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1.  High levels of Notch signaling down-regulate Numb and Numblike 
The Journal of Cell Biology  2006;175(4):535-540.
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.
doi:10.1083/jcb.200602009
PMCID: PMC2064589  PMID: 17116748
2.  Dishevelled limits Notch signalling through inhibition of CSL 
Development (Cambridge, England)  2012;139(23):4405-4415.
Notch and Wnt are highly conserved signalling pathways that are used repeatedly throughout animal development to generate a diverse array of cell types. However, they often have opposing effects on cell-fate decisions with each pathway promoting an alternate outcome. Commonly, a cell receiving both signals exhibits only Wnt pathway activity. This suggests that Wnt inhibits Notch activity to promote a Wnt-ON/Notch-OFF output; but what might underpin this Notch regulation is not understood. Here, we show that Wnt acts via Dishevelled to inhibit Notch signalling, and that this crosstalk regulates cell-fate specification in vivo during Xenopus development. Mechanistically, Dishevelled binds and directly inhibits CSL transcription factors downstream of Notch receptors, reducing their activity. Furthermore, our data suggest that this crosstalk mechanism is conserved between vertebrate and invertebrate homologues. Thus, we identify a dual function for Dishevelled as an inhibitor of Notch signalling and an activator of the Wnt pathway that sharpens the distinction between opposing Wnt and Notch responses, allowing for robust cell-fate decisions.
doi:10.1242/dev.081885
PMCID: PMC3509734  PMID: 23132247
Dishevelled; Notch; Wnt; Signalling crosstalk; Xenopus
3.  The Drosophila Importin-α3 Is Required for Nuclear Import of Notch In Vivo and It Displays Synergistic Effects with Notch Receptor on Cell Proliferation 
PLoS ONE  2013;8(7):e68247.
The Notch signaling pathway controls diverse cell-fate specification events throughout development. The versatility of this pathway to influence different aspects of development comes from its multiple levels of regulation. Upon ligand-induced Notch activation, the Notch intracellular domain (Notch-ICD) is released from the membrane and translocates to the nucleus, where it transduces Notch signals by regulating the transcription of downstream target genes. But the exact mechanism of translocation of Notch-ICD into the nucleus is not clear. Here, we implicate Importin-α3 (also known as karyopherin-α3) in the nuclear translocation of Notch-ICD in Drosophila. Our present analyses reveal that Importin-α3 can directly bind to Notch-ICD and loss of Importin-α3 function results in cytoplasmic accumulation of the Notch receptor. Using MARCM (Mosaic Analysis with a Repressible Cell Marker) technique, we demonstrate that Importin-α3 is required for nuclear localization of Notch-ICD. These results reveal that the nuclear transport of Notch-ICD is mediated by the canonical Importin-α3/Importin-β transport pathway. In addition, co-expression of both Notch-ICD and Importin-α3 displays synergistic effects on cell proliferation. Taken together, our results suggest that Importin-α3 mediated nuclear import of Notch-ICD may play important role in regulation of Notch signaling.
