Journal articles on the topic 'NOTCH 1 Intracellular Domain Indicate'
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1
Kitagawa, Motoo, Toshinao Oyama, Taichi Kawashima, Barry Yedvobnick, Anumeha Kumar, Kenji Matsuno, and Kenichi Harigaya. "A Human Protein with Sequence Similarity to DrosophilaMastermind Coordinates the Nuclear Form of Notch and a CSL Protein To Build a Transcriptional Activator Complex on Target Promoters." Molecular and Cellular Biology 21, no. 13 (July 1, 2001): 4337–46. http://dx.doi.org/10.1128/mcb.21.13.4337-4346.2001.
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ABSTRACT Mastermind (Mam) has been implicated as an important positive regulator of the Notch signaling pathway by genetic studies usingDrosophila melanogaster. Here we describe a biochemical mechanism of action of Mam within the Notch signaling pathway. Expression of a human sequence related to Drosophila Mam (hMam-1) in mammalian cells augments induction of Hairy Enhancer of split (HES) promoters by Notch signaling. hMam-1 stabilizes and participates in the DNA binding complex of the intracellular domain of human Notch1 and a CSL protein. Truncated versions of hMam-1 that can maintain an association with the complex behave in a dominant negative fashion and depress transactivation. Furthermore,Drosophila Mam forms a similar complex with the intracellular domain of Drosophila Notch andDrosophila CSL protein during activation of Enhancer of split, the Drosophila counterpart ofHES. These results indicate that Mam is an essential component of the transcriptional apparatus of Notch signaling.
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Blokzijl, Andries, Camilla Dahlqvist, Eva Reissmann, Anna Falk, Annalena Moliner, Urban Lendahl, and Carlos F. Ibáñez. "Cross-talk between the Notch and TGF-β signaling pathways mediated by interaction of the Notch intracellular domain with Smad3." Journal of Cell Biology 163, no. 4 (November 24, 2003): 723–28. http://dx.doi.org/10.1083/jcb.200305112.
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The Notch and transforming growth factor-β (TGF-β) signaling pathways play critical roles in the control of cell fate during metazoan development. However, mechanisms of cross-talk and signal integration between the two systems are unknown. Here, we demonstrate a functional synergism between Notch and TGF-β signaling in the regulation of Hes-1, a direct target of the Notch pathway. Activation of TGF-β signaling up-regulated Hes-1 expression in vitro and in vivo. This effect was abrogated in myogenic cells by a dominant-negative form of CSL, an essential DNA-binding component of the Notch pathway. TGF-β regulated transcription from the Hes-1 promoter in a Notch-dependent manner, and the intracellular domain of Notch1 (NICD) cooperated synergistically with Smad3, an intracellular transducer of TGF-β signals, to induce the activation of synthetic promoters containing multimerized CSL- or Smad3-binding sites. NICD and Smad3 were shown to interact directly, both in vitro and in cells, in a ligand-dependent manner, and Smad3 could be recruited to CSL-binding sites on DNA in the presence of CSL and NICD. These findings indicate that Notch and TGF-β signals are integrated by direct protein–protein interactions between the signal-transducing intracellular elements from both pathways.
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Chandiran, Karthik, and Lisa M. Minter. "Notch-1 regulation of microRNAs alter the expression of proinflammatory cytokines and Th1 response." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 127.1. http://dx.doi.org/10.4049/jimmunol.196.supp.127.1.
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Abstract Notch signaling is required for activation and differentiation of T cells. Upon its S3 cleavage by gamma secretase, Notch intracellular domain triggers the expression of various molecules depending on its binding partners. The data published from our lab states that Notch1 can induce Th1 immune response and regulates the expression of IFNγ by modulating the expression of the transcription factor T-bet. However the detailed mechanism behind Notch1 mediated Th1 differentiation is not clear. Here we report that Notch1 can regulate Th1 differentiation by targeting microRNAs. miR-29 is a family of micro RNAs (miR29a,b,c) that targets ifng and both its transcription factors tbx21 and eomes directly by binding to their respective 3′ UTRs. By using novel gamma secretase inhibitors (GSIs) we show that Notch can modulate the expression of miR-29. Overexpression of Notch1 in T cell hybridoma cell lines further validates Notch1- mediated mirR-29 regulation. These results indicate a novel regulatory mechanism of T cell differentiation through Notch1 mediated microRNA expression.
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Koshizaka, Masaya, Minoru Takemoto, Seiya Sato, Hirotake Tokuyama, Masaki Fujimoto, Emiko Okabe, Ryoichi Ishibashi, et al. "An Angiotensin II Type 1 Receptor Blocker Prevents Renal Injury via Inhibition of the Notch Pathway in Ins2 Akita Diabetic Mice." Experimental Diabetes Research 2012 (2012): 1–10. http://dx.doi.org/10.1155/2012/159874.
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Recently, it has been reported that the Notch pathway is involved in the pathogenesis of diabetic nephropathy. In this study, we investigated the activation of the Notch pathway in Ins2 Akita diabetic mouse (Akita mouse) and the effects of telmisartan, an angiotensin II type1 receptor blocker, on the Notch pathway. The intracellular domain of Notch1 (ICN1) is proteolytically cleaved from the cell plasma membrane in the course of Notch activation. The expression of ICN1 and its ligand, Jagged1, were increased in the glomeruli of Akita mice, especially in the podocytes. Administration of telmisartan significantly ameliorated the expression of ICN1 and Jagged1. Telmisartan inhibited the angiotensin II-induced increased expression of transforming growth factorβand vascular endothelial growth factor A which could directly activate the Notch signaling pathway in cultured podocytes. Our results indicate that the telmisartan prevents diabetic nephropathy through the inhibition of the Notch pathway.
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Nobta, Masuhiro, Tomoo Tsukazaki, Yasuaki Shibata, Chang Xin, Takeshi Moriishi, Seiji Sakano, Hiroyuki Shindo, and Akira Yamaguchi. "Critical Regulation of Bone Morphogenetic Protein-induced Osteoblastic Differentiation by Delta1/Jagged1-activated Notch1 Signaling." Journal of Biological Chemistry 280, no. 16 (February 4, 2005): 15842–48. http://dx.doi.org/10.1074/jbc.m412891200.
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Functional involvement of the Notch pathway in osteoblastic differentiation has been previously investigated using the truncated intracellular domain, which mimics Notch signaling by interacting with the DNA-binding protein CBF-1. However, it is unclear whether Notch ligands Delta1 and Jagged1 also induce an identical cellular response in osteoblastic differentiation. We have shown that both Delta1 and Jagged1 were expressed concomitantly with Notch1 in maturating osteoblastic cells during bone regeneration and that overexpressed and immobilized recombinant Delta1 and Jagged1 alone did not alter the differentiated state of MC3T3-E1 and C2C12 cells. However, they augmented bone morphogenetic protein-2 (BMP2)-induced alkaline phosphatase activity and the expression of several differentiation markers, except for osteocalcin, and ultimately enhanced calcified nodule andin vivoectopic bone formation of MC3T3-E1. In addition, both ligands transmitted signal through the CBF-1-dependent pathway and stimulated the expression of HES-1, a direct target of Notch pathway. To test the necessity of Notch signaling in BMP2-induced differentiation, Notch signaling was inhibited by the dominant negative extracellular domain of Notch1, specific inhibitor, or small interference RNA. These treatments decreased alkaline phosphatase activity as well as the expression of other differentiation markers and inhibited the promoter activity of Id-1, a target gene of the BMP pathway. These results indicate the functional redundancy between Delta1 and Jagged1 in osteoblastic differentiation whereby Delta1/Jagged1-activated Notch1 enhances BMP2-induced differentiation through the identical signaling pathway. Furthermore, our data also suggest that functional Notch signaling is essential not only for BMP2-induced osteoblast differentiation but also for BMP signaling itself.
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Kusano, Shuichi, and Nancy Raab-Traub. "An Epstein-Barr Virus Protein Interacts with Notch." Journal of Virology 75, no. 1 (January 1, 2001): 384–95. http://dx.doi.org/10.1128/jvi.75.1.384-395.2001.
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ABSTRACT The Epstein-Barr virus (EBV) BamHI A mRNAs were originally identified in cDNA libraries from nasopharyngeal carcinoma, where they are expressed at high levels. The RNAs are differentially spliced to form several open reading frames and also contain the BARF0 open reading frame at the 3′ end. One cDNA, RK-BARF0, included a potential endoplasmic reticulum-targeting signal peptide sequence. The RK-BARF0 protein is shown here to interact with the Notch4 ligand binding domain, using yeast two-hybrid screening, coimmunoprecipitation, and confocal microscopy. This interaction induces translocation of a portion of the full-length unprocessed Notch4 to the nucleus by using the Notch nuclear localization signal. These effects of RK-BARF0 on Notch intracellular location indicate that EBV possibly modulates Notch signaling. Unprocessed Notch4 was also detected in immunoprecipitated complexes from EBV-infected cells by using a rabbit antiserum raised against a BARF0-specific peptide. This finding provides additional evidence for expression of RK-BARF0 and its interaction with Notch during EBV infection. In EBV-infected, EBNA2-negative cells, RK-BARF0 induced the expression of EBV latent membrane protein 1 (LMP1), and this induction was dependent on the RK-BARF0/Notch interaction domain. The activation of LMP1 expression by RK-BARF0 may be responsible for expression of LMP1 in EBV latent infections in the absence of EBNA2.
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Kim, Su Hwan, Jin Hyun Kang, Tae Ki Uhm, Do Jin Kim, and Il Yup Chung. "Notch signaling promotes MUC5AC expression through epidermal growth factor receptor-extracellular regulated kinase pathway (163.1)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 163.1. http://dx.doi.org/10.4049/jimmunol.186.supp.163.1.
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Abstract Airway mucus hypersecretion and goblet cell hyperplasia plays an important role in the pathogenesis of several major airway inflammatory diseases. MUC5AC is a major component of the mucus produced by airway epithelial cells and considered to be a marker of mucus cell hyperplasia. In the study we investigate the involvement of Notch signaling in EGF-mediated expression of MUC5AC in human epithelial NCI-H292 cells. EGF stimulated the generation of Notch intracellular domain (NICD) in a RBP-Jκ-dependent manner. Treatment with γ-secretase inhibitor (GSI) or introduction of small interfering RNA directed against Notch1 reduced EGF-induced expression of MUC5AC mRNA and protein. The inhibitory effect of both GSI and Notch1 silencing on MUC5AC expression was accompanied by reduced generation of NICD and reduced expression of a Notch downstream target, Hes-1. Blockage of the Notch signaling with the two agents resulted in a decrease in ERK phosphorylation induced by EGF stimulation. ERK inhibitor inhibited MUC5AC production. These results indicate that ERK activation is necessary for Notch signaling to regulate the EGFR-mediated MUC5AC expression. Collectively, these results suggest that Notch signaling regulates the EGF-induced expression of MUC5AC through modulation of ERK activity.
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Hoebeke, Inge, Magda De Smedt, Inge Van de Walle, Katia Reynvoet, Greet De Smet, Jean Plum, and Georges Leclercq. "Overexpression of HES-1 is not sufficient to impose T-cell differentiation on human hematopoietic stem cells." Blood 107, no. 7 (April 1, 2006): 2879–81. http://dx.doi.org/10.1182/blood-2005-05-1815.
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Abstract By retroviral overexpression of the Notch-1 intracellular domain (ICN) in human CD34+ hematopoietic stem cells (HSCs), we have shown previously that Notch-1 signaling promotes the T-cell fate and inhibits the monocyte and B-cell fate in several in vitro and in vivo differentiation assays. Here, we investigated whether the effects of constitutively active Notch-1 can be mimicked by overexpression of its downstream target gene HES1. Upon HES-1 retroviral transduction, human CD34+ stem cells had a different outcome in the differentiation assays as compared to ICN-transduced cells. Although HES-1 induced a partial block in B-cell development, it did not inhibit monocyte development and did not promote T/NK-cell-lineage differentiation. On the contrary, a higher percentage of HES-1-transduced stem cells remained CD34+. These experiments indicate that HES-1 alone is not able to substitute for Notch-1 signaling to induce T-cell differentiation of human CD34+ hematopoietic stem cells.
