibus (GEO) and made statistical computing to discover differentially expressed genes (DEGs). The screened IA-related gene NOTCH3 was determined by bioinformatic data mining. We verified the IA-related indicators of NOTCH3. Association was found between IA and the NOTCH3 SNPs rs779314594, rs200504060 and rs2285981. Levels of NOTCH3 mRNA were lower in IA tissue than in control tissue, but higher in peripheral blood neutrophils from IA patients than in neutrophils from controls. Levels of NOTCH3 protein were lower in IA tissue than in cerebral artery tissue. NOTCH3 also

Methods and Results-Using Doppler echocardiography, histology, immunohistochemistry, quantitative gene expression analysis, and cell culture assays, we examined the effect of a hypercholesterolemic diet supplemented with vitamin D on mice heterozygous for null mutations in the Notch1 receptor or the effector transcription factor gene RBPJk. After 16 weeks on the hyperlipidemic diet, calcific aortic disease was detected in heterozygous RBPJk mice. Analysis of valve leaflets revealed macrophage infiltration, enhanced collagen deposition, proosteogenic protein expression, and calcification. Heterozygous null Notch1 mice displayed milder histopathologic changes and did not develop any significant hemodynamic disturbance. Valvular disease correlated with reduced expression of the Notch target gene Hey1 in valves of RBPJk heterozygous mice fed the hyperlipidemic diet. Consistent with the in vivo data, Notch signaling inhibition in porcine valve interstitial cells led to downregulation of HEY1 ...

Angiogenesis is a coordinated process tightly regulated by the balance between Delta-like-4 (DLL4) and Jagged-1 (JAG1) in endothelial cells. Here we show that galectin-3 (gal-3), a glycan-binding protein secreted by cancer cells under hypoxic conditions, triggers sprouting angiogenesis, assisted by hypoxic changes in the glycosylation status of endothelial cells that enhance binding to gal-3. Galectin-3s proangiogenic functions were found to be predominantly dependent on the Notch ligand JAG1. Differential direct binding to JAG1 was shown by surface plasmon resonance assay. Upon binding to Notch ligands, gal-3 preferentially increased JAG1 protein half-life over DLL4 and preferentially activated JAG1/Notch-1 signaling in endothelial cells. JAG1 overexpression in Lewis lung carcinoma cells accelerated tumor growth in vivo, but this effect was prevented in Lgals3-/- mice. Our findings establish gal-3 as a molecular regulator of the JAG1/Notch-1 signaling pathway and have direct implications for the

Figure 4. Regulation of Notch1 by Fyn and Srcasm. A, Fyn downregulates Notch1 transcript levels. qRT-PCR was done on cDNA from FVB/N F1 hybrid, K14-Fyn Y528F F1 hybrid SCIS, and SCCs from Fyn Y528F and Fyn Y528F/SrcasmDN mice to determine Notch1 transcript levels. Three biopsies of each type were analyzed. Normalized mean and SD are shown. *, P , 0.01, compared with FVB. B, Fyn downregulates levels of Srcasm transcript. qRT-PCR was done on cDNA from FVB/N skin, K14-Fyn Y528F FVB/N F1 SCIS, and K14-Fyn Y528F FVB/N F1 SCC to determine Srcasm transcript levels as in A. *, P = 0.041; **, P = 0.029, compared with FVB. C, Srcasm regulates Fyn-dependent Notch inhibition and phospho-STAT3 phosphorylation. Protein lysates from C57BL/6 and FVB/N F1 hybrid skin and SCIS or SCC lesions from FVB/N F1 hybrid lines were subjected to Western blotting with the indicated antibodies; pY416-activated SFKs. Fyn/Srcasm1 exhibited a weak phenotype; Fyn/Srcasm2 exhibited no phenotype. D, SrcasmDN does not revert ...