doi:10.1371/journal.pone.0068247
PMCID: PMC3698139  PMID: 23840889
4.  Notch gain of function inhibits chondrocyte differentiation via Rbpj-dependent suppression of Sox9 
Abstract
Notch signaling plays a critical role during development by directing the binary cell fate decision between progenitors and differentiated cells. Previous studies have shown sustained Notch activation in cartilage leads to chondrodysplasia. Genetic evidence indicates that Notch regulates limb bud mesenchymal stem cell differentiation into chondrocytes via an Rbpj-dependent Notch pathway. However, it is still unknown how Notch governs chondrogenesis in the axial skeleton where Notch serves a primary patterning function. We hypothesized that both Rbpj-dependent and Rbpj-independent Notch signaling mechanisms might be involved. Cartilage-specific Notch gain-of-function (GOF) mutant mice display chondrodysplasia accompanied by loss of Sox9 expression in vertebrae. To evaluate the contribution of an Rbpj-dependent Notch signaling to this phenotype, we deleted Rbpj on the Notch GOF background. These mice showed persistent spine abnormalities characterized by “butterfly” vertebrae suggesting that removal of Rbpj does not fully rescue the axial skeleton deformities caused by Notch GOF. However, Sox9 protein level was restored in Rbpj-deficient Notch GOF mice compared with Notch GOF mutants, demonstrating that regulation of Sox9 expression is canonical or Rbpj-dependent. To further understand the molecular basis of this regulation, we performed chromatin immunoprecipitation (ChIP) assays and detected the recruitment of the Rbpj/NICD transcription complex to Rbpj-binding sites upstream of the Sox9 promoter. The association of the Rbpj/NICD complex with the Sox9 promoter is associated with transcriptional repression of Sox9 in a cellular model of chondrocyte differentiation. Hence, Notch negatively regulates chondrocyte differentiation in the axial skeleton by suppressing Sox9 transcription, and Rbpj-independent Notch signaling mechanisms may also contribute to axial skeletogenesis.
doi:10.1002/jbmr.1770
PMCID: PMC3548081  PMID: 22991339
CHONDRODYSPLASIA; AXIAL SKELETON; GENETIC MOUSE MODEL; NOTCH SIGNALING; SOX9; CHONDROCYTE
5.  Notch Gain of Function Inhibits Chondrocyte Differentiation via Rbpj-Dependent Suppression of Sox9 
Notch signaling plays a critical role during development by directing the binary cell fate decision between progenitors and differentiated cells. Previous studies have shown sustained Notch activation in cartilage leads to chondrodysplasia. Genetic evidence indicates that Notch regulates limb bud mesenchymal stem cell differentiation into chondrocytes via an Rbpj-dependent Notch pathway. However, it is still unknown how Notch governs chondrogenesis in the axial skeleton where Notch serves a primary patterning function. We hypothesized that both Rbpj-dependent and Rbpj-independent Notch signaling mechanisms might be involved. Cartilage specific Notch gain-of-function (GOF) mutant mice display chondrodysplasia accompanied by loss of Sox9 expression in vertebrae. To evaluate the contribution of an Rbpj-dependent Notch signaling to this phenotype, we deleted Rbpj on the Notch GOF background. These mice showed persistent spine abnormalities characterized by “butterfly” vertebrae suggesting that removal of Rbpj does not fully rescue the axial skeleton deformities caused by Notch GOF. However, Sox9 protein level was restored in Rbpj deficient Notch GOF mice compared to Notch GOF mutants, demonstrating that regulation of Sox9 expression is canonical or Rbpj-dependent. To further understand the molecular basis of this regulation, we performed chromatin immunoprecipitation (ChIP) assays and detected the recruitment of the Rbpj/NICD transcription complex to Rbpj-binding sites upstream of the Sox9 promoter. The association of the Rbpj/NICD complex with the Sox9 promoter is associated with transcriptional repression of Sox9 in a cellular model of chondrocyte differentiation. Hence, Notch negatively regulates chondrocyte differentiation in the axial skeleton by suppressing Sox9 transcription, and Rbpj-independent Notch signaling mechanisms may also contribute to axial skeletogenesis.
doi:10.1002/jbmr.1770
PMCID: PMC3548081  PMID: 22991339
Chondrodysplasia; axial skeleton; genetic mouse model; Notch signaling; Sox9; Chondrocyte
6.  Notch activity in the nervous system: to switch or not switch? 
Neural Development  2009;4:36.