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Kindler, Thomas, Melanie G. Cornejo, Claudia Scholl, Jianing Liu, Dena S. Leeman, J. Erika Haydu, Stefan Fröhling, Benjamin H. Lee, and D. Gary Gilliland. "K-RasG12D–induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 mutations and are sensitive to γ-secretase inhibitors." Blood 112, no. 8 (October 15, 2008): 3373–82. http://dx.doi.org/10.1182/blood-2008-03-147587.
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Abstract To study the impact of oncogenic K-Ras on T-cell leukemia/lymphoma development and progression, we made use of a conditional K-RasG12D murine knockin model, in which oncogenic K-Ras is expressed from its endogenous promoter. Transplantation of whole bone marrow cells that express oncogenic K-Ras into wild-type recipient mice resulted in a highly penetrant, aggressive T-cell leukemia/lymphoma. The lymphoblasts were composed of a CD4/CD8 double-positive population that aberrantly expressed CD44. Thymi of primary donor mice showed reduced cellularity, and immunophenotypic analysis demonstrated a block in differentiation at the double-negative 1 stage. With progression of disease, approximately 50% of mice acquired Notch1 mutations within the PEST domain. Of note, primary lymphoblasts were hypersensitive to γ-secretase inhibitor treatment, which is known to impair Notch signaling. This inhibition was Notch-specific as assessed by down-regulation of Notch1 target genes and intracellular cleaved Notch. We also observed that the oncogenic K-Ras-induced T-cell disease was responsive to rapamycin and inhibitors of the RAS/MAPK pathway. These data indicate that patients with T-cell leukemia with K-Ras mutations may benefit from therapies that target the NOTCH pathway alone or in combination with inhibition of the PI3K/AKT/MTOR and RAS/MAPK pathways.
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Wallberg, Annika E., Kia Pedersen, Urban Lendahl, and Robert G. Roeder. "p300 and PCAF Act Cooperatively To Mediate Transcriptional Activation from Chromatin Templates by Notch Intracellular Domains In Vitro." Molecular and Cellular Biology 22, no. 22 (November 15, 2002): 7812–19. http://dx.doi.org/10.1128/mcb.22.22.7812-7819.2002.
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ABSTRACT Ligand activation of Notch receptors leads to release of the intracellular receptor domain (Notch IC), which translocates to the nucleus and interacts with the DNA-binding protein RBP-Jκ to control expression of specific target genes. A number of proteins have been shown to interact with Notch ICs and to modulate target gene activation, but the precise function of and interplay between these factors is not known. This report investigates the Notch IC-interacting proteins, p300, PCAF, and Mastermind-like 1 (MAML1), in an in vitro transcription system with purified factors and naked DNA or chromatin templates. MAML1, RBP-Jκ, and Notch IC are all required for optimal transcription from DNA, whereas transcription from chromatin requires, in addition, p300, which interacts with MAML1. The transcriptional activity of p300 requires acetyl coenzyme A, indicating that it functions as a histone acetyltransferase when mediating Notch IC function. PCAF is unable to promote transcription on its own but enhances Notch IC-mediated transcription from chromatin in conjunction with p300. These data define a critical role for p300 in the potentiation of Notch IC function by MAML1 and PCAF, provide the first evidence for cooperativity between PCAF and p300 in Notch IC function, and also indicate direct effects of RBP-Jκ, Notch IC, and MAML1 on the general transcription machinery.
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Kamińska, Alicja, Sylwia Marek, Laura Pardyak, Małgorzata Brzoskwinia, Barbara Bilinska, and Anna Hejmej. "Crosstalk between Androgen-ZIP9 Signaling and Notch Pathway in Rodent Sertoli Cells." International Journal of Molecular Sciences 21, no. 21 (November 5, 2020): 8275. http://dx.doi.org/10.3390/ijms21218275.
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Our recent study demonstrated altered expression of Notch ligands, receptors, and effector genes in testes of pubertal rats following reduced androgen production or signaling. Herein we aimed to explore the role of nuclear androgen receptor (AR) and membrane androgen receptor (Zrt- and Irt-like protein 9; ZIP9) in the regulation of Notch pathway activation in rodent Sertoli cells. Experiments were performed using TM4 and 15P-1 Sertoli cell lines and rat primary Sertoli cells (PSC). We found that testosterone (10−8 M–10−6 M) increased the expression of Notch1 receptor, its active form Notch1 intracellular domain (N1ICD) (p < 0.05, p < 0.01, p < 0.001), and the effector genes Hey1 (p < 0.05, p < 0.01, p < 0.001) and Hes1 (p < 0.05, p < 0.001) in Sertoli cells. Knockdown of AR or ZIP9 as well as antiandrogen exposure experiments revealed that (i) action of androgens via both AR and ZIP9 controls Notch1/N1ICD expression and transcriptional activity of recombination signal binding protein (RBP-J), (ii) AR-dependent signaling regulates Hey1 expression, (iii) ZIP9-dependent pathway regulates Hes1 expression. Our findings indicate a crosstalk between androgen and Notch signaling in Sertoli cells and point to cooperation of classical and non-classical androgen signaling pathways in controlling Sertoli cell function.
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Chang, Chunkang, Chengming Fei, Juan Guo, Youshan Zhao, Shucheng Gu, and Xiao Li. "Activation of Notch-Hes Signaling Pathway Impairs Osteogenic Differentiation and Hematopoietic Stem Cell-Supporting Capacitiy of Mesenchymal Stromal Cells Derived from Myelodysplastic Syndromes Patients." Blood 126, no. 23 (December 3, 2015): 2857. http://dx.doi.org/10.1182/blood.v126.23.2857.2857.
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Abstract Ineffective hematopoiesis is a major characteristic of myelodysplastic syndromes (MDS). Bone marrow mesenchymal stromal cells(BMMSCs) and their progeny (i.e., osteoblasts, adipocytes, and reticular cells), which are considered as main cellular components of the bone marrow niche, have been shown to physiologically support hematopoiesis, but their contribution to the pathogenesis of MDS is controversially discussed. In this study, we examined the osteogenic differentiation and hematopoietic stem cell-supporting capacitiy of BMMSCs in patients with MDS (n=67) and healthy donors (n=22). After 21 days osteogenic induction differentiation, osteogenesis potential of BMMSCs was significantly reduced in cases with RARS(83.3%), RCMD(75.0%), RAEB I(44.4%), RAEB II (40%), indicated by cytochemical stainings and reduced expressions of Runx2. Moreover, we observed that in co-cultures with normal hematopoietic stem cells(HSCs) and MDS-BMMSCs, the colony number (CFU-GM、BFU-E and CFU-GEMM) was significantly lower in the presence of MDS-BMMSCs in comparison to the normal counterpart. Furthermore, in MDS-BMMSCs, we detected increased mRNA expression of several members of the Notch pathway, including Delta-like-1, Jagged-1, Notch1, Notch2, Hes1 and Hes5. Basically, the Notch-Hes pathway is the main regulator of the microenvironment dependent hematopoietic stem cell fate. Therefore we investigated if the activation of Notch-Hes pathway affected their osteogenesis and hematopoietic stem cell-supporting capacitiy of BMMSCs. By overexpression of Notch1 intracellular domain (NICD) in BMMSCs from healthy donors, we confirmed that Notch signaling negatively regulated BMMSCs osteogenesis through inhibition of Runx2 transcriptional activity. Importantly, treatment with the Notch1 inhibitor DAPT reversed the osteogenic differentiation and improved the hematopoiesis supporting capacitiy of MDS-BMMSCs. Taken together, our findings suggest that the ineffective hematopoiesis typical of MDS may be partly due to the impaired osteogenic differentiation of BMMSCs, and the activation of Notch-Hes signaling is involved in the impaired osteogenic differentiation and diminished hematopoietic stem cell-supporting capacitiy of MDS-BMMSCs, restoring the adequate Notch-Hes signaling could represent a potential therapeutic approach to MDS. Disclosures No relevant conflicts of interest to declare.
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Jeffries, Shawn, David J. Robbins, and Anthony J. Capobianco. "Characterization of a High-Molecular-Weight Notch Complex in the Nucleus of Notchic-Transformed RKE Cells and in a Human T-Cell Leukemia Cell Line." Molecular and Cellular Biology 22, no. 11 (June 1, 2002): 3927–41. http://dx.doi.org/10.1128/mcb.22.11.3927-3941.2002.
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ABSTRACT Notch genes encode a family of transmembrane proteins that are involved in many cellular processes, such as differentiation, proliferation, and apoptosis. It is well established that all four Notch genes can act as oncogenes; however, the mechanism by which Notch proteins transform cells remains unknown. Previously, we reported that both nuclear localization and transcriptional activation are required for neoplastic transformation of RKE cells. Furthermore, we identified cyclin D1 as a direct transcriptional target of constitutively active Notch molecules. In an effort to understand the mechanism by which Notch functions in the nucleus, we sought to determine if Notch formed stable complexes using size exclusion chromatography. Herein, we report that the Notch intracellular domain (Nic) forms distinct high-molecular-weight complexes in the nuclei of transformed RKE cells. The largest complex is approximately 1.5 MDa and contains both endogenous CSL (for CBF1, Suppressor of Hairless, and Lag-1) and Mastermind-Like-1 (Maml). Nic molecules that do not have the high-affinity binding site for CSL (RAM) retain the ability to associate with CSL in a stable complex through interactions involving Maml. However, Maml does not directly bind to CSL. Furthermore, Maml can rescue ΔRAM transcriptional activity on a CSL-dependent promoter. These results indicate that deletion of the RAM domain does not equate to CSL-independent signaling. Moreover, in SUP-T1 cells, Nic exists exclusively in the largest Nic-containing complex. SUP-T1 cells are derived from a T-cell leukemia that harbors the t(7;9)(q34;q34.3) translocation and constitutively express Nic. Taken together, our data indicate that complex formation is likely required for neoplastic transformation by Notchic.
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Jundt, Franziska, Oezlem Acikgoez, Robert Preissner, and Bernd Dorken. "Manipulation of the Notch Pathway by γ-Secretase Inhibitors as a Novel Therapeutic Approach in Multiple Myeloma." Blood 104, no. 11 (November 16, 2004): 645. http://dx.doi.org/10.1182/blood.v104.11.645.645.
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Abstract Notch receptors expressed on hematopoietic stem cells interact with their ligands on bone marrow stromal cells. Thereby they control cell fate decisions and survival. We recently demonstrated that Notch has a pathogenetic role in multiple myeloma (MM), where tight interactions between neoplastic plasma cells and their microenvironment are essential for tumor cell growth (Blood.2004; 103:3511–3515). Our data provided evidence that Notch1 and Notch2 were highly expressed in cultured and primary MM cells, whereas nonneoplastic counterparts showed low to undetectable levels of Notch. Furthermore, our functional data indicated that activating the Notch pathway in MM cells by the Notch ligand Jagged1 potently induced tumor cell growth and suggested that these interactions contribute to myelomagenesis in vivo. In this study, we blocked Notch by novel γ-secretase inhibitors in cultured MM cells. γ-secretase catalyzes the release of the intracellular domain of Notch that subsequently translocates to the nucleus to activate expression of downstream target genes. Inhibition of γ-secretase activity is currently investigated as a therapeutic strategy in Alzheimer’s disease, because γ-secretase similarly cleaves amyloid precursor proteins to release Aβ peptides, accumulation of which is causally related to Alzheimer’s disease. To identify novel γ-secretase inhibitors that might block Notch in MM cells, we analyzed two million compounds by 3D in silico screening. Thereby structurally known inhibitors were compared with compounds from data banks. Novel structurally related compounds (27) were tested by in vitro assays. Specific inhibition of Notch signaling was controlled by RT-PCR of the down-regulated Notch target gene Hes-1 in MM cells. In addition, tumor cell proliferation of MM cells was efficiently blocked by 3 out of 27 newly identified inhibitors in a dose-dependent manner. Currently, we investigate whether these compounds also have potent anti-tumor activity against MM cells in mouse models in vivo. If so, interruption of Notch signaling by newly identified inhibitors might be a novel therapeutic principle to control the proliferation capacity of MM.
