The notch 3 intracellular domain represses notch 1-mediated activation through Hairy/Enhancer of split (HES) promoters

Development ◽  
1999 ◽  
Vol 126 (17) ◽  
pp. 3925-3935 ◽  
Author(s):  
P. Beatus ◽  
J. Lundkvist ◽  
C. Oberg ◽  
U. Lendahl
Keyword(s):  
Cellular Differentiation ◽  
Notch Signaling Pathway ◽  
Notch Receptor ◽  
Current View ◽  
Notch 1 ◽  
Intracellular Domain ◽  
Ligand Activation ◽  
Notch 3 ◽  
Notch Receptors ◽  
Enhancer Of Split

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.

scholarly journals Delta-1 Activation of Notch-1 Signaling Results inHES-1 Transactivation

1998 ◽  
Vol 18 (12) ◽  
pp. 7423-7431 ◽  
Author(s):  
Sophie Jarriault ◽  
Odile Le Bail ◽  
Estelle Hirsinger ◽  
Olivier Pourquié ◽  
Frédérique Logeat ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Drosophila Melanogaster ◽  
Notch Signaling ◽  
Cell Fate ◽  
Notch Signaling Pathway ◽  
Notch Receptor ◽  
Cell Fate Determination ◽  
Notch 1 ◽  
Promoter Construct ◽  
Enhancer Of Split

ABSTRACT The Notch receptor is involved in many cell fate determination events in vertebrates and invertebrates. It has been shown inDrosophila melanogaster that Delta-dependent Notch signaling activates the transcription factor Suppressor of Hairless, leading to an increased expression of the Enhancer of Splitgenes. Genetic evidence has also implicated the kuzbaniangene, which encodes a disintegrin metalloprotease, in the Notch signaling pathway. By using a two-cell coculture assay, we show here that vertebrate Dl-1 activates the Notch-1 cascade. Consistent with previous data obtained with active forms of Notch-1 aHES-1-derived promoter construct is transactivated in cells expressing Notch-1 in response to Dl-1 stimulation. Impairing the proteolytic maturation of the full-length receptor leads to a decrease in HES-1 transactivation, further supporting the hypothesis that only mature processed Notch is expressed at the cell surface and activated by its ligand. Furthermore, we observed that Dl-1-inducedHES-1 transactivation was dependent both on Kuzbanian and RBP-J activities, consistent with the involvement of these two proteins in Notch signaling in Drosophila. We also observed that exposure of Notch-1-expressing cells to Dl-1 results in an increased level of endogenous HES-1 mRNA. Finally, coculture of Dl-1-expressing cells with myogenic C2 cells suppresses differentiation of C2 cells into myotubes, as previously demonstrated for Jagged-1 and Jagged-2, and also leads to an increased level of endogenousHES-1 mRNA. Thus, Dl-1 behaves as a functional ligand for Notch-1 and has the same ability to suppress cell differentiation as the Jagged proteins do.

Gene expression analysis of Notch signaling pathway receptors in colon cancer stem cells.

2012 ◽  
Vol 30 (30_suppl) ◽  
pp. 29-29
Author(s):  
Ioannis Papasotiriou ◽  
Panagiotis Apostolou ◽  
Maria Toloudi ◽  
Marina Chatziioannou ◽  
Eleni Ioannou
Keyword(s):  
Gene Expression ◽  
Notch Signaling ◽  
Signaling Pathway ◽  
Expression Analysis ◽  
Protein Level ◽  
Gene Expression Analysis ◽  
Notch Signaling Pathway ◽  
Notch 1 ◽  
Notch 3 ◽  
Notch Receptors