The Notch pathway is instrumental for cell fate diversification during development. Pioneer studies conducted in Drosophila and more recent work performed in vertebrates have shown that in the nervous system, Notch is reiteratively employed when cells choose between two alternative fates, a process referred to as a binary fate decision. While the early (neural versus epidermal) fate decisions mainly involve an inhibitory effect of Notch on the neural fate, late fate decisions (choice between different subtypes of neural cells) have been proposed to involve a binary switch activity whereby Notch would be instructive for one fate and inhibitory for the other. We re-examine this binary switch model in light of two recent findings made in the vertebrate nervous system. First, in the zebrafish epiphysis, Notch is required to resolve a mixed identity through the inhibition of one specific fate. Second, in the murine telencephalon, Notch regulates the competence of neural progenitors to respond to the JAK/STAT pathway, thereby allowing for the induction of an astrocyte fate. In neither case is Notch instructive for the alternative fate, but rather cooperates with another signalling pathway to coordinate binary fate choices. We also review current knowledge on the molecular cascades acting downstream of Notch in the context of neural subtype diversification, a crucial issue if one is to determine Notch function as an instructive, permissive or inhibitory signal in the various cellular contexts where it is implicated. Finally, we speculate as to how such a 'non-switch' activity could contribute to the expansion of neuronal subtype diversity.
doi:10.1186/1749-8104-4-36
PMCID: PMC2761386  PMID: 19799767
7.  Characterization of Notch1 Antibodies That Inhibit Signaling of Both Normal and Mutated Notch1 Receptors 
PLoS ONE  2010;5(2):e9094.
Background
Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.
Principal Findings
Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC50 values as low as 5±3 nM and 0.13±0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR “class I” point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare “class II” or “class III” mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors.
Conclusions/Significance
Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.
doi:10.1371/journal.pone.0009094
PMCID: PMC2817004  PMID: 20161710
8.  Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells 
Breast Cancer Research  2004;6(6):R605-R615.
Introduction
Notch signaling has been implicated in the regulation of cell-fate decisions such as self-renewal of adult stem cells and differentiation of progenitor cells along a particular lineage. Moreover, depending on the cellular and developmental context, the Notch pathway acts as a regulator of cell survival and cell proliferation. Abnormal expression of Notch receptors has been found in different types of epithelial metaplastic lesions and neoplastic lesions, suggesting that Notch may act as a proto-oncogene. The vertebrate Notch1 and Notch4 homologs are involved in normal development of the mammary gland, and mutated forms of these genes are associated with development of mouse mammary tumors.
Methods
In order to determine the role of Notch signaling in mammary cell-fate determination, we have utilized a newly described in vitro system in which mammary stem/progenitor cells can be cultured in suspension as nonadherent 'mammospheres'. Notch signaling was activated using exogenous ligands, or was inhibited using previously characterized Notch signaling antagonists.
Results
Utilizing this system, we demonstrate that Notch signaling can act on mammary stem cells to promote self-renewal and on early progenitor cells to promote their proliferation, as demonstrated by a 10-fold increase in secondary mammosphere formation upon addition of a Notch-activating DSL peptide. In addition to acting on stem cells, Notch signaling is also able to act on multipotent progenitor cells, facilitating myoepithelial lineage-specific commitment and proliferation. Stimulation of this pathway also promotes branching morphogenesis in three-dimensional Matrigel cultures. These effects are completely inhibited by a Notch4 blocking antibody or a gamma secretase inhibitor that blocks Notch processing. In contrast to the effects of Notch signaling on mammary stem/progenitor cells, modulation of this pathway has no discernable effect on fully committed, differentiated, mammary epithelial cells.
Conclusion
These studies suggest that Notch signaling plays a critical role in normal human mammary development by acting on both stem cells and progenitor cells, affecting self-renewal and lineage-specific differentiation. Based on these findings we propose that abnormal Notch signaling may contribute to mammary carcinogenesis by deregulating the self-renewal of normal mammary stem cells.
doi:10.1186/bcr920
PMCID: PMC1064073  PMID: 15535842
mammary gland development; mammary progenitor cells; mammary stem cells; Notch
9.  Notch Directly Regulates Gata3 Expression during T Helper 2 Cell Differentiation 
Immunity  2007;27(1):100-110.