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Zhu, Xiaolong, Sha Ding, Cong Qiu, Yanna Shi, Lin Song, Yueyue Wang, Yuewen Wang, et al. "SUMOylation Negatively Regulates Angiogenesis by Targeting Endothelial NOTCH Signaling." Circulation Research 121, no. 6 (September 2017): 636–49. http://dx.doi.org/10.1161/circresaha.117.310696.
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Rationale: The highly conserved NOTCH (neurogenic locus notch homolog protein) signaling pathway functions as a key cell–cell interaction mechanism controlling cell fate and tissue patterning, whereas its dysregulation is implicated in a variety of developmental disorders and cancers. The pivotal role of endothelial NOTCH in regulation of angiogenesis is widely appreciated; however, little is known about what controls its signal transduction. Our previous study indicated the potential role of post-translational SUMO (small ubiquitin-like modifier) modification (SUMOylation) in vascular disorders. Objective: The aim of this study was to investigate the role of SUMOylation in endothelial NOTCH signaling and angiogenesis. Methods and Results: Endothelial SENP1 (sentrin-specific protease 1) deletion, in newly generated endothelial SENP1 (the major protease of the SUMO system)–deficient mice, significantly delayed retinal vascularization by maintaining prolonged NOTCH1 signaling, as confirmed in cultured endothelial cells. An in vitro SUMOylation assay and immunoprecipitation revealed that when SENP1 associated with N1ICD (NOTCH1 intracellular domain), it functions as a deSUMOylase of N1ICD SUMOylation on conserved lysines. Immunoblot and immunoprecipitation analyses and dual-luciferase assays of natural and SUMO-conjugated/nonconjugated NOTCH1 forms demonstrated that SUMO conjugation facilitated NOTCH1 cleavage. This released N1ICD from the membrane and stabilized it for translocation to the nucleus where it functions as a cotranscriptional factor. Functionally, SENP1-mediated NOTCH1 deSUMOylation was required for NOTCH signal activation in response to DLL4 (Delta-like 4) stimulation. This in turn suppressed VEGF (vascular endothelial growth factor) receptor signaling and angiogenesis, as evidenced by immunoblotted signaling molecules and in vitro angiogenesis assays. Conclusions: These results establish reversible NOTCH1 SUMOylation as a regulatory mechanism in coordinating endothelial angiogenic signaling; SENP1 acts as a critical intrinsic mediator of this process. These findings may apply to NOTCH-regulated biological events in nonvascular tissues and provide a novel therapeutic strategy for vascular diseases and tumors.
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Zanotti, Stefano, and Ernesto Canalis. "Notch and the Skeleton." Molecular and Cellular Biology 30, no. 4 (December 7, 2009): 886–96. http://dx.doi.org/10.1128/mcb.01285-09.
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ABSTRACT Notch receptors are transmembrane receptors that regulate cell fate decisions. There are four Notch receptors in mammals. Upon binding to members of the Delta and Jagged family of transmembrane proteins, Notch is cleaved and the Notch intracellular domain (NICD) is released. NICD then translocates to the nucleus, where it associates with the CBF-1, Suppressor of Hairless, and Lag-2 (CSL) and Mastermind-Like (MAML) proteins. This complex activates the transcription of Notch target genes, such as Hairy Enhancer of Split (Hes) and Hes-related with YRPF motif (Hey). Notch signaling is critical for the regulation of mesenchymal stem cell differentiation. Misexpression of Notch in skeletal tissue indicates a role as an inhibitor of skeletal development and postnatal bone formation. Overexpression of Notch inhibits endochondral bone formation and osteoblastic differentiation, causing severe osteopenia. Conditional inactivation of Notch in the skeleton causes an increase in cancellous bone volume and enhanced osteoblastic differentiation. Notch ligands are expressed in the hematopoietic stem cell niche and are critical for the regulation of hematopoietic stem cell self-renewal. Dysregulation of Notch signaling is the underlying cause of diseases affecting the skeletal tissue, including Alagille syndrome, spondylocostal dysostosis, and possibly, osteosarcoma.
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Hsu, Kai-Wen, Rong-Hong Hsieh, Yan-Hwa Wu Lee, Chi-Hong Chao, Kou-Juey Wu, Min-Jen Tseng, and Tien-Shun Yeh. "The Activated Notch1 Receptor Cooperates with α-Enolase and MBP-1 in Modulating c-myc Activity." Molecular and Cellular Biology 28, no. 15 (May 19, 2008): 4829–42. http://dx.doi.org/10.1128/mcb.00175-08.
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ABSTRACT The Notch signal pathway plays multifaceted roles to promote or suppress tumorigenesis. The Notch1 receptor intracellular domain (N1IC), the activated form of the Notch1 receptor, activates the c-myc proto-oncogene. The complex of N1IC and transcription factor YY1 binds to the human c-myc promoter to enhance c-myc expression in a CBF1-independent manner. Here we demonstrated that N1IC interacted with the c-Myc-regulating proteins α-enolase and c-myc promoter binding protein 1 (MBP-1). Both α-enolase and MBP-1 suppressed the N1IC-enhanced activity of the c-myc promoter in a CBF1-independent manner. The YY1 response element in front of the P2 c-myc promoter was essential and sufficient for the modulation of c-myc by N1IC and α-enolase or MBP-1. Furthermore, N1IC, YY1, and α-enolase or MBP-1 but not CBF1 bound to the c-myc promoter through associating with the YY1 response element. Hemin-induced erythroid differentiation was suppressed by N1IC in K562 cells. This suppression was relieved by the expression of α-enolase and MBP-1. In addition, both α-enolase and MBP-1 suppressed the N1IC-enhanced colony-forming ability through c-myc. These results indicate that the activated Notch1 receptor and α-enolase or MBP-1 cooperate in controlling c-myc expression through binding the YY1 response element of the c-myc promoter to regulate tumorigenesis.
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Maillard, Ivan, Seth E. Pross, Olga Shestova, Hong Sai, Jon C. Aster, Avinash Bhandoola, and Warren S. Pear. "Canonical Notch Signaling Is Dispensable for the Maintenance of Adult Hematopoietic Stem Cells." Blood 106, no. 11 (November 16, 2005): 267. http://dx.doi.org/10.1182/blood.v106.11.267.267.
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Abstract Canonical Notch signaling operates through a highly conserved pathway that regulates the differentiation and homeostasis of hematopoietic cells. Ligand-receptor binding initiates proteolytic release of the Notch intracellular domain (ICN) which migrates to the nucleus, binds the transcription factor CSL/RBPJk and activates target genes through the recruitment of transcriptional coactivators of the Mastermind-like family (MAML). Notch signaling is essential for the emergence of hematopoietic stem cells (HSCs) during fetal life, but its effects on adult HSCs are controversial. In gain-of-function experiments, activation of Notch signaling in adult HSCs increased their self-renewal potential in vitro and in vivo. However, loss-of-function studies have provided conflicting results as to the role of physiological Notch signaling in HSC maintenance and homeostasis. To address this question, we expressed DNMAML1, a GFP-tagged pan-inhibitor of Notch signaling, in mouse HSCs. We have shown previously that DNMAML1 interferes with the formation of the ICN/CSL/MAML transcriptional activation complex and blocks signaling from all four Notch receptors (Notch1-4) (Maillard, Blood 2004). Transfer of DNMAML1-transduced bone marrow (BM) as compared to control GFP-transduced BM into lethally irradiated recipients gave rise to similar long-term stable expression of GFP for at least 6 months after transplant. DNMAML1 and GFP-transduced cells contributed equally to all hematopoietic lineages, except to the T cell and marginal zone B cell lineages, which are Notch-dependent. Expression of DNMAML1 did not affect the size of the BM progenitor compartment (Lin negative, Sca-1 positive, c-Kit high, or LSK cells), or the proportion of LSK cells that were negative for Flt3 and L-Selectin expression (containing long-term HSCs). The stem cell function of DNMAML1-transduced LSK cells was further assessed with in vivo competitive repopulation assays in lethally irradiated recipients. DNMAML1 and GFP-transduced LSK cells competed equally well with wild-type BM, as judged by their contribution to the myeloid lineage up to 4 months post-transplant, through two successive rounds of transplantation. Our data indicate that canonical Notch signaling is dispensable for the maintenance of stem cell function in adult HSCs.
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Hsieh, J. J., T. Henkel, P. Salmon, E. Robey, M. G. Peterson, and S. D. Hayward. "Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2." Molecular and Cellular Biology 16, no. 3 (March 1996): 952–59. http://dx.doi.org/10.1128/mcb.16.3.952.
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The Notch/Lin-12/Glp-1 receptor family participates in cell-cell signaling events that influence cell fate decisions. Although several Notch homologs and receptor ligands have been identified, the nuclear events involved in this pathway remain incompletely understood. A truncated form of Notch, consisting only of the intracellular domain (NotchIC), localizes to the nucleus and functions as an activated receptor. Using both an in vitro binding assay and a cotransfection assay based on the two-hybrid principle, we show that mammalian NotchIC interacts with the transcriptional repressor CBF1, which is the human homolog of Drosophila Suppressor of Hairless. Cotransfection assays using segments of mouse NotchIC and CBF1 demonstrated that the N-terminal 114-amino-acid region of mouse NotchIC contains the CBF1 interactive domain and that the cdc10/ankyrin repeats are not essential for this interaction. This result was confirmed in immunoprecipation assays in which the N-terminal 114-amino-acid segment of NotchIC, but not the ankyrin repeat region, coprecipitated with CBF1. Mouse NotchIC itself is targeted to the transcriptional repression domain (aa179 to 361) of CBF1. Furthermore, transfection assays in which mouse NotchIC was targeted through Gal4-CBF1 or through endogenous cellular CBF1 indicated that NotchIC transactivates gene expression via CBF1 tethering to DNA. Transactivation by NotchIC occurs partially through abolition of CBF1-mediated repession. This same mechanism is used by Epstein-Barr virus EBNA2. Thus, mimicry of Notch signal transduction is involved in Epstein-Barr virus-driven immortalization.
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Nakamura, Makoto, Lizi Wu, Satoru Kojika, James D. Griffin, and Kanji Sugita. "Activation of Notch1 Signaling Suppresses Granulocytic Differentiation and Maintains a Part of Myeloid Progenitor Cells At the Immature Stage." Blood 118, no. 21 (November 18, 2011): 2375. http://dx.doi.org/10.1182/blood.v118.21.2375.2375.