29 Background: The Notch signaling pathway is not only involved in cell differentiation, but also in carcinogenesis. Four different receptors are included in this pathway (Notch-1, Notch-2, Notch-3, and Notch-4) and each is encoded by a different gene. Recent data suggest that the activation of Notch-1 induces epithelial to mesenchymal transition (EMT) consistent with cancer stem cell (CSC) phenotype. It raises therefore the question of whether there is an interaction between the receptors. The present study aims to find out the gene expression patterns of Notch receptors in Colon CSCs, under the suppression of Notch-1. Methods: The experiments were performed in Colon CSCs provided by CELPOGEN. The stemness of these cells was tested both in gene and protein level with qPCR and flow cytometry assays for the stemness markers Oct4-CD44. The gene suppressed with RNAi assays and the knockdown was evaluated with molecular biology methods. The gene expression analysis was performed after RT-qPCR assays, by using the comparative Ct method (ΔΔCt) for the calculation of relative quantitation. Results: The Colon CSCs expressed Oct4-CD44 both in gene and protein level, indicating the stemness status. The knockdown of Notch-1 up to 75%, led to a reduction of gene expression in Notch-2 and Notch-3 receptors, while no significant difference was observed in Notch-4. The Notch-2 expression was decreased up to 90% while that of Notch-3 was at the same level with Notch-1. Conclusions: This research study attempts to find out any possible correlation between the Notch receptors in Colon CSCS. The experimental data indicate that Notch-1 interacts with Notch-2 and Notch-3 by affecting heavily their gene expression. It is therefore necessary to study if the other receptors are capable for EMT and then to find out which factors are involved. Further studies to a greater range of samples need to be performed, in order to use these data at clinical level.

scholarly journals POS0042 NOTCH 1 INHIBITION INCREASES OSTEOCLAST PROGENITOR ACTIVITY IN THE MOUSE MODEL OF RHEUMATOID ARTHRITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 226.3-226
Author(s):  
M. Filipović ◽  
A. Šućur ◽  
D. Flegar ◽  
Z. Jajić ◽  
M. Ikić Matijašević ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Notch Signaling ◽  
Neutralizing Antibodies ◽  
Osteoclast Differentiation ◽  
Notch Receptor ◽  
Notch 1 ◽  
Collagen Induced Arthritis ◽  
Notch Receptors

Background:Osteoclasts mediate periarticular and systemic bone loss in rheumatoid arthritis (RA). Osteoclast progenitor cells (OCPs) derived from the myeloid lineage are susceptible to regulation through Notch signaling. Murine bone marrow and splenic OCPs, identified as CD45+Ly6G-CD3-B220-NK1.1-CD11blo/+CD115+CCR2+ cells, are specifically increased in arthritis. We previously identified an increased frequency of OCPs expressing Notch receptors in arthritic mice.Objectives:Several studies suggested that Notch signaling modulation affects the course of experimental arthritis. We aimed to determine the effects of Notch receptor signaling inhibition on OCP activity and arthritis severity in murine collagen-induced arthritis (CIA).Methods:Male C57/Bl6 and DBA mice were immunized with chicken type II collagen and treated with i.p. injections of anti-Notch 1 neutralizing antibodies (1mg/kg). Notch receptor 1 through 4 expression on OCPs was analyzed by flow cytometry in periarticular bone marrow (PBM) and spleen (SPL). Gene expression of Notch receptors, ligands and transcription targets as well as osteoclast differentiation genes RANK, cFos and cFms was determined by qPCR from tissues and sorted OCPs. FACS sorted OCPs were stimulated by osteoclastogenic factors (M-CSF and RANKL), in control, IgG, Jagged (Jag)1 or Delta-like (DLL)1 coated wells, with or without anti-Notch 1 antibodies. Research was approved by the Ethics Committee.Results:We confirmed the expression of Notch receptors on OCPs by flow cytometry with Notch 1 and 2 being most abundantly expressed (around 25% and 40% positive OCPs in PBM and 35% and 20% in SPL respectively), with a significant increase of Notch 2 expression in arthritis. Seeding OCPs on DLL1 coated wells significantly increased while seeding on Jag1 coated wells significantly decreased osteoclastogenesis as reflected on the number of TRAP+ osteoclasts and expression of osteoclast differentiation genes. The addition of anti-Notch 1 antibodies to ligand-stimulated OCPs resulted in an increased number of TRAP+ osteoclasts, partially reversing Jag1 inhibition. In vivo treatment with anti-Notch 1 antibodies did not affect total OCP frequency, but increased expression of Notch 4 both in PBM and SPL as seen by flow cytometry and qPCR. Additionally, anti-Notch 1 treatment stimulated Notch transcription factors HES and HEY. Both PBM and SPL cultured OCPs from anti-Notch 1 treated mice produced a higher number of large TRAP+ osteoclasts, doubling the area covered with osteoclasts in the latter compared to untreated mice. Increased osteoclastogenesis in vitro was further confirmed by an increased expression of osteoclast differentiation genes in the treated group.Conclusion:Our results confirm that Notch signaling may represent an important therapeutic target for the regulation of osteoclast activity in arthritis. Both in vitro and in vivo anti-Notch 1 neutralizing antibodies enhanced osteoclastogenesis in CIA model, implying an inhibitory role of Notch 1 signaling in osteoclast differentiation. As Notch 2 expression is increased on OCPs of arthritic mice, we next plan to determine the effects of Notch 2 neutralization on osteoclast activity and arthritis severity.References:[1]Ikić Matijašević M, Flegar D, Kovačić N, Katavić V, Kelava T, Šućur A, et al. Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis. Clin Exp Immunol. 2016;186(3):321–35.[2]Šućur A, Filipović M, Flegar D, Kelava T, Šisl D, Lukač N, et al. Notch receptors and ligands in inflammatory arthritis – a systematic review. Immunology Letters 2020 Vol. 223, p. 106–14.Acknowledgements:The work has been supported by Croatian Science Foundation projects IP-2018-01-2414, UIP-2017-05-1965 and DOK-2018-09-4276.Disclosure of Interests:None declared.