SUMMARY
Notch signaling plays multiple roles to direct diverse decisions regarding cell fate during T cell development. During helper T (Th) cell differentiation, Notch is involved in generating optimal Th2 cell responses. Here, we present data investigating how Notch mediates Th2 cell differentiation. Notch showed a CD4+ T cell intrinsic role in promoting IL-4 expression that required GATA-3. In the absence of Notch signals, Gata3 expression was markedly diminished. Introduction of an activated allele of Notch1 into CD4+ T cells led to the specific and direct upregulation of a developmentally regulated Gata3 transcript that included the exon 1a sequences. Furthermore, Notch acted in parallel with GATA-3 to synergistically activate IL-4 expression. Together, these data implicate Gata3 as a direct transcriptional Notch target that acts in concert with Notch signaling to generate optimal Th2 cell responses.
doi:10.1016/j.immuni.2007.04.018
PMCID: PMC2801546  PMID: 17658278
10.  OSM-11 Facilitates LIN-12 Notch Signaling during Caenorhabditis elegans Vulval Development  
PLoS Biology  2008;6(8):e196.
Notch signaling is critical for cell fate decisions during development. Caenorhabditis elegans and vertebrate Notch ligands are more diverse than classical Drosophila Notch ligands, suggesting possible functional complexities. Here, we describe a developmental role in Notch signaling for OSM-11, which has been previously implicated in defecation and osmotic resistance in C. elegans. We find that complete loss of OSM-11 causes defects in vulval precursor cell (VPC) fate specification during vulval development consistent with decreased Notch signaling. OSM-11 is a secreted, diffusible protein that, like previously described C. elegans Delta, Serrate, and LAG-2 (DSL) ligands, can interact with the lineage defective-12 (LIN-12) Notch receptor extracellular domain. Additionally, OSM-11 and similar C. elegans proteins share a common motif with Notch ligands from other species in a sequence defined here as the Delta and OSM-11 (DOS) motif. osm-11 loss-of-function defects in vulval development are exacerbated by loss of other DOS-motif genes or by loss of the Notch ligand DSL-1, suggesting that DOS-motif and DSL proteins act together to activate Notch signaling in vivo. The mammalian DOS-motif protein Deltalike1 (DLK1) can substitute for OSM-11 in C. elegans development, suggesting that DOS-motif function is conserved across species. We hypothesize that C. elegans OSM-11 and homologous proteins act as coactivators for Notch receptors, allowing precise regulation of Notch receptor signaling in developmental programs in both vertebrates and invertebrates.
Author Summary
The classic view of Notch receptor activation involves receptor binding to transmembrane Notch ligands that contain a conserved DSL (Delta, Serrate, and LAG-2) domain. Here, we find that the Caenorhabditis elegans OSM-11 protein is a novel ligand of the well-characterized Notch signal transduction pathway and plays a role in cell fate specification during development. OSM-11 is a secreted, diffusible protein whose loss decreases Notch signaling in vivo. OSM-11 and related C. elegans proteins do not contain a DSL domain, but contain a conserved motif we have named DOS (Delta and OSM-11) that is also found in the extracellular domain of known Notch ligands in organisms other than C. elegans. The functional mammalian homolog of OSM-11 is the secreted protein Deltalike1 (Dlk1), also known as Preadipocyte Factor 1 (PREF1), which plays a poorly defined role in Notch signaling regulating obesity and other developmental decisions. This suggests that Notch ligands are split into two complementary coligand families that act together to regulate Notch signaling in developmental contexts. In addition to regulating development, DOS ligands play roles in osmotic stress and C. elegans behavior, suggesting previously unsuspected roles for Notch signaling across species.