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Abstract Abstract 2375 The Notch signaling pathway is evolutionally conserved and has crucial roles in the control of fate decision and differentiation in numerous cell types. Although Notch1 is continuously expressed in differentiation myeloid cells, the role of Notch1 signaling in regulating differentiation remains controversial. Here, we enhanced and/or suppressed Notch signaling in myeloblasts and then determined the effects of Notch signaling modulation on granulocytic differentiation. Specifically, we first transduced myeloblastic 32D and HL60 cells with retroviruses that express the intracellular domain of Notch1 (ICN1; pMSCV-ICN1-IRES-GFP) to activate Notch1 signaling, or alternatively expressed a dominant-negative form of Mastermind-like 1 (DNMAML1; pMSCV-DNMAML1-GFP) to inhibit Notch signaling. Then the transduced (GFP+) and untransduced (GFP-) cells were induced into granulocytic differentiation using G-CSF for 32D cells and ATRA for HL60 cells. The degree of granulocytic differentiation was then assessed by flow cytometric analysis of surface expression of CD11b, a marker of granulocytic lineage. We found that the percentage of differentiated cells (CD11bhigh for 32D and CD11b+ for HL60) in the ICN1 expressing (GFP+) group was significantly (p < 0.05) lower than that in the control (GFP-) group for most time-points that we examined, whereas the difference in the proportion of differentiated cells between DNMAML1 expressing (GFP+) and control (GFP-) groups was not statistically significant. These data suggest that forced activation of Notch1 signaling inhibits granulocytic differentiation, whereas endogenous Notch1 signaling appears not to have a major role in granulocytic maturation in these cell lines. Next, We further studied the role of Notch1 signaling in granulopoiesis by first activating Notch1 signaling in 32D cells by stable expression of exogenous ICN1 followed by Notch inhibition via DNMAML1 expression within the same cells. The subsequent four sub-populations of 32D cells termed as Vec/GFP (control), Vec/DNMAML1 (cells with endogenous Notch signaling blocked by DNMAML1, ICN1/GFP (cells with activating Notch1), and ICN1/DNMAML1 (cells with activating Notch1 followed by Notch signaling inhibition) were then cultured with G-CSF and evaluated for differentiation by CD11b staining. We found that on days 6 and 8 after the induction of differentiation, the proportion of differentiated (CD11bhigh) cells in ICN1/GFP was significantly (p < 0.05) lower than those in the other sub-populations, supporting that ICN1 inhibits granulocytic differentiation of 32D cells. On the other hand, the difference in the proportion of differentiated cells between the other 3 sub-populations was not statistically significant at any time-points, suggesting that DNMAML1 reverses the phenotype induced by activated Notch1 and that endogenous Notch1 signaling may have no effect on granulocytic maturation. Real-time RT-PCR analysis of cytoplasmic expression of myeloperoxidase (MPO) indicated that the MPO expression reached maximal level by day 2 in control (Vec/GFP) cells, the peak was delayed until day 5 in ICN1/GFP cells. The peak expression was observed on day 3 in both Vec/DNMAML1 and ICN1/DNMAML1 cells, indicating that ICN1 represses or delays granulocytic differentiation and that DNMAML1 partially neutralizes such a phenotype. Furthermore, morphological analysis, viable cell count, and cell cycle analysis revealed that a subset of ICN1/GFP cells remained myeloblastic with proliferative capacity after the induction of granulocytic differentiation, supporting the idea that ICN1 inhibits granulocytic differentiation. Our data indicate that Notch1 signaling activation suppresses granulocytic differentiation, and maintains a part of myeloid progenitor cells at the immature stage. Therefore, it suggests that aberrant Notch1 signaling could support the granulocytic transformation and the maintenance of the malignant phenotype. Disclosures: Griffin: Novartis Pharmaceuticals: Consultancy, Research Funding.
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Callahan, Robert, Barry A. Chestnut, and Ahmed Raafat. "Original Research: Featured Article: Imatinib mesylate (Gleevec) inhibits Notch and c-Myc signaling: Five-day treatment permanently rescues mammary development." Experimental Biology and Medicine 242, no. 1 (August 22, 2016): 53–67. http://dx.doi.org/10.1177/1535370216665175.
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Wap-Int3 transgenic females expressing the Notch4 intracellular domain (designated Int3) from the whey acidic protein promoter exhibit two phenotypes in the mammary gland: blockage of lobuloalveolar development and lactation, and tumor development with 100% penetrance. Previously, we have shown that treatment of Wap-Int3 tumor bearing mice with Imatinib mesylate (Gleevec) is associated with complete regression of the tumor. In the present study, we show that treatment of Wap-Int3 mice during day 1 through day 6 of pregnancy with Gleevec leads to the restoration of their lobuloalveolar development and ability to lactate in subsequent pregnancies in absence of Gleevec treatment. In addition, these mice do not develop mammary tumors. We investigated the mechanism for Gleevec regulation of Notch signaling and found that Gleevec treatment results in a loss of Int3 protein but not of Int3 mRNA in HC11 mouse mammary epithelial cells expressing Int3. The addition of MG-132, a proteasome inhibitor, shows increased ubiquitination of Int3 in the presence of Gleevec. Thus, Gleevec affects the stability of Int3 by promoting the degradation of Int3 via E3 ubiquitin ligases targeting it for the proteasome degradation. Gleevec is a tyrosine kinase inhibitor that acts on c-Kit and PDGFR. Therefore, we investigated the downstream substrate kinase GSK3β to ascertain the possible role that this kinase might play in the stability of Int3. Data show that Gleevec degradation of Int3 is GSK3β dependent. We have expanded our study of the effects Gleevec has on tumorigenesis of other oncogenes. We have found that anchorage-independent growth of HC11-c-Myc cells as well as tumor growth in nude mice is inhibited by Gleevec treatment. As with Int3, Gleevec treatment appears to destabilize the c-Myc protein but not mRNA. These results indicate that Gleevec could be a potential therapeutic drug for patients bearing Notch4 and/or c-Myc positive breast carcinomas.
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Lin, Michelle I., Charles W. Carspecken, Audrey Uong, Emily Price, and Leonard I. Zon. "Genetic Interaction Between Angiopoietin-Like Proteins 1 and 2 and Notch Signaling During HSC Development." Blood 116, no. 21 (November 19, 2010): 573. http://dx.doi.org/10.1182/blood.v116.21.573.573.
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Abstract Abstract 573 Members of the angiopoietin-like protein (angptl) family have recently been shown to stimulate ex vivo expansion of mouse and human hematopoietic stem cells (HSCs). The requirement of angptls for HSCs during development and their mechanism of action have yet to be determined. To first recapitulate the effects of exogenous angptl on HSC expansion, we made a stable transgenic zebrafish line, Tg(hsp70:angptl2), that overexpresses angptl2 upon heatshock induction. Heatshocked Tg embryos showed a significant increase in cmyb- and runx1-positive HSCs in the aorta-gonad-mesonephros (AGM) region, the site of definitive hematopoiesis at 36 hours post-fertilization (hpf), suggesting that angptl2 can sufficiently expand definitive HSCs during development. We then assessed the effects of angptl1 and/or angptl2 morpholino (MO) knockdown. Single MO knockdown resulted in a decrease in cmyb- and runx1-positive HSCs in the AGM whereas embryos injected with combined angptl1 and 2 MOs (double morphants) exhibited more severe phenotype in which HSCs were completely absent. Angptl regulation of HSC development may occur by stimulating the production or specification of a patent hemogenic endothelium, as the double morphants displayed a disruption of vascular specification at 28 hpf, with decreased expression of the arterial marker ephrinB2 but increased ectopic expression of the venous marker, flt4 in the dorsal aorta. These double morphants also exhibited disrupted intersegmental blood vessel sprouting. Because proper patterning of the developing blood vessels is a prerequisite for subsequent HSC formation, these results strongly suggest an early downstream effect of angptl signaling on hemogenic endothelium specification. To dissect the mechanism of angptl signaling, we asked whether there is a genetic interaction between notch and angptl signaling since the observed phenotype in the angptl double morphants resembled that in the notch mutant mindbomb, mib. We first monitored notch signaling using a transgenic notch reporter zebrafish line that expresses EGFP when the active component of notch signaling, notch intracellular domain (NICD), is expressed. MO knockdown of angptls 1 and 2 resulted in an absence of notch signaling, particularly in the vasculature. Interestingly, overexpressing angptl2 by crossing Tg(hsp70:angptl2) into the mib mutant showed a significant rescue of the HSC phenotype, implying that angptl2 acts downstream of mib signaling. When MOs against angptls 1 and 2 were then injected into the NICD overexpressing Tg embryos, we found that NICD restored HSC formation in the angptl double morphants. Together, these data corroborate the hypothesis that angptls can regulate notch signaling by acting downstream of notch ligand-receptor interaction and upstream of NICD during definitive hematopoiesis. To further probe at the downstream signaling of angptls since their receptor is currently unknown, we found that both angptls can stimulate activation of akt in cultured endothelial cells. To investigate whether akt is involved in angptl signaling during development, we injected the constitutively active myr-akt into angptl double morphants and observed rescue of both HSC and vascular defects. This strongly indicates that akt is the key signaling component downstream of angptl signaling during these biological processes. Injection of myr-akt can also rescue HSC defects in mib, suggesting that notch signaling also requires akt activation for definitive HSC formation. Taken together, our data demonstrate that the angptls are required for HSC development through notch and akt signaling, by coordinating the production of a functional hemogenic endothelium. Disclosures: Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
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Huntsman, Heather D., Daisuke Araki, Richard H. Smith, and Andre Larochelle. "Hypoxia and Notch Signaling Synergistically Expand Long-Term Repopulating Human Hematopoietic Stem/Progenitor Cells." Blood 132, Supplement 1 (November 29, 2018): 2557. http://dx.doi.org/10.1182/blood-2018-99-119388.
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Abstract Ex vivo culture of human hematopoietic stem/progenitor cells (HSPCs) under either low O2 tension or Notch signal activation driven by Delta1 ligand (Delta1) have been independently shown to primarily expand short-term HSPCs. In contrast, we have previously demonstrated a significant (5-fold) expansion of long-term repopulating HSPCs upon combination of these two strategies, suggesting synergy between Notch and hypoxia signaling pathways. Understanding the molecular mechanisms underlying this synergy in HSPCs could provide new insights to further enhance expansion by combined activation of these pathways. Given the stoichiometric relationship between the number of Notch receptors (Notch 1-4) and Notch signaling intensity, we assessed whether hypoxia regulated Notch receptor expression. G-CSF mobilized human CD34+ cells from 3 healthy subjects were cultured for 0, 4, 24, or 48hrs in the presence of cytokines (SCF, FLT3L, TPO), under hypoxic (1.5-2.0% O2) or normoxic (21% O2) conditions. Using flow cytometry, Notch 3 expression was not detected in CD34+ cells and no differences in the expression level of any of the other Notch receptor isoforms (Notch 1, 2 and 4) were observed regardless of O2 tension. Another unique aspect of Notch signaling is that the receptor must undergo enzymatic cleavage to initiate signal transduction. These cleavage events culminate in the nuclear translocation of the intracellular domain of notch (ICDN) where it binds to Notch promoter regions, leading to the expression of Notch regulated genes. It has been suggested that a shift in energy metabolism from aerobic to glycolytic may lead to an enhancement of these cleavage events. To directly measure the differences in Notch cleavage events, CD34+ cells from 2 healthy donors were cultured for 24 hours under hypoxic or normoxic conditions, followed by a 1-hour exposure to Delta1. Cells were then collected, processed and stained with antibodies specific to the cleaved ICDN of Notch 1, 2, and 4. Using ImageStream analysis, preliminary data suggest that hypoxia accelerates the rate of Notch 2 receptor intracellular signaling by as much as 2-fold at this early time point. Another potential mechanism by which hypoxia may modulate Notch signaling is via protein-protein interaction of the ICDN and HIF1α, a master transcriptional regulator of cellular response to hypoxia. To assess this possible interaction, CD34+ cells were cultured in chamber slides as described above. After exposure to Delta1, cells were fixed, permeabilized, and labeled with antibodies specific for cleaved ICDN and HIF1α. Ten cells per condition (i.e. normoxia or hypoxia, +/- Delta1) were imaged with a Zeiss 780 inverted confocal microscope. Using Imaris and Interactive Data Language (IDL) software, all high resolution images were analyzed to determine the subcellular localization of each protein. As expected, exposure to Delta1 increased the levels of cleaved ICDN, and hypoxia increased the levels of HIF1α detected. Interestingly, the combination of hypoxia and Delta1 exposure resulted in a significant increase in the ICDN-HIF1α Pearson's coefficient of colocalization (PCC = 0.51) compared to control groups without Delta1 (PCC [normoxia] = 0.17, p<0.0001; PCC [hypoxia] = 0.26, p<0.05). Finally, to assess whether the observed ICDN-HIF1α interaction led to a recruitment of HIF1α to Notch promoter regions and thereby upregulate Notch-driven genes, ChIP-qPCR was performed on cells cultured under the same conditions described above. Samples were fixed, lysed, and immunoprecipitated through an anti-HIF1α column. Selected protein/DNA conjugates were then eluted and qPCR was performed on the captured DNA fragments. In cultures exposed to hypoxia and Delta1, ChiP-qPCR results indicated a significant enhancement of HIF1α /promoter interaction at the Hey-2 gene, a major downstream target of HIF1α. No DNA from this region was detected in the normoxia+Delta1 control sample (0.02% of input vs. below the level of background, p<0.05). Taken together, these data suggest that the synergy between hypoxia and Notch in human HSPCs may be driven by an enhancement of Notch signaling events, a protein-protein interaction between ICDN and HIF1α, and a recruitment of HIF1α to the promoter region of Hey-2, a gene traditionally thought to be regulated by Notch alone. Experiments are ongoing to further understand these mechanisms and their relationship to HSPC expansion. Disclosures No relevant conflicts of interest to declare.