Functional diversity ofnotchfamily genes in fetal lung development

2004 ◽  
Vol 286 (5) ◽  
pp. L1075-L1083 ◽  
Author(s):  
Yanping Kong ◽  
Jonathon Glickman ◽  
Meera Subramaniam ◽  
Aliakbar Shahsafaei ◽  
K. P. Allamneni ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Neural Tissue ◽  
Fetal Lung ◽  
Branching Morphogenesis ◽  
Notch Receptor ◽  
Neuroendocrine Cells ◽  
Mouse Lung ◽  
Notch 1 ◽  
Embryonic Mouse ◽  
Notch 3

In Drosophila, developmental signaling via the transmembrane Notch receptor modulates branching morphogenesis and neuronal differentiation. To determine whether the notch gene family can regulate mammalian organogenesis, including neuroendocrine cell differentiation, we evaluated developing murine lung. After demonstrating gene expression for notch-1, notch-2, notch-3, and the Notch ligands jagged-1 and jagged-2 in embryonic mouse lung, we tested whether altering expression of these genes can modulate branching morphogenesis. Branching of embryonic day (E) 11.5 lung buds increased when they were treated with notch-1 antisense oligodeoxynucleotides in culture compared with the corresponding sense controls, whereas notch-2, notch-3, jagged-1, or jagged-2 antisense oligos had no significant effect. To assess cell differentiation, we immunostained lung bud cultures for the neural/neuroendocrine marker PGP9.5. Antisense to notch-1 or jagged-1 markedly increased numbers of PGP9.5-positive neuroendocrine cells alone without affecting neural tissue, whereas only neural tissue was promoted by notch-3 antisense in culture. There was no significant effect on cell proliferation or apoptosis in these antisense experiments. Cumulatively, these observations suggest that interactions between distinct Notch family members can have diverse tissue-specific regulatory functions during development, arguing against simple functional redundancy.

scholarly journals Numb regulates the balance between Notch recycling and late-endosome targeting inDrosophilaneural progenitor cells

2016 ◽  
Vol 27 (18) ◽  
pp. 2857-2866 ◽  
Author(s):  
Seth A. Johnson ◽  
Diana Zitserman ◽  
Fabrice Roegiers
Keyword(s):  
Notch Signaling ◽  
Asymmetric Cell Division ◽  
Adaptor Protein ◽  
Notch Signaling Pathway ◽  
Notch Receptor ◽  
Recycling Endosome ◽  
Notch Receptors ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Sensory Organ Precursor