TY - JOUR. T1 - Turn it down a notch. AU - Carrieri, Francesca. AU - Dale, Jacqueline Kim. N1 - This work was supported by a Wellcome Trust PhD studentship to FAC and a Wellcome Trust Strategic award [097945/Z/11/Z]. PY - 2017/1/18. Y1 - 2017/1/18. N2 - In the developing vertebrate embryo, segmentation initiates very early and in a species-specific manner through the formation of repeated segments,termed somites, that form on either side of the neural tube along the anterior to posterior axis. The periodicity of somite formation is regulated by a molecular oscillator, called the segmentation clock, driving cyclic gene expression in the unsegmented paraxial mesoderm, from which somites derive. Three signalling pathways have been proposed to underlie the molecular mechanism of the oscillator: Wnt, Fgf and Notch. In particular, the Notch signalling pathway has been demonstrated to be an essential piece in the intricate somitogenesis regulation puzzle. Notch is required to synchronize oscillations ...

This proposal describes the approach that Zeger-Abrams Inc. (ZA) plans to use to implement prototype models (one each) of a Low-Band (designated as 30-600 MHz) and Mid-Band (designated as 400-4,000 MHz) Cosite Notch Filter (CNF). Each CNF will filter out selected narrow spectral portions from the output of a jammer power amplifier while allowing it to pass the rest of its designated frequency range with very low loss. Each CNF will produce four such notches that are independently tunable with center frequency accurately located at the frequencies of friendly force receivers and with accurately selectable notch widths. The Low-Band notch width will be selectable from 45 kHz to 8 MHz. The Mid-Band notch width will be selectable from 1 MHz to 8 MHz ...

Definition of Acetabular notch in the Financial Dictionary - by Free online English dictionary and encyclopedia. What is Acetabular notch? Meaning of Acetabular notch as a finance term. What does Acetabular notch mean in finance?

Notch regulation of myogenic versus endothelial fates of cells that migrate from the somite to the limb[1] Multipotent Pax3-positive (Pax3(+)) cells in the somites give rise to skeletal muscle and to cells of the vasculature. We had previously proposed that this cell-fate choice depends on the equilibrium between Pax3 and Foxc2 expression. In this study, we report that the Notch pathway promotes vascular versus skeletal muscle cell fates. ...We now demonstrate that in addition to the inhibitory role of Notch signaling on skeletal muscle cell differentiation, the Notch pathway affects the Pax3:Foxc2 balance and promotes the endothelial versus myogenic cell fate, before migration to the limb, in multipotent Pax3(+) cells in the somite of the mouse embryo. Limb Development , Notch ...

BACKGROUND: notch receptors are critical determinants of cell fate in a variety of organisms. Notch signaling is involved in the chondrogenic specification of neural crest cells. Aberrant Notch activity has been implicated in numerous human diseases including cancers; however its role in chondrogenic tumors has not been clarified. METHOD: tissue samples from a case of primary chondrosarcoma of the maxilla and its recurrent tumor were examined immunohistochemically for Notch1-4 and their ligands (Jagged1, Jagged2 and Delta1) expression. RESULTS: both primary and recurrent tumors were histopathologically diagnosed as conventional hyaline chondrosarcoma (WHO Grade I). Hypercellular tumor areas strongly expressed Notch3 and Jagged1 in spindle and pleomorphic cells suggesting up-regulation of these protein molecules at sites of tumor proliferation. Expression patterns were distinct with some overlap. Differentiated malignant and atypical chondrocytes demonstrated variable expression levels of ...