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Spengler, Katrin, Nderim Kryeziu, Silke Große, Alexander S. Mosig, and Regine Heller. "VEGF Triggers Transient Induction of Autophagy in Endothelial Cells via AMPKα1." Cells 9, no. 3 (March 11, 2020): 687. http://dx.doi.org/10.3390/cells9030687.
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AMP-activated protein kinase (AMPK) is activated by vascular endothelial growth factor (VEGF) in endothelial cells and it is significantly involved in VEGF-induced angiogenesis. This study investigates whether the VEGF/AMPK pathway regulates autophagy in endothelial cells and whether this is linked to its pro-angiogenic role. We show that VEGF leads to AMPKα1-dependent phosphorylation of Unc-51-like kinase 1 (ULK1) at its serine residue 556 and to the subsequent phosphorylation of the ULK1 substrate ATG14. This triggers initiation of autophagy as shown by phosphorylation of ATG16L1 and conjugation of the microtubule-associated protein light chain 3B, which indicates autophagosome formation; this is followed by increased autophagic flux measured in the presence of bafilomycin A1 and by reduced expression of the autophagy substrate p62. VEGF-induced autophagy is transient and probably terminated by mechanistic target of rapamycin (mTOR), which is activated by VEGF in a delayed manner. We show that functional autophagy is required for VEGF-induced angiogenesis and may have specific functions in addition to maintaining homeostasis. In line with this, inhibition of autophagy impaired VEGF-mediated formation of the Notch intracellular domain, a critical regulator of angiogenesis. Our study characterizes autophagy induction as a pro-angiogenic function of the VEGF/AMPK pathway and suggests that timely activation of autophagy-initiating pathways may help to initiate angiogenesis.
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Mangum, Kevin D., Emily J. Freeman, Justin C. Magin, Joan M. Taylor, and Christopher P. Mack. "Transcriptional and posttranscriptional regulation of the SMC-selective blood pressure-associated gene, ARHGAP42." American Journal of Physiology-Heart and Circulatory Physiology 318, no. 2 (February 1, 2020): H413—H424. http://dx.doi.org/10.1152/ajpheart.00143.2019.
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We previously showed that ARHGAP42 is a smooth muscle cell (SMC)-selective, RhoA-specific GTPase activating protein that regulates blood pressure and that a minor allele single nucleotide variation within a DNAse hypersensitive regulatory element in intron1 (Int1DHS) increased ARHGAP42 expression by promoting serum response factor binding. The goal of the current study was to identify additional transcriptional and posttranscriptional mechanisms that control ARHGAP42 expression. Using deletion/mutation, gel shift, and chromatin immunoprecipitation experiments, we showed that recombination signal binding protein for immunoglobulin κ-J region (RBPJ) and TEA domain family member 1 (TEAD1) binding to a conserved core region was required for full IntDHS transcriptional activity. Importantly, overexpression of the notch intracellular domain (NICD) or plating SMCs on recombinant jagged-1 increased IntDHS activity and endogenous ARHGAP42 expression while siRNA-mediated knockdown of TEAD1 inhibited ARHGAP42 mRNA levels. Re-chromatin immunoprecipitation experiments indicated that RBPJ and TEAD1 were bound to the Int1DHS enhancer at the same time, and coimmunoprecipitation assays indicated that these factors interacted physically. Our results also suggest TEAD1 and RBPJ bound cooperatively to the Int1DHS and that the presence of TEAD1 promoted the recruitment of NICD by RBPJ. Finally, we showed that ARHGAP42 expression was inhibited by micro-RNA 505 (miR505) which interacted with the ARHGAP42 3′-untranslated region (UTR) to facilitate its degradation and by AK124326, a long noncoding RNA that overlaps with the ARHGAP42 transcription start site on the opposite DNA strand. Since siRNA-mediated depletion of AK124326 was associated with increased H3K9 acetylation and RNA Pol-II binding at the ARHGAP42 gene, it is likely that AK124326 inhibits ARHGAP42 transcription. NEW & NOTEWORTHY First, RBPJ and TEAD1 converge at an intronic enhancer to regulate ARHGAP42 expression in SMCs. Second, TEAD1 and RBPJ interact physically and bind cooperatively to the ARHGAP42 enhancer. Third, miR505 interacts with the ARHGAP42 3′-UTR to facilitate its degradation. Finally, LncRNA, AK124326, inhibits ARHGAP42 transcription.
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Maeda, Takahiro, Taha Merghoub, Lin Dong, Manami Maeda, Robin Hobbs, Johannes Zakrzewski, Marcel van den Brink, et al. "LRF/Pokemon Plays a Pivotal Role in B Versus T Lymphoid Lineage Fate Decision at the Early Lymphoid Progenitor Stage by Opposing Notch1 Signaling." Blood 108, no. 11 (November 16, 2006): 778. http://dx.doi.org/10.1182/blood.v108.11.778.778.
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Abstract LRF (Leukaemia/Lymphoma Related Factor, formerly described as Pokemon, FBI-1 and OCZF, encoded by the Zbtb7a gene) is a transcriptional repressor that belongs to the POK (POZ/BTB and Krüppel) protein family. We recently reported that LRF is a proto-oncogene, which is highly expressed in Non-Hodgkin’s Lymphoma tissues (Nature. 433, 278–85). To elucidate its function in fetal and adult lymphopoiesis, we analyzed LRF knockout mice (Zbtb7a−/−) and the conditional knockout mutants (Zbtb7a flox/−Mx-1cre+), respectively. Zbtb7a −/− mice are embryonic lethal due to severe anemia around 16.5 d.p.c. Absolute number of mature B cells was markedly decreased in 15.5 d.p.c Zbtb7a−/− fetal livers (FL), while total number of the earliest immature B cells (Lin-AA4.1+CD19−B220+) was comparable to wild type (WT) littermates. Flow-Sorted Zbtb7a−/− FL Hematopoietic Stem Cells (FL-HSCs) did not give rise to ProB cells in vitro in an OP9 cell culture system. Furthermore, competitive repopulation assay suggested that the defect in B cell development in Zbtb7a−/− FL was cell-autonomous. Conditional inactivation of LRF in adult mice resulted in a significant decrease of B220+ B cells in the peripheral blood (PB). Absolute numbers of both ProB and PreB cells in the BM were drastically reduced in Zbtb7a flox/−Mx-1cre+ mice after pIpC injections, while PreProB cells were rather accumulated. Zbtb7a flox/−Mx-1cre+ PreProB cells did not give rise to ProB cells in vitro in OP9 cell culture. Unexpectedly, Zbtb7a flox/−Mx-1cre+ PreProB cells ectopically expressed T cell genes (e.g. pTCRα, Notch1, Notch3, Hes1, Gata3, TCF1), while they lacked B-cell specific gene expression (e.g. E2A, Ebf1, Pax5, Rag, VpreB1). In agreement with this finding, Zbtb7a flox/−Mx-1cre+ PreProB cells efficiently differentiated into DP-T cells upon 6 days of culture on OP9-DL1 cells, which overexpress Notch1 ligand Delta-like1. We did not observe a gross defect in the T cell compartment in PB and Thymus of Zbtb7a flox/−Mx-1cre+ mice. However, we observed an accumulation of CD4/8 double positive (DP) T cells in their BM. DP-T cells consisted of nearly 30% of the BM mononuclear cells (MNCs) in Zbtb7a flox/−Mx-1cre+ mice. Since the phenotype of LRF conditional knockout mice is reminiscent of that of ICN1 (Intracellular domain of Notch1) overexpression in the mouse BM, we hypothesized that LRF could oppose Notch1 signaling pathway at the early lymphoid progenitor stage. We found that pTCRα, a Notch1 target gene, is highly up-regulated in Zbtb7a flox/−Mx-1cre+ CLPs and that LRF transcriptionally represses mouse pTCRα promoter activity. Our finding strongly indicates that LRF loss at the early lymphoid progenitor stage causes aberrant de-repression of T-cell specific genes, which results in the block of B cell development and generation of T cells in the BM. We therefore propose that LRF is essential in instructing the early lymphoid progenitors into B cell lineage by repressing T cell-instructive signal produced by Notch.
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D'Avola, Annalisa, Alison Yeomans, Samantha Drennan, Matthew Rose-Zerilli, Jonathan C. Strefford, Freda K. Stevenson, Andrew J. Steele, Graham Packham, and Francesco Forconi. "Global and MYC-Specific Translation Is Enhanced in Activated Chronic Lymphocytic Leukemia Cells Carrying NOTCH1 C.7541_7542delct Mutations." Blood 128, no. 22 (December 2, 2016): 970. http://dx.doi.org/10.1182/blood.v128.22.970.970.
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Abstract Introduction: mRNA translation is increased in activated tumor cells of the aggressive form of Chronic Lymphocytic Leukemia (CLL), typically unmutated (U) immunoglobulin gene heavy-chain variable region (IGHV) with a strong sIgM signaling capacity (Yeomans et al, Blood 2016). C-MYC protein is a master regulator of cell performance and its expression is controlled at both transcriptional and translational levels. C-MYC protein is over-expressed in the proliferation centers of CLL and high c-MYC mRNA expression is associated with poor prognosis. In leukemic cell lines, c-MYC is an essential mediator and direct target of NOTCH1. Pro-activating c.7541_7542delCTmutations in NOTCH1 PEST domain of chromosome 9 exon 34 (NOTCH1ΔCT) are enriched in U-CLL with high sIgM levels/signaling capacity and associate with poorer prognosis in CLL (D'Avola et al, Blood, 2016), likely due to accumulation of more stable NOTCH1 protein and enhanced signaling in tissue activated CLL cells (Arruga et al, Leukemia, 2014). Aims and Methods: We investigated the consequences of NOTCH1ΔCT on global mRNA and c-MYC translation using a novel flow cytometry-based O-propargyl-puromycin (OPP) incorporation assay ('Click-iT' assay) and by c-MYC-specific immunoblotting in U-CLL. Since prolonged culture of CLL cells in vitro in the absence of stimuli led to spontaneous inactivation of NOTCH1 pathway, CpG-mediated TLR9 induction was used as a tool for activation of CLL cells in vitro. Cycloheximide (CHX) was used as a negative control for mRNA translation. For this study, 2 cohorts were investigated: i) a test "CLLΔCT cohort" of U-CLL with NOTCH1ΔCT (variant allele frequency [VAF] by droplet digital PCR, range 42.6-48.9%, median 47% of the CD19+CD5+ CLL cell population), but no additional genetic lesion other than 13q deletion, and ii) a control "CLLWT cohort" of U-CLL with no NOTCH1ΔCT (VAF<1% in all cases) or additional genetic lesion other than 13q deletion. CLL cells were incubated with 7.5 μg/ml CpG-ODN 2006 for 24 hours and assays were performed at baseline, 3 and 24 hours. NOTCH1 pathway γ-secretase inhibition was performed with DAPT GSi. Results: The CLLΔCT cohort had higher sIgM levels (range 31-372 MFI, median 81 MFI) and signaling capacity (Fab'2 anti-IgM induced intracellular calcium mobilization sIgM [iCa2+] range 47-54%, median 51) than the CLLWT cohort (sIgM levels range 19-288 MFI, median 47 MFI; IgM iCa2+ range 2-78%, median 25%). Following TLR9-mediated cell activation, the CLLΔCT cohort had sustained NICD (NOTCH1-intracellular cleaved domain) protein accumulation for up to 24 hours and expressed higher NOTCH1 target gene HES1 (hairy enhancer of split) transcript levels than in the CLLWT cohort. These data indicated NOTCH1 canonical pathway sustainment in the CLLΔCT upon activation. Global mRNA translation after 24 hours in the presence of CpG was 11.5 fold higher than that without CpG in the CLLΔCT cohort and only 4 fold higher in the CLLWT cohort, revealing significantly higher levels of translation in CLLΔCT than in CLLWT (p=0.03). CpG-induced global mRNA translation in the CLLWT cohort was similar to that in the CLLΔCT cohort treated with CHX. By using CpG-induced global mRNA translation in the presence of CHX inhibitor as background levels for each group, DAPT GSiat 2.5 to 10 μM showed from 47% to 63% inhibition of the residual CpG-induced global translation in CLLΔCT (p<0.05), but no effect in CLLWT. Remarkably, c-MYC mRNA translation after 3 hour culture with CpG was higher in CLLΔCT than in CLLWT (p= 0.02), and a similar trend was maintained in the cases investigated at 24 hour. Treatment of CLLΔCT cells with DAPT GSi decreased expression of c-MYC in a dose-dependent manner. Conclusion: NOTCH1ΔCT mutations associate with a very aggressive clinical behavior in CLL. These results now indicate that pro-activating mutations of NOTCH1 pathway associate with increased global mRNA translation and c-MYC expression. They highlight a mechanism by which NOTCH1 pathway may induce c-MYC overexpression in CLL, likely leading to increased proliferation and survival. The association of increased NOTCH1 variant allele frequency with sIgM levels and signaling capacity indicate that these mechanisms are predominant in the less anergic subgroup of U-CLL and make NOTCH1 mediated c-MYC translation an attractive target for therapeutic inhibition. Disclosures Steele: Portola Pharmaceuticals: Honoraria. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding.