The Notch signaling pathway plays essential roles in both animal development and human disease. Regulation of Notch receptor levels in membrane compartments has been shown to affect signaling in a variety of contexts. Here we used steady-state and pulse-labeling techniques to follow Notch receptors in sensory organ precursor cells in Drosophila. We find that the endosomal adaptor protein Numb regulates levels of Notch receptor trafficking to Rab7-labeled late endosomes but not early endosomes. Using an assay we developed that labels different pools of Notch receptors as they move through the endocytic system, we show that Numb specifically suppresses a recycled Notch receptor subpopulation and that excess Notch signaling in numb mutants requires the recycling endosome GTPase Rab11 activity. Our data therefore suggest that Numb controls the balance between Notch receptor recycling and receptor targeting to late endosomes to regulate signaling output after asymmetric cell division in Drosophila neural progenitors.

scholarly journals Jun Blockade of Erythropoiesis: Role for Repression of GATA-1 by HERP2

2004 ◽  
Vol 24 (17) ◽  
pp. 7779-7794 ◽  
Author(s):  
Kamaleldin E. Elagib ◽  
Mang Xiao ◽  
Isa M. Hussaini ◽  
Lorrie L. Delehanty ◽  
Lisa A. Palmer ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Cellular Differentiation ◽  
Erythroid Differentiation ◽  
Notch Signaling Pathway ◽  
Physical Interaction ◽  
Related Factor ◽  
Regulatory Pathway ◽  
Hematopoietic Progenitors ◽  
Enhancer Of Split ◽  
Gata Family

ABSTRACT Although Jun upregulation and activation have been established as critical to oncogenesis, the relevant downstream pathways remain incompletely characterized. In this study, we found that c-Jun blocks erythroid differentiation in primary human hematopoietic progenitors and, correspondingly, that Jun factors block transcriptional activation by GATA-1, the central regulator of erythroid differentiation. Mutagenesis of c-Jun suggested that its repression of GATA-1 occurs through a transcriptional mechanism involving activation of downstream genes. We identified the hairy-enhancer-of-split-related factor HERP2 as a novel gene upregulated by c-Jun. HERP2 showed physical interaction with GATA-1 and repressed GATA-1 transcriptional activation. Furthermore, transduction of HERP2 into primary human hematopoietic progenitors inhibited erythroid differentiation. These results thus define a novel regulatory pathway linking the transcription factors c-Jun, HERP2, and GATA-1. Furthermore, these results establish a connection between the Notch signaling pathway, of which the HERP factors are a critical component, and the GATA family, which participates in programming of cellular differentiation.

scholarly journals NOTCH3 signaling is essential for NF-κB activation in TLR-activated macrophages

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Susana López-López ◽  
Eva María Monsalve ◽  
María José Romero de Ávila ◽  
Julia González-Gómez ◽  
Natalia Hernández de León ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Macrophage Activation ◽  
Notch Signaling Pathway ◽  
P38 Map Kinase ◽  
Notch Receptor ◽  
Dependent Manner ◽  
Activated Macrophages ◽  
Notch Receptors ◽  
Inflammatory Processes ◽  
Tlr4 Activation

Abstract Macrophage activation by Toll receptors is an essential event in the development of the response against pathogens. NOTCH signaling pathway is involved in the control of macrophage activation and the inflammatory processes. In this work, we have characterized NOTCH signaling in macrophages activated by Toll-like receptor (TLR) triggering and determined that DLL1 and DLL4 are the main ligands responsible for NOTCH signaling. We have identified ADAM10 as the main protease implicated in NOTCH processing and activation. We have also observed that furin, which processes NOTCH receptors, is induced by TLR signaling in a NOTCH-dependent manner. NOTCH3 is the only NOTCH receptor expressed in resting macrophages. Its expression increased rapidly in the first hours after TLR4 activation, followed by a gradual decrease, which was coincident with an elevation of the expression of the other NOTCH receptors. All NOTCH1, 2 and 3 contribute to the increased NOTCH signaling detected in activated macrophages. We also observed a crosstalk between NOTCH3 and NOTCH1 during macrophage activation. Finally, our results highlight the relevance of NOTCH3 in the activation of NF-κB, increasing p65 phosphorylation by p38 MAP kinase. Our data identify, for the first time, NOTCH3 as a relevant player in the control of inflammation.