A regulated expression of 394 known genes and 295 ESTs was tedected. The sensitivity and realiability of detection by microarrays was shown by confirming the expression pattern for 20 genes by radioactive quantitative RT-PCR. Extensive functional classification of regulated genes was performed. The most interesting finding was concomitant activation of TGF-β, Wnt and Notch signaling pathways, confirmed by strong upregulation of their target genes by PCR. The TGF-β pathway is activated by stimulated production of the growth factor itself, while the exact mechanism of Wnt and Notch activation remains elusive. We showed BMP-2 stimulated expression of Hey1, a direct Notch target gene, in mouse MC3T3 and C2C12 cells, in human mesenchymal cells and in mouse calvaria. Small interfering RNA-mediated inhibition of Hey1 induction led to an increase in osteoblast matrix mineralization, suggesting that Hey1 is a negative regulator of osteoblast maturation. This negative regulation is apparently achieved ...

Diabetic, non-healing wounds are a major clinical problem with considerable morbidity and associated financial costs. However, mechanisms by which diabetes impedes tissue repair mechanisms remain unclear. Previous studies have suggested decreased tissue levels of growth factors, including keratinocyte growth factor, VEGF, PDGF, excess protease activity, decreased angiogenesis, altered inflammation, or an increased microbial load as possible contributing factors for the impaired wound healing observed in diabetes mellitus (Galkowska et al, 2006; Brem & Tomic-Canic, 2007; Grice et al, 2010; Gardner et al, 2013; Eming et al, 2014; Pastar et al, 2014; Lindley et al, 2016; Quinn et al, 2016; Ramirez et al, 2018). In this study, we discovered that the Notch pathway activity is elevated in fibroblasts of human diabetic ulcers and diabetic murine wounds, but not in normal murine acute wounds and non-diabetic ischemic wounds. Furthermore, we uncovered the Notch1 pathway as an important molecular ...

INTRODUCTION: The liver is a multi-purposeful organ, susceptible to injury. Following acute injury, cells such as hepatocytes, regenerate, while latent hepatic progenitor cell (HPC) become active after chronic liver injury. Some studies suggest Fibroblast growth factors (FGF) may activate HPCs similarly to embryonic liver stem cells. In addition, Notch signaling is critical in bile duct development and injury. With progenitor cell transplantation as an alternative to tissue transplantation, we hypothesize that FGF and Notch signaling pathways induce proliferation of HPC after chronic liver injury.; MATERIALS AND METHODS: Wild-type mice were fed either regular or 0.1% 3,5-diethoxycarbonyl dihydrocollidine (DDC) chow to induce injury. Transgenic, dominant-negative RosartTA;tet(o)sFGFR2b+/- mice were given doxycycline 2 days prior and throughout DDC injury. mRNA and immunostaining of FGF and Notch receptors and ligands were performed. Cells isolated from transgenic mice were fluoresecently sorted ...

Gamma-secretase activity is vital for the transmembrane cleavage of Notch receptors and the subsequent migration of their intracellular domains to the nucleus. Notch overexpression has been associated with breast, colon, cervical and prostate cancers. We tested the effect of three different gamma-secretase inhibitors (GSIs) in breast cancer cells. One inhibitor (GSI1) was lethal to breast cancer cell lines at concentrations of 2 muM and above but had a minimal effect on the non-malignant breast lines. GSI1 was also cytotoxic for a wide variety of cancer cell lines in the NCI60 cell screen. GSI1 treatment resulted in a marked decrease in gamma-secretase activity and downregulation of the Notch signalling pathway with no effects on expression of the gamma-secretase components or ligands. Flow cytometric and western blot analyses indicated that GSI1 induces a G2/M arrest leading to apoptosis, through downregulation of Bcl-2, Bax and Bcl-XL. GSI1 also inhibited proteasome activity. Thus, the ...

Developmental patterning requires juxtacrine signaling in order to tightly coordinate the fates of neighboring cells. Recent work has shown that Notch and Delta, the canonical metazoan juxtacrine signaling receptor and ligand, mutually inactivate each other in the same cell. This cis-interaction generates mutually exclusive sending and receiving states in individual cells. It generally remains unclear, however, how this mutual inactivation and the resulting switching behavior can impact developmental patterning circuits. Here we address this question using mathematical modeling in the context of two canonical pattern formation processes: boundary formation and lateral inhibition. For boundary formation, in a model motivated by Drosophila wing vein patterning, we find that mutual inactivation allows sharp boundary formation across a broader range of parameters than models lacking mutual inactivation. This model with mutual inactivation also exhibits robustness to correlated gene expression ...