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Ross, David A., and Tom Kadesch. "The Notch Intracellular Domain Can Function as a Coactivator for LEF-1." Molecular and Cellular Biology 21, no. 22 (November 15, 2001): 7537–44. http://dx.doi.org/10.1128/mcb.21.22.7537-7544.2001.
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ABSTRACT Notch signaling commences with two ligand-mediated proteolysis events that release the Notch intracellular domain, NICD, from the plasma membrane. NICD then translocates into the nucleus and interacts with the DNA binding protein CSL to activate transcription. We found that NICD expression also potentiates activity of the transcription factor LEF-1. NICD stimulation of LEF-1 activity was context dependent and occurred on a subset of promoters distinct from those activated by β-catenin. Importantly, the effect of NICD does not appear to be mediated through canonical components of the Wnt signaling pathway or downstream components of the Notch pathway. In vitro assays show a weak association between the C-terminal transactivation domain of NICD and the high-mobility group domain of LEF-1, suggesting that the two proteins interact in vivo. Our data therefore describe a new nuclear target of Notch signaling and a new coactivator for LEF-1.
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Tagami, Shinji, Masayasu Okochi, Kanta Yanagida, Akiko Ikuta, Akio Fukumori, Naohiko Matsumoto, Yoshiko Ishizuka-Katsura, et al. "Regulation of Notch Signaling by Dynamic Changes in the Precision of S3 Cleavage of Notch-1." Molecular and Cellular Biology 28, no. 1 (October 29, 2007): 165–76. http://dx.doi.org/10.1128/mcb.00863-07.
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ABSTRACT Intramembrane proteolysis by presenilin-dependent γ-secretase produces the Notch intracellular cytoplasmic domain (NCID) and Alzheimer disease-associated amyloid-β. Here, we show that upon Notch signaling the intracellular domain of Notch-1 is cleaved into two distinct types of NICD species due to diversity in the site of S3 cleavage. Consistent with the N-end rule, the S3-V cleavage produces stable NICD with Val at the N terminus, whereas the S3-S/S3-L cleavage generates unstable NICD with Ser/Leu at the N terminus. Moreover, intracellular Notch signal transmission with unstable NICDs is much weaker than that with stable NICD. Importantly, the extent of endocytosis in target cells affects the relative production ratio of the two types of NICD, which changes in parallel with Notch signaling. Surprisingly, substantial amounts of unstable NICD species are generated from the Val→Gly and the Lys→Arg mutants, which have been reported to decrease S3 cleavage efficiency in cultured cells. Thus, we suggest that the existence of two distinct types of NICD points to a novel aspect of the intracellular signaling and that changes in the precision of S3 cleavage play an important role in the process of conversion from extracellular to intracellular Notch signaling.
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Kothari, Alok, Hamza Celik, Andrew Martens, Cates Mallaney, Elizabeth L. Ostrander, Elizabeth Eultgen, and Grant A. Challen. "Pathways for Oncogenesis in T-Cell Acute Lymphoblastic Leukemia Driven By DNA Methylation and Notch Signaling." Blood 126, no. 23 (December 3, 2015): 1226. http://dx.doi.org/10.1182/blood.v126.23.1226.1226.
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Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia predominantly found in children that has a worse prognosis than other forms of pediatric leukemia with few options for relapsed disease. These issues lead to a need for new targeted therapies. Many of the genes critical for normal T-cell differentiation are aberrantly expressed when patients develop T-ALL, and this knowledge can serve as the basis for rationale design of targeted therapies. In this list are DNA methyltransferase 3A (DNMT3A), a protein that is responsible for de novo DNA methylation, critical for the function of hematopoietic stem cells (HSCs), and NOTCH1, a protein involved in T-cell signaling and regulating cell fate decisions. Patients that with DNMT3A loss-of-functions mutations driving T-ALL have a worse prognosis than their counterparts with normal DNMT3A function. Previous attempts to directly target the action of NOTCH1 have not led to success, specifically gamma secretase inhibitors, so we aim to define the epigenetic regulation of this pathway and the co-operation between DNMT3A and NOTCH1 mutations may open the door for new therapies in patients with the poorest outcomes. We show here that ablation of Dnmt3a in murine HSCs using Mx1-Cre (Mx1-CRE:Dnmt3afl/fl) results in abnormal T-cell development, leading to an accumulation of progenitors at the double negative 2 (DN2) stage (CD4-, CD8-, c-Kit+, CD44+, CD25+) in a proportion of mice (but not all). In affected mice, although there is a significant accumulation of these progenitors, the downstream population of mature T-cells is essentially unchanged compared to wild-type mice. This developmental arrest is associated with increased expression of the Notch1 signaling pathway. Initially suspected as the mechanism given the native action of Dnmt3a, was differential DNA methylation of Notch1 and its downstream targets such as Hes1 and c-Myc, however, through performed bisulfite sequencing of the promoters for those genes and discovered that there was no difference in DNA methylation between wild-type and Dnmt3a-null thymic progenitor cells. The cellular difference underlying accumulation of this Dnmt3a-null thymic progenitor population appears to be increased resistance to apoptosis, and molecular studies are ongoing to explore the molecular mechanism behind this. The observations that thymic progenitor cells lacking Dnmt3a have modified expression of Notch1, and mutations of both DNMT3A and NOTCH1 have been found in T-ALL patient samples, indicates potential co-operation between these two pathways in T-ALL oncogenesis. To test the biological co-operation between these pathways in vivo, we introduced activated Notch1 intracellular domain (NICD) into wild-type and Dnmt3a-null hematopoietic progenitor cells and assessed the latency to T-ALL by bone marrow transplantation. Introducing NICD into a Dnmt3a-null background significantly accelerated T-ALL oncogenesis (median survival wild-type NICD = 72-days, median survival Dnmt3a-null NICD = 46-days, p < 0.001, Figure 1). To explore the molecular differences leading to enhanced T-ALL pathogenesis in a Dnmt3a-null background, we performed RNA-Seq on T-ALL blasts and found that murine leukemia samples with the combination of Dnmt3a loss-of-function and constitutive Notch1 activation show increased in Jak/Stat pathway signaling compared to T-ALL blasts with native Dnmt3a and normal double positive T-cells (CD8+, CD4+). We hypothesize that Jak/Stat pathway inhibitors could improve outcomes for T-ALL patients with combination DNMT3A loss-of-function and activating NOTCH1 mutations. Ruxolitinib is a well-known JAK1/JAK2 inhibitor used in the treatment of myelofibrosis where it has produced robust results. Given the alterations in Jak/Stat signaling, we plan to induce leukemia by elimination of Dnmt3a using the Cre based model, followed by viral introduction of the Notch1 mutant and then treat mice with ruxolitinib and compare survival. It is our hypothesis that the drug will increase latency to leukemia in this quick to develop murine model. Using developed drugs to target new pathways in T-ALL has the potential to offer new strategies to patients with difficult to treat relapsed disease. Disclosures No relevant conflicts of interest to declare.
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Oswald, Franz, Birgitt Täuber, Thomas Dobner, Soizic Bourteele, Ulrike Kostezka, Guido Adler, Susanne Liptay, and Roland M. Schmid. "p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1." Molecular and Cellular Biology 21, no. 22 (November 15, 2001): 7761–74. http://dx.doi.org/10.1128/mcb.21.22.7761-7774.2001.
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ABSTRACT Notch-1 belongs to a family of transmembrane receptor proteins that direct the decisions as to various cell fates. After ligand binding, a proteolytic cleavage step occurs and the intracellular part of Notch-1, Notch-1-IC, translocates into the nucleus, where it targets the DNA binding protein RBP-Jκ/CBF1. RBP-Jκ mediates repression through recruitment of a histone deacetylase-containing complex. The Notch-1-IC/RBP-Jκ complex overcomes repression and activates the transcription of Notch target genes. We have identified a novel domain in Notch-1-IC, the EP domain, which is indispensable for full transcriptional activation. This transactivation domain is localized adjacent to the ankyrin repeats of Notch-1-IC. In cotransfection experiments, Notch-1-IC-mediated transcriptional activation was inhibited by E1A12S and p53, two proteins, which interfere with the function of the common coactivator p300. Protein-protein interaction assays demonstrated the association of Notch-1-IC and the CH3 region of p300. In addition, the interaction of mammalian Notch-1-IC with p300 was destabilized after deletion of the EP domain of Notch-1-IC. Based on physical interaction with Notch-1-IC and coactivator functions of p300, we propose a model for Notch-1-mediated gene regulation via p300.
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Beatus, P., J. Lundkvist, C. Oberg, and U. Lendahl. "The notch 3 intracellular domain represses notch 1-mediated activation through Hairy/Enhancer of split (HES) promoters." Development 126, no. 17 (September 1, 1999): 3925–35. http://dx.doi.org/10.1242/dev.126.17.3925.
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The Notch signaling pathway is important for cellular differentiation. The current view is that the Notch receptor is cleaved intracellularly upon ligand activation. The intracellular Notch domain then translocates to the nucleus, binds to Suppressor of Hairless (RBP-Jk in mammals), and acts as a transactivator of Enhancer of Split (HES in mammals) gene expression. In this report we show that the Notch 3 intracellular domain (IC), in contrast to all other analysed Notch ICs, is a poor activator, and in fact acts as a repressor by blocking the ability of the Notch 1 IC to activate expression through the HES-1 and HES-5 promoters. We present a model in which Notch 3 IC interferes with Notch 1 IC-mediated activation at two levels. First, Notch 3 IC competes with Notch 1 IC for access to RBP-Jk and does not activate transcription when positioned close to a promoter. Second, Notch 3 IC appears to compete with Notch 1 IC for a common coactivator present in limiting amounts. In conclusion, this is the first example of a Notch IC that functions as a repressor in Enhancer of Split/HES upregulation, and shows that mammalian Notch receptors have acquired distinct functions during evolution.
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Schroeter, Eric H., Jeffrey A. Kisslinger, and Raphael Kopan. "Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain." Nature 393, no. 6683 (May 1998): 382–86. http://dx.doi.org/10.1038/30756.
