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 ...
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.. ...
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 ...
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 ...
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 ...
Author Summary Multicellular development requires tightly regulated spatial pattern formation, frequently including the generation of sharp differences over short length scales. Classic examples include boundary formation in the Drosophila wing veins and lateral inhibition patterning in the differentiation of sensory cells. These processes and a diverse variety of others are mediated by the Notch signaling system which allows neighboring cells to exchange information, via interaction between the Notch receptor on one cell and its ligands such as Delta, on another. Interestingly, recent evidence has shown that Notch and Delta within the same cell (in cis) also interact, mutually inactivating each other. However, the significance of this interaction for pattern formation has remained unclear. Here we show, by analytical and computational modeling, how this cis interaction intrinsically generates a difference-promoting logic that optimizes the system for use in fine-grained pattern formation. Specifically,
Objective(s): NOTCH signaling pathway is well known for its role in cell fate, cell survival, cell differentiation, and apoptosis. Some of the NOTCH signaling genes are critical for endometrial function and implantation in animals and appear to play a similar role in humans. The purpose of the current study was to investigate the potential roles of some main components of the NOTCH family in human endometrium during implantation period in common gynecological diseases.Materials and Methods: Endometrial NOTCH receptors NOTCH1, 3, 4 and ligand JAG1, 2 and survivin mRNA expression were investigated using the Q-PCR technique and the amount of the JAG1, 2 proteins was also determined by Western blot. Samples were obtained from 12 patients with endometriosis, 12 patients with repeated implantation failure (RIF), 12 patients with Polycystic Ovary Syndrome (PCOS) and 10 healthy fertile women as a control group. Data were analyzed using SPSS version 18. Group comparisons were performed by one-way ANOVA or
Signaling through the Notch1 receptor is essential for the control of numerous developmental processes during embryonic life as well as in adult tissue homeostasis and disease. Since the outcome of Notch1 signaling is highly context-dependent, and its precise physiological and pathological role in many organs is unclear, it is of great interest to localize and identify the cells that receive active Notch1 signals in vivo. Here, we report the generation and characterization of a BAC-transgenic mouse line, N1-Gal4VP16, that when crossed to a Gal4-responsive reporter mouse line allowed the identification of cells undergoing active Notch1 signaling in vivo. Analysis of embryonic and adult N1-Gal4VP16 mice demonstrated that the activation pattern of the transgene coincides with previously observed activation patterns of the endogenous Notch1 receptor. Thus, this novel reporter mouse line provides a unique tool to specifically investigate the spatial and temporal aspects of Notch1 signaling in vivo. ...
In this study, we formally tested the hypothesis that ADAM10 regulates B cell development. The generation and analysis of B cell-specific ADAM10 knockout mice revealed that ADAM10 critically regulates development of the entire MZB lineage by initiating Notch2 signaling.. The rate-limiting step in Notch2 signaling is cleavage within the receptors negative regulatory region (NRR) located in the membrane-proximal portion of the extracellular domain. The structure of the NRR prevents ligand-independent Notch cleavage. Mutations in the NRR can allow cleavage in the absence of ligand, leading to constitutive Notch signaling. In the case of Notch1, this leads to the formation of T cell acute lymphocytic leukemia (Kopan and Ilagan, 2009). Brou et al. (2000) and Mumm et al. (2000) identified the Notch1 cleavage site in the NRR between Ala-1710 and Val-1711, just 13 amino acids upstream of the transmembrane domain. These studies, in combination with more recent reports, have demonstrated that ADAM10, ...
In this study, the research team investigated NOTCH1 mutations in keratinocyte lines derived from OSCC biopsies that had been subjected to whole exome sequencing.. One line, SJG6, was found to have truncating mutations in both NOTCH1 alleles, resulting in loss of NOTCH1 expression.. Overexpression of the NOTCH1 intracellular domain in SJG6 cells promoted cell adhesion and differentiation, while suppressing proliferation, migration and clonal growth, consistent with the previously reported tumour suppressive function of NOTCH1 in OSCC.. Dr. Fiona M. Watt from the Centre for Stem Cells and Regenerative Medicine, Kings College London; Tower Wing, Guys Hospital, London, UK said "Notch1 is a heterodimeric and multifunctional transmembrane receptor that regulates key cellular processes, including cell fate determination, maintenance of stem cells, cell survival, proliferation and apoptosis.". Notch1 is a heterodimeric and multifunctional transmembrane receptor that regulates key cellular processes, ...
In this study, we have shown that ligand-induced Notch activation in multipotent hematopoietic precursor cells regulates lineage expression in a density-dependent manner. Our data suggest that the enhanced formation of B and T precursors occurs in the presence of lower densities of Delta1ext-IgG because of the effects on multipotent precursors. In addition, higher densities of ligand lead mainly to T cell differentiation because of the inhibition of early B cell and myeloid differentiation by multipotent precursors and the promotion of T cell differentiation by lymphoid committed precursors. These results represent the first evidence that a single Notch ligand can enhance the development of cells that adopt either a B or a T cell fate.. Previous studies of cell fate outcomes induced by Delta1 have used cell-expressed ligands and were thus unable to address quantitative differences in ligand expression (11, 21, 26). However, our studies suggest that lower densities of Delta1 induce Notch ...
Notch receptors are frequently deregulated in several human malignancies including human breast cancer. Activation of Notch has been reported to cause mammary carcinomas in mice. However, the...