The Notch signaling pathway is one of the main signaling pathways that mediates direct contact between cells, and is essential for normal development. It regulates various cellular processes, including cell proliferation, apoptosis, migration, invasion, angiogenesis and metastasis. It additionally serves an important function in tumor progression. Non‑coding RNAs mainly include small microRNAs, long non‑coding RNAs and circular RNAs. At present, a large body of literature supports the biological significance of non‑coding RNAs in tumor progression. It is also becoming increasingly evident that cross‑talk exists between Notch signaling and non‑coding RNAs. The present review summarizes the current knowledge of Notch‑mediated gastrointestinal cancer cell processes, and the effect of the crosstalk between the three major types of non‑coding RNAs and the Notch signaling pathway on the fate of gastrointestinal cancer cells ...

Neurogenesis is restricted in the adult mammalian brain; most neurons are neither exchanged during normal life nor replaced in pathological situations. We report that stroke elicits a latent neurogenic program in striatal astrocytes in mice. Notch1 signaling is reduced in astrocytes after stroke, and attenuated Notch1 signaling is necessary for neurogenesis by striatal astrocytes. Blocking Notch signaling triggers astrocytes in the striatum and the medial cortex to enter a neurogenic program, even in the absence of stroke, resulting in 850 ± 210 (mean ± SEM) new neurons in a mouse striatum. Thus, under Notch signaling regulation, astrocytes in the adult mouse brain parenchyma carry a latent neurogenic program that may potentially be useful for neuronal replacement strategies. ...

Many studies have aimed to clarify the relationship between Notch1 signaling and papillary thyroid carcinoma (PTC), but the results have been inconsistent to date. In the present study, a systematic review and meta-analysis were performed to analyze the relationship between Notch1 signaling and the clinical characteristics of PTC. Literature databases, including PubMed (Medline), Embase and China National Knowledge Infrastructure, were searched for relevant studies from inception to April 2018. A total of five studies, including 421 patients with PTC from China and South Korea, were included in the meta-analysis. The results revealed that the upregulation of Notch1 signaling was positively correlated with lymph node metastasis in patients with PTC (OR = 3.25, 95% CI 1.14-9.23, P = 0.03). Additionally, positive correlations were found between Notch1 signaling and tumor size (OR = 4.34, 95% CI 1.66-11.38, P = 0.003), capsular invasion (OR = 3.49, 95% CI 1.90-6.41, P | 0.0001) and clinical stage of PTC (OR

For security against pathogens, it is essential that na?ve CD4+ T cells differentiate into specific effector T helper (Th) cell subsets following activation by antigen presented by dendritic cells (DCs). An instructive role for Notch ligand expressing DCs in the induction of Th cell differentiation is usually further challenged by evidence for the involvement of Notch signaling in differentiation of Th9, Th17, regulatory T cells, and follicular Th cells. In this review, we will discuss the two opposing models, referred to as the instructive and the unbiased amplifier model. We spotlight both the function of different Notch receptors on CD4+ T cells and the impact of Notch ligands on antigen-presenting cells. (5). Th2 cells control helminth infections and are implicated in allergic immune responses NMS-P515 such as allergic asthma. They are potent suppliers of Th2 cytokines that induce IgE synthesis (IL-4), recruit eosinophils (IL-5), and cause smooth muscle hyperreactivity and goblet cell ...

NOTCH3 - NOTCH3 Mutant (C568Y), Myc-DDK-tagged ORF clone of Homo sapiens notch 3 (NOTCH3) as transfection-ready DNA available for purchase from OriGene - Your Gene Company.