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Nakazawa, Minako, Hiroyasu Ishii, Hiroyuki Aono, Miwa Takai, Takahiro Honda, Satoko Aratani, Akiyoshi Fukamizu, et al. "Role of notch-1 intracellular domain in activation of rheumatoid synoviocytes." Arthritis & Rheumatism 44, no. 7 (2001): 1545–54. http://dx.doi.org/10.1002/1529-0131(200107)44:7<1545::aid-art278>3.0.co;2-q.
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Crow, Jacob J., and Allan R. Albig. "Notch family members follow stringent requirements for intracellular domain dimerization at sequence-paired sites." PLOS ONE 15, no. 11 (November 24, 2020): e0234101. http://dx.doi.org/10.1371/journal.pone.0234101.
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Notch signaling is essential for multicellular life, regulating core functions such as cellular identity, differentiation, and fate. These processes require highly sensitive systems to avoid going awry, and one such regulatory mechanism is through Notch intracellular domain dimerization. Select Notch target genes contain sequence-paired sites (SPS); motifs in which two Notch transcriptional activation complexes can bind and interact through Notch’s ankyrin domain, resulting in enhanced transcriptional activation. This mechanism has been mostly studied through Notch1, and to date, the abilities of the other Notch family members have been left unexplored. Through the utilization of minimalized, SPS-driven luciferase assays, we were able to test the functional capacity of Notch dimers. Here we show that the Notch 2 and 3 NICDs also exhibit dimerization-induced signaling, following the same stringent requirements as seen with Notch1. Furthermore, our data suggested that Notch4 may also exhibit dimerization-induced signaling, although the amino acids required for Notch4 NICD dimerization appear to be different than those required for Notch 1, 2, and 3 NICD dimerization. Interestingly, we identified a mechanical difference between canonical and cryptic SPSs, leading to differences in their dimerization-induced regulation. Finally, we profiled the Notch family members’ SPS gap distance preferences and found that they all prefer a 16-nucleotide gap, with little room for variation. In summary, this work highlights the potent and highly specific nature of Notch dimerization and refines the scope of this regulatory function.
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Chu, Jianlin, Shawn Jeffries, Jason E. Norton, Anthony J. Capobianco, and Emery H. Bresnick. "Repression of Activator Protein-1-mediated Transcriptional Activation by the Notch-1 Intracellular Domain." Journal of Biological Chemistry 277, no. 9 (December 5, 2001): 7587–97. http://dx.doi.org/10.1074/jbc.m111044200.
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Chapman, Gavin, Lining Liu, Cecilia Sahlgren, Camilla Dahlqvist, and Urban Lendahl. "High levels of Notch signaling down-regulate Numb and Numblike." Journal of Cell Biology 175, no. 4 (November 20, 2006): 535–40. http://dx.doi.org/10.1083/jcb.200602009.
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Inhibition of Notch signaling by Numb is critical for many cell fate decisions. In this study, we demonstrate a more complex relationship between Notch and the two vertebrate Numb homologues Numb and Numblike. Although Numb and Numblike at low levels of Notch signaling negatively regulated Notch, high levels of Notch signaling conversely led to a reduction of Numb and Numblike protein levels in cultured cells and in the developing chick central nervous system. The Notch intracellular domain but not the canonical Notch downstream proteins Hes 1 and Hey 1 caused a reduction of Numb and Numblike. The Notch-mediated reduction of Numblike required the PEST domain in the Numblike protein and was blocked by the proteasome inhibitor MG132. Collectively, these observations reveal a reciprocal negative regulation between Notch and Numb/Numblike, which may be of relevance for stabilizing asymmetric cell fate switches and for tumor development.
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Engel, Michael E., Hong Nguyen, Jolene Mariotti, Aubrey A. Hunt, and Scott Hiebert. "Myeloid Translocation Gene (MTG)-16 Binds Intracellular Domains of Notch Receptors to Coordinate Notch-Dependent Cell Fate Specification." Blood 114, no. 22 (November 20, 2009): 257. http://dx.doi.org/10.1182/blood.v114.22.257.257.
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Abstract Abstract 257 The Notch signaling pathway regulates gene expression programs to control the specification of cell fate in diverse tissues. In response to ligand binding, the intracellular domain of Notch receptors is cleaved by the γ-secretase complex and then translocates to the nucleus. There it binds the transcriptional repressor CSL, triggering its conversion to an activator of Notch target gene expression. The events that control this conversion are poorly understood. We show that the transcriptional co-repressor, MTG16, interacts with both CSL and the intracellular domains of Notch receptors. The MTG16 NHR3 domain contributes to CSL binding, while N-ICD binding sites lie within the PST1 and PST2 domains. The Notch intracellular domain disrupts the MTG16—CSL interaction, suggesting a pivotal role in regulating the Notch transcription complex. Using co-culture of Lin-/Sca-1+/c-Kit+ (LSK) cells on OP9-DL1 stromal cells, we show that ex vivo fate specification in response to Notch signal activation is altered in Mtg16 (−/−) hematopoietic progenitors. While Notch signal activation specifies lymphoid fate in Mtg16 (+/+) LSK cells, Mtg16 (−/−) LSK cells display cell surface marker expression reminiscent of myeloid differentiation. We used this lineage allocation assay to assess the contribution of MTG16 to cell fate determination. Lymphoid fate specification is restored by MTG16WT expression in Mtg16 (−/−) LSK cells. However, an MTG16 mutant deficient in N1-ICD binding is defective in this assay, suggesting this region is important to Notch-dependent lineage allocation. These data suggest that MTG family proteins interface with critical components of the Notch transcription complex and intimate a functional relationship between MTG proteins and Notch signaling in normal and malignant hematopoiesis. Disclosures: No relevant conflicts of interest to declare.
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Zucchetto, Antonella, Chiara Caldana, Federico Pozzo, Silvia Rasi, Carmela Ciardullo, Dania Benedetti, Erika Tissino, et al. "NOTCH1 Mutations Are Associated with High CD49d Expression in Chronic Lymphocytic Leukemia." Blood 124, no. 21 (December 6, 2014): 1978. http://dx.doi.org/10.1182/blood.v124.21.1978.1978.
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Abstract Mutations of NOTCH1 have emerged as one of the most frequent somatic alterations in chronic lymphocytic leukemia (CLL), affecting up to 10–15% of patients. These mutations (~80% are 7544-7545delCT frameshift deletions) generate a truncated protein that accumulates in the cell and activates the downstream NOTCH1 signaling which is implicated in apoptosis resistance and increased survival of CLL cells. CD49d (α4 integrin chain) is one of the most relevant negative prognosticator in CLL, expressed by ~40% of CLL cases, and associated with aggressive/accelerated clinical courses, whose key role in CLL cell microenvironmental interactions has been thoroughly investigated. Literature data indicate that NOTCH1 has a role in activating the integrin signaling in several cell models. Given the higher CD49d expression characterizing trisomy 12 CLL, a CLL subset in which the NOTCH1 pathway is more often activated by NOTCH1 mutations, this study was aimed at investigating the contribution of NOTCH1 in the regulation of CD49d expression in CLL. The 7544-7545delCT NOTCH1 mutations were investigated by ARMS-PCR in 1027 CLL cases, all characterized for CD49d expression and for the cytogenetic profiles by FISH. NOTCH1 mutated cases were 158/1027 (15%), with a higher prevalence (36.7% of NOTCH1 mutated cases) in the trisomy 12 cytogenetic group. Analysis of CD49d expression highlighted a very strong association between the presence of NOTCH1 mutations and CD49d expression (p<0.0001). In particular, high CD49d expression (>30% of positive cells) was found in 102/158 (64.5%) NOTCH1 mutated cases as compared to 285/869 (32.8%) NOTCH1 wild-type cases. Of note, excluding trisomy 12 CLL, again NOTCH1 mutated CLL (100/865, 11.6%) displayed a significantly higher frequency of CD49d+ cases (52%) as compared to NOTCH1 wild-type CLL (25.7%) (p<0.0001). We next analyzed the percentage of mutated NOTCH1 DNA in the context of the CLL clone by a quantitative real-time PCR (QRT-PCR) approach set up by us to quantify the delCT NOTCH1 mutation. Using the 10% cut-off value to discriminate between cases with high (high NOTCH1) and low (low NOTCH1) mutation burden, 90/138 (67.7%) and 43/138 (32.3%) CLL cases were classified as low NOTCH1 and high NOTCH1, respectively. A higher prevalence of CD49d+ cases was found in the high NOTCH1 group (79%) as compared to the low NOTCH1 group (58%) (p=0.03). Moreover, a significant association between CD49d expression and a high NOTCH1 mutation burden was observed also excluding trisomy 12 CLL, with 69% of CD49d+ cases in the high NOTCH1 group, compared to 41% of CD49d+cases in the low NOTCH1 group (p=0.03). The association between NOTCH1 mutations and CD49d expression was next confirmed by next-generation sequencing results using the flow cytometrically sorted (>99% purity) CD49d- and CD49d+ components from 8 CLL cases characterized by both CD49d bimodal expression, and the presence of 7544-7545delCT NOTCH1 mutations at the subclonal level. In 7/8 cases, the CD49d+ component showed a higher NOTCH1 mutation burden compared to the CD49d- component, this difference reaching statistical significance in 4/7 cases. Of note, a similar clustering of mutations could not be observed in the CD49d- and CD49d+ components of other CLL cases characterized by bimodal CD49d expression and subclonal mutations of SF3B1 (n=1), BIRC3 (n=2) or TP53 (n=2). To verify whether NOTCH1 accumulation, as occurring in NOTCH1 mutated CLL, may influence CD49d expression, MEC-1 cells were transfected with a vector containing either a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or a NICD carrying a missense mutation (c.5304G>A) generating a stop codon at the beginning of the sequence, as control. The higher levels of both NOTCH1 transcript (fold increase over control=2.2) and protein (fold increase over control=1.3) characterizing mutated-NICD MEC-1 cells, was paralleled by higher levels of CD49d expression (mean fluorescence intensity=23.300 versus 12.400) in these cells. Altogether our data demonstrate a direct correlation between NOTCH1 mutations and CD49d expression also outside the trisomy 12 CLL group, and suggest that accumulation of NOTCH1 may be directly or indirectly responsible for the up-regulation of CD49d expression. Disclosures No relevant conflicts of interest to declare.
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Fassa, Angeliki, Loukia Parisiadou, Nikolaos K. Robakis, and Spiros Efthimiopoulos. "Novel Processing of Notch 1 within Its Intracellular Domain by a Cysteine Protease." Neurodegenerative Diseases 4, no. 2-3 (2007): 148–55. http://dx.doi.org/10.1159/000101839.
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Ressel, L., R. W. Else, A. Poli, and D. J. Argyle. "Aberrant Subcellular Immunolocalization of NOTCH-1 Activated Intracellular Domain in Feline Mammary Tumours." Journal of Comparative Pathology 150, no. 4 (May 2014): 366–72. http://dx.doi.org/10.1016/j.jcpa.2013.11.213.
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Varnum-Finney, B., L. Wu, M. Yu, C. Brashem-Stein, S. Staats, D. Flowers, J. D. Griffin, and I. D. Bernstein. "Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling." Journal of Cell Science 113, no. 23 (December 1, 2000): 4313–18. http://dx.doi.org/10.1242/jcs.113.23.4313.
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Cell-cell interactions mediated by Notch and its ligands are known to effect many cell fate decisions in both invertebrates and vertebrates. However, the mechanisms involved in ligand induced Notch activation are unknown. Recently it was shown that, in at least some cases, endocytosis of the extracellular domain of Notch and ligand by the signaling cell is required for signal induction in the receptive cell. These results imply that soluble ligands (ligand extracellular domains) although capable of binding Notch would be unlikely to activate it. To test the potential activity of soluble Notch ligands, we generated monomeric and dimeric forms of the Notch ligand Delta-1 by fusing the extracellular domain to either a series of myc epitopes (Delta-1(ext-myc)) or to the Fc portion of human IgG-1 (Delta-1(ext-IgG)), respectively. Notch activation, assayed by inhibition of differentiation in C2 myoblasts and by HES1 transactivation in U20S cells, occurred when either Delta-1(ext-myc) or Delta-1(ext-IgG) were first immobilized on the plastic surface. However, Notch was not activated by either monomeric or dimeric ligand in solution (non-immobilized). Furthermore, both non-immobilized Delta-1(ext-myc) and Delta-1(ext-IgG) blocked the effect of immobilized Delta. These results indicate that Delta-1 extracellular domain must be immobilized to induce Notch activation in C2 or U20S cells and that non-immobilized Delta-1 extracellular domain is inhibitory to Notch function. These results imply that ligand stabilization may be essential for Notch activation.