Notch receptors modulate transcriptional targets following the proteolytic release of the Notch intracellular domain (NotchIC). Phosphorylated forms of NotchIC have been identified within the nucleus and have been associated with CSL members, as well as correlated with regions of the receptor that are required for activity. Genetic studies have suggested that Shaggy, the Drosophila homolog of glycogen synthase kinase-3ß (GSK3ß) may act as a positive modulator of the Notch signaling. GSK3ß is a serine/threonine kinase and is a component of the Wnt/wingless signaling cascade. GSK3ß is able to bind and phosphorylate Notch1IC in vitro, and attenuation of GSK3ß activity reduces phosphorylation of NotchIC in vivo. Functionally, ligand-activated signaling through the endogenous Notch1 receptor is reduced in GSK3ß null fibroblasts, implying a positive role for GSK3ß in mammalian Notch signaling. As a possible mechanistic explanation of the effect of GSK3ß on Notch signaling, it was observed that ...
NOTCH ligands DLL1, DLL4, JAG1 and JAG2 undergo ubiquitination and endocytosis after binding NOTCH2 in trans. Integrity of the intracellular domain of DLL1 was shown to be essential for the successful release of NOTCH2 intracellular domain, NICD2, in response to DLL1 binding (Shimizu et al. 2002). In Drosophila, ubiquitination of Delta and Serrate ligands is performed by either Mindbomb or Neuralized ubiquitin ligase. In mammals, there are two Mindbomb homologues, MIB1 and MIB2 and two Neuralized homologues, NEURL (also known as NEUR1) and NEURL1B (also known as NEUR2). Although both Mib1 and Mib2 ubiquitinate Delta (Koo et al. 2005), only Mib1 was shown to be essential for normal development in mice, with Mib1 deficient mice exhibiting typical Notch deficiency phenotypes (Koo et al. 2007). This could be due to different expression patterns of Mib1 and Mib2. While Mib1 is abundantly expressed in embryos and adult tissues, Mib2 expression is limited to adult tissues only (Koo et al. 2005). Mouse ...
NOTCH ligands DLL1, DLL4, JAG1 and JAG2 undergo ubiquitination and endocytosis after binding NOTCH2 in trans. Integrity of the intracellular domain of DLL1 was shown to be essential for the successful release of NOTCH2 intracellular domain, NICD2, in response to DLL1 binding (Shimizu et al. 2002). In Drosophila, ubiquitination of Delta and Serrate ligands is performed by either Mindbomb or Neuralized ubiquitin ligase. In mammals, there are two Mindbomb homologues, MIB1 and MIB2 and two Neuralized homologues, NEURL (also known as NEUR1) and NEURL1B (also known as NEUR2). Although both Mib1 and Mib2 ubiquitinate Delta (Koo et al. 2005), only Mib1 was shown to be essential for normal development in mice, with Mib1 deficient mice exhibiting typical Notch deficiency phenotypes (Koo et al. 2007). This could be due to different expression patterns of Mib1 and Mib2. While Mib1 is abundantly expressed in embryos and adult tissues, Mib2 expression is limited to adult tissues only (Koo et al. 2005). Mouse ...
Notch signaling can regulate cell-to-cell communication and control cell fate decisions in a variety of cell types. In the vascular endothelium, signaling between Dll4 and Notch1 in tip and stalk cells, respectively, prevents stalk cell sprouting.16 This Notch-dependent homotypic interaction helps to pattern blood vessels. In this study, we asked how Notch signaling might facilitate heterotypic interactions between mural cells and endothelial cells that help shape the vasculature. Previous work has suggested an important role for pericytes in modulating tube formation,50,51 and an in vitro study by our laboratory demonstrated that mural cell-expressed Notch3 affects angiogenesis.30 Here, we show that under physiological conditions, Notch3 deletion decreases vascularization in the mouse retina at early developmental stages. These angiogenic defects are not a result of enhanced regression, which points to growth retardation as the likely cause. The decline in angiogenesis is associated with a ...
Asymmetric cell division is a conserved mechanism by which cell fate diversity is generated during Metazoan development. How one cell can generate two daughter cells with different identities and how defects in this asymmetry can contribute to cancer are the fundamental questions we are addressing in Drosophila. We are investigating this process in the context of asymmetric cell division of neural precursor cells, called Sensory Organ Precursor (SOP). These latter undergo four rounds of asymmetric divisions, in which mother cells generate distinct daughters via the unequal segregation of the cell-fate determinants Numb and Neuralized at mitosis. At each division binary cell fate decision are regulated by Delta-Notch dependent cell-cell signalling. Numb is an endocytic protein that can bind to Notch and a four pass transmembrane protein named Sanpodo (Spdo), a protein required for Notch activation in SOP lineage, thereby preventing Notch activation in this cell. Neur acts in SOPs and pIIb cells ...
Asymmetric cell division is a conserved mechanism by which cell fate diversity is generated during Metazoan development. How one cell can generate two daughter cells with different identities and how defects in this asymmetry can contribute to cancer are the fundamental questions we are addressing in Drosophila. We are investigating this process in the context of asymmetric cell division of neural precursor cells, called Sensory Organ Precursor (SOP). These latter undergo four rounds of asymmetric divisions, in which mother cells generate distinct daughters via the unequal segregation of the cell-fate determinants Numb and Neuralized at mitosis. At each division binary cell fate decision are regulated by Delta-Notch dependent cell-cell signalling. Numb is an endocytic protein that can bind to Notch and a four pass transmembrane protein named Sanpodo (Spdo), a protein required for Notch activation in SOP lineage, thereby preventing Notch activation in this cell. Neur acts in SOPs and pIIb cells ...