scholarly journals SEL-10 Is an Inhibitor of Notch Signaling That Targets Notch for Ubiquitin-Mediated Protein Degradation

2001 ◽  
Vol 21 (21) ◽  
pp. 7403-7415 ◽  
Author(s):  
Guangyu Wu ◽  
Svetlana Lyapina ◽  
Indranil Das ◽  
Jinhe Li ◽  
Mark Gurney ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Protein Degradation ◽  
Notch Receptor ◽  
Dominant Negative ◽  
Intracellular Domain ◽  
Notch Receptors ◽  
Intracellular Domains ◽  
Wd40 Repeats ◽  
In Cells

ABSTRACT Notch receptors and their ligands play important roles in both normal animal development and pathogenesis. We show here that the F-box/WD40 repeat protein SEL-10 negatively regulates Notch receptor activity by targeting the intracellular domain of Notch receptors for ubiquitin-mediated protein degradation. Blocking of endogenous SEL-10 activity was done by expression of a dominant-negative form containing only the WD40 repeats. In the case of Notch1, this block leads to an increase in Notch signaling stimulated by either an activated form of the Notch1 receptor or Jagged1-induced signaling through Notch1. Expression of dominant-negative SEL-10 leads to stabilization of the intracellular domain of Notch1. The Notch4 intracellular domain bound to SEL-10, but its activity was not increased as a result of dominant-negative SEL-10 expression. SEL-10 bound Notch4 via the WD40 repeats and bound preferentially to a phosphorylated form of Notch4 in cells. We mapped the region of Notch4 essential for SEL-10 binding to the C-terminal region downstream of the ankyrin repeats. When this C-terminal fragment of Notch4 was expressed in cells, it was highly labile but could be stabilized by the expression of dominant-negative SEL-10. Ubiquitination of Notch1 and Notch4 intracellular domains in vitro was dependent on SEL-10. Although SEL-10 interacts with the intracellular domains of both Notch1 and Notch4, these proteins respond differently to interference with SEL-10 function. Thus, SEL-10 functions to promote the ubiquitination of Notch proteins; however, the fates of these proteins may differ.

scholarly journals Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification

2010 ◽  
Vol 30 (7) ◽  
pp. 1852-1863 ◽  
Author(s):  
Michael E. Engel ◽  
Hong N. Nguyen ◽  
Jolene Mariotti ◽  
Aubrey Hunt ◽  
Scott W. Hiebert
Keyword(s):  
Gene Expression ◽  
Notch Signaling ◽  
Cell Fate ◽  
Ex Vivo ◽  
Notch Signaling Pathway ◽  
Notch Receptor ◽  
Intracellular Domain ◽  
Fate Specification ◽  
Dependent Cell ◽  
Critical Components

ABSTRACT The Notch signaling pathway regulates gene expression programs to influence the specification of cell fate in diverse tissues. In response to ligand binding, the intracellular domain of the Notch receptor 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 corepressor, MTG16, interacts with both CSL and the intracellular domains of Notch receptors, suggesting a pivotal role in regulation of the Notch transcription complex. The Notch1 intracellular domain disrupts the MTG16-CSL interaction. Ex vivo fate specification in response to Notch signal activation is impaired in Mtg16 −/− hematopoietic progenitors, and restored by MTG16 expression. An MTG16 derivative lacking the binding site for the intracellular domain of Notch1 fails to restore Notch-dependent cell fate. These data suggest that MTG16 interfaces with critical components of the Notch transcription complex to affect Notch-dependent lineage allocation in hematopoiesis.

scholarly journals 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

2001 ◽  
Vol 21 (13) ◽  
pp. 4337-4346 ◽  
Author(s):  
Motoo Kitagawa ◽  
Toshinao Oyama ◽  
Taichi Kawashima ◽  
Barry Yedvobnick ◽  
Anumeha Kumar ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Signaling Pathway ◽  
Mammalian Cells ◽  
Sequence Similarity ◽  
Notch Signaling Pathway ◽  
Dominant Negative ◽  
Intracellular Domain ◽  
Human Sequence ◽  
Pathway Expression ◽  
Enhancer Of Split

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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