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Cheng, Jing, Qianfu Wu, Rong Lv, Li Huang, Banglong Xu, Xianbao Wang, Aihua Chen, and Fei He. "MicroRNA-449a Inhibition Protects H9C2 Cells Against Hypoxia/Reoxygenation-Induced Injury by Targeting the Notch-1 Signaling Pathway." Cellular Physiology and Biochemistry 46, no. 6 (2018): 2587–600. http://dx.doi.org/10.1159/000489686.
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Background/Aims: The present study aimed to detect the expression of miR-449a and investigate the effect of miR-449a on cell injury in cardiomyocytes subjected to hypoxia/ reoxygenation (H/R) and its underlying mechanisms. Methods: The expression of miR-449a was determined using reverse transcription–polymerase chain reaction in both neonatal rat ventricular myocytes and H9C2 cells. For gain-of-function and loss-of-function studies, H9C2 cells were transfected with either miR-449a mimics or miR-449a inhibitor. The target gene of miR-449a was confirmed by a dual-luciferase reporter assay. Apoptosis was analyzed by both flow cytometry using Annexin V and propidium iodide and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL). Necrosis was confirmed by the detection of lactate dehydrogenase release. The cell viability was measured using the methylthiotetrazole method. The protein levels of Notch-1, Notch-1 intracellular domain, hairy and enhancer of split-1 (Hes-1), and apoptosis-related genes were measured by Western blot analysis. Results: MiR-449a was significantly upregulated in both neonatal rat ventricular myocytes and H9C2 cells subjected to H/R. However, H/R-induced cell apoptosis and necrosis were markedly reduced by miR-449a inhibition. By targeting Notch-1, miR-449a regulated the Notch-1/ Hes-1 signaling pathway. The blockade of the Notch signaling pathway partly abolished the protective effect of miR-449a suppression against H/R injury, whereas the overexpression of Notch-1 intracellular domain partly reversed the effect of miR-449a overexpression on H/R-induced cell injury. Conclusions: The present study suggested that miR-449a inhibition protected H9C2 cells against H/R-induced cell injury by targeting the Notch-1 signaling pathway, providing a novel insight into the molecular basis of myocardial ischemia–reperfusion injury and a potential therapeutic target.
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Wu, Lizi, Tao Sun, Karla Kobayashi, Ping Gao, and James D. Griffin. "Identification of a Family of Mastermind-Like Transcriptional Coactivators for Mammalian Notch Receptors." Molecular and Cellular Biology 22, no. 21 (November 1, 2002): 7688–700. http://dx.doi.org/10.1128/mcb.22.21.7688-7700.2002.
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ABSTRACT The molecular mechanisms by which Notch receptors induce diverse biological responses are not fully understood. We recently cloned a mammalian homologue of the Mastermind gene of Drosophila melanogaster, MAML1 (Mastermind-like-1 molecule) and determined that it functions as a transcriptional coactivator for Notch receptors. In this report, we characterize two additional genes in this Mastermind-like gene family: MAML2 and MAML3. The three MAML genes are widely expressed in adult tissues but exhibit distinct expression patterns in mouse early spinal cord development. All MAML proteins localize to nuclear bodies, share a conserved basic domain in their N termini that binds to the ankyrin repeat domain of Notch, and contain a transcriptional activation domain in their C termini. Moreover, as determined by using coimmunoprecipitation assays, each MAML protein was found to be capable of forming a multiprotein complex with the intracellular domain of each Notch receptor (ICN1 to -4) and CSL in vivo. However, MAML3 bound less efficiently to the ankyrin repeat domain of Notch1. Also, in U20S cells, whereas MAML1 and MAML2 functioned efficiently as coactivators with each of the Notch receptors to transactivate a Notch target HES1 promoter construct, MAML3 functioned more efficiently with ICN4 than with other forms of ICN. Similarly, MAML1 and MAML2 amplified Notch ligand (both Jagged2 and Delta1)-induced transcription of the HES-1 gene, whereas MAML3 displayed little effect. Thus, MAML proteins may modify Notch signaling in different cell types based on their own expression levels and differential activities and thereby contribute to the diversity of the biological effects resulting from Notch activation.
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Matsuno, K., R. J. Diederich, M. J. Go, C. M. Blaumueller, and S. Artavanis-Tsakonas. "Deltex acts as a positive regulator of Notch signaling through interactions with the Notch ankyrin repeats." Development 121, no. 8 (August 1, 1995): 2633–44. http://dx.doi.org/10.1242/dev.121.8.2633.
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We present a molecular and genetic analysis which elucidates the role of deltex in the Notch signaling pathway. Using the yeast ‘interaction trap’ assay, we define the protein regions responsible for heterotypic interactions between Deltex and the intracellular domain of Notch as well as uncover homotypic interaction among Deltex molecules. The function of the Deltex-Notch interaction domains is examined by in vivo expression studies. Taken together, data from overexpression of Deltex fragments and from studies of physical interactions between Deltex and Notch, suggest that Deltex positively regulates the Notch pathway through interactions with the Notch ankyrin repeats. Experiments involving cell cultures indicate that the Deltex-Notch interaction prevents the cytoplasmic retention of the Suppressor of Hairless protein, which otherwise is sequestered in the cytoplasm via association with the Notch ankyrin repeats and translocates to the nucleus when Notch binds to its ligand Delta. On the basis of these findings, we propose a model wherein Deltex regulates Notch activity by antagonizing the interaction between Notch and Suppressor of Hairless.
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Keilani, Serene, DeLacy Healey, and Kiminobu Sugaya. "Reelin regulates differentiation of neural stem cells by activation of notch signaling through Disabled-1 tyrosine phosphorylation." Canadian Journal of Physiology and Pharmacology 90, no. 3 (March 2012): 361–69. http://dx.doi.org/10.1139/y2012-001.
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We have previously reported the cross-talk between Reelin and Notch-1 signaling pathways, which are 2 major pathways that regulate brain development. We found that Reelin activated Notch-1 signaling, leading to the expression of brain lipid binding protein (BLBP) and the formation of radial glial cells in human neural progenitor cells (hNPCs). In the current study, we investigated the molecular mechanisms by which Reelin activates Notch-1. We show that Reelin-stimulated Notch-1 activation is dependent on Reelin signaling. The induction of Disabled-1 (Dab-1) tyrosine phosphorylation, and the subsequent activation of Src family kinases, were found to be essential steps for the activation of Notch-1 signaling by Reelin. Reelin treatment increased the interaction between Dab-1 and Notch-1 intracellular domain (NICD), and enhanced NICD translocation to the nucleus. This study advances our knowledge of the regulation of Notch-1 activation by Reelin signaling in hNPCs, as an approach to understanding cell fate determination, differentiation, and neurogenesis during brain development.
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De Strooper, Bart, Wim Annaert, Philippe Cupers, Paul Saftig, Katleen Craessaerts, Jeffrey S. Mumm, Eric H. Schroeter, et al. "A presenilin-1-dependent γ-secretase-like protease mediates release of Notch intracellular domain." Nature 398, no. 6727 (April 1999): 518–22. http://dx.doi.org/10.1038/19083.
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Zhang, C., J. Chang, W. Sonoyama, S. Shi, and C. Y. Wang. "Inhibition of Human Dental Pulp Stem Cell Differentiation by Notch Signaling." Journal of Dental Research 87, no. 3 (March 2008): 250–55. http://dx.doi.org/10.1177/154405910808700312.
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Notch signaling plays a critical role in development and cell fate specification. Notch receptors and ligands have been found to be expressed in dental epithelium or mesenchyme in the developing tooth, suggesting that Notch signaling may regulate odontogenesis. Post-natal human dental pulp stem cells (DPSCs) isolated from the dental pulp have characteristics of mesenchymal stem cells and can differentiate into odontoblasts. In this study, we examined whether Notch signaling regulated the odontoblastic differentiation of DPSCs. We found that over-expression of the Notch ligand, Jagged-1, activated the Notch signaling pathway in DPSCs. Jagged-1 inhibited the odontoblastic differentiation of DPSCs in vitro. Jagged-1-expressing DPSCs could not form mineralized tissues in vivo. Moreover, over-expression of the constitutively activated Notch1 intracellular domain (Notch-ICD) also inhibited odontoblastic differentiation of DPSCs. Taken together, our results demonstrate that Notch signaling can inhibit the odontoblastic differentiation of DPSCs.
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Der Vartanian, Audrey, Aymeric Audfray, Bilal Al Jaam, Mathilde Janot, Sébastien Legardinier, Abderrahman Maftah, and Agnès Germot. "ProteinO-Fucosyltransferase 1 Expression Impacts Myogenic C2C12 Cell Commitment via the Notch Signaling Pathway." Molecular and Cellular Biology 35, no. 2 (November 10, 2014): 391–405. http://dx.doi.org/10.1128/mcb.00890-14.
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The Notch signaling pathway plays a crucial role in skeletal muscle regeneration in mammals by controlling the transition of satellite cells from quiescence to an activated state, their proliferation, and their commitment toward myotubes or self-renewal. O-fucosylation on Notch receptor epidermal growth factor (EGF)-like repeats is catalyzed by the proteinO-fucosyltransferase 1 (Pofut1) and primarily controls Notch interaction with its ligands. To approach the role of O-fucosylation in myogenesis, we analyzed a murine myoblastic C2C12 cell line downregulated forPofut1expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown ofPofut1affected the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7+/MyoD−cells and earlier myogenic program entrance were observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue ofPofut1expression in knockdown cells restored Notch signaling activation and a normal course in C2C12 differentiation. Our results establish the critical role of Pofut1 on Notch pathway activation during myogenic differentiation.
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Bertagna, Angela, Dima Toptygin, Ludwig Brand, and Doug Barrick. "The effects of conformational heterogeneity on the binding of the Notch intracellular domain to effector proteins: a case of biologically tuned disorder." Biochemical Society Transactions 36, no. 2 (March 20, 2008): 157–66. http://dx.doi.org/10.1042/bst0360157.
Full textAbstract:
Cell-fate decisions in metazoans are frequently guided by the Notch signalling pathway. Notch signalling is orchestrated by a type-1 transmembrane protein, which, upon interacting with extracellular ligands, is proteolytically cleaved to liberate a large intracellular domain [NICD (Notch intracellular domain)]. NICD enters the nucleus where it binds the transcription factor CSL (CBF1/suppressor of Hairless/Lag-1) and activates transcription of Notch-responsive genes. In the present paper, the interaction between the Drosophila NICD and CSL will be examined. This interaction involves two separate binding regions on NICD: the N-terminal tip of NICD {the RAM [RBP-Jκ (recombination signal-binding protein 1 for Jκ)-associated molecule] region} and an ankyrin domain ∼100 residues away. CD studies show that the RAM region of NICD lacks α-helical and β-sheet secondary structure, and also lacks rigid tertiary structure. Fluorescence studies show that the tryptophan residues in RAM are highly solvated and are quenched by solvent. To assess the impact of this apparent disorder on the bivalent binding of NICD to CSL, we modelled the region between the RAM and ANK (ankyrin repeat)-binding regions using polymer statistics. A WLC (wormlike chain) model shows that the most probable sequence separation between the two binding regions is ∼50 Å (1 Å=0.1 nm), matching the separation between these two sites in the complex. The WLC model predicts a substantial enhancement of ANK occupancy via effective concentration, and suggests that the linker length between the two binding regions is optimal for bivalent interaction.