The C. elegans OSM-11 protein acts with DSL ligands to activate Notch signaling in cell fate specification and defines a conserved family of potential Notch co-ligands.
doi:10.1371/journal.pbio.0060196
PMCID: PMC2504490  PMID: 18700817
11.  Impact of Notch Signaling on Inflammatory Responses in Cardiovascular Disorders 
Notch signaling is a major pathway in cell fate decisions. Since the first reports showing the major role of Notch in embryonic development, a considerable and still growing literature further highlights its key contributions in various pathological processes during adult life. In particular, Notch is now considered as a major player in vascular homeostasis through the control of key cellular functions. In parallel, confounding evidence emerged that inflammatory responses regulate Notch signaling in vitro in endothelial cells, smooth muscle cells or vascular infiltrating cells and in vivo in vascular and inflammatory disorders and in cardiovascular diseases. This review presents how inflammation influences Notch in vascular cells and, reciprocally, emphasizes the functional role of Notch on inflammatory processes, notably by regulating key cell functions (differentiation, proliferation, apoptosis/survival, activation). Understanding how the disparity of Notch receptors and ligands impacts on vasculature biology remains critical for the design of relevant and adequate therapeutic strategies targeting Notch in this major pathological context.
doi:10.3390/ijms14046863
PMCID: PMC3645668  PMID: 23531541
cardiovascular diseases; endothelial cells; inflammation; Notch signaling; signaling pathways; vascular cells
12.  Essential Role of Endothelial Notch1 in Angiogenesis 
Circulation  2005;111(14):1826-1832.
Background
Notch signaling influences binary cell fate decisions in a variety of tissues. The Notch1 receptor is widely expressed during embryogenesis and is essential for embryonic development. Loss of global Notch1 function results in early embryonic lethality, but the cell type responsible for this defect is not known. Here, we identify the endothelium as the primary target tissue affected by Notch1 signaling.
Methods and Results
We generated an endothelium-specific deletion of Notch1 using Tie2Cre and conditional Notch1flox/flox mice. Mutant embryos lacking endothelial Notch1 died at approximately embryonic day 10.5 with profound vascular defects in placenta, yolk sac, and embryo proper, whereas heterozygous deletion had no effect. In yolk sacs of mutant embryos, endothelial cells formed a primary vascular plexus indicative of intact vasculogenesis but failed to induce the secondary vascular remodeling required to form a mature network of well-organized large and small blood vessels, which demonstrates a defect in angiogenesis. These vascular defects were also evident in the placenta, where blood vessels failed to invade the placental labyrinth, and in the embryo proper, where defective blood vessel maturation led to pericardial and intersomitic hemorrhage. Enhanced activation of caspase-3 was detected in endothelial and neural cells of mutant mice, which resulted in enhanced apoptotic degeneration of somites and the neural tube.
Conclusions
These findings recapitulate the vascular phenotype of global Notch1-/- mutants and indicate an essential cell-autonomous role of Notch1 signaling in the endothelium during vascular development. These results may have important clinical implications with regard to Notch1 signaling in adult angiogenesis.
doi:10.1161/01.CIR.0000160870.93058.DD
PMCID: PMC2633594  PMID: 15809373
vasculature; genetics; defects; angiogenesis; endothelium
13.  Ligand-Independent Traffic of Notch Buffers Activated Armadillo in Drosophila 
PLoS Biology  2009;7(8):e1000169.
Full-length Notch receptor binds to the Wnt pathway effector β-catenin and mediates its endocytosis and degradation, demonstrating a novel mechanism by which Notch may modulate Wnt pathway activity.
Notch receptors act as ligand-dependent membrane-tethered transcription factors with a prominent role in binary cell fate decisions during development, which is conserved across species. In addition there is increasing evidence for other functions of Notch, particularly in connection with Wnt signalling: Notch is able to modulate the activity of Armadillo/ß-catenin, the effector of Wnt signalling, in a manner that is independent of its transcriptional activity. Here we explore the mechanism of this interaction in the epithelium of the Drosophila imaginal discs and find that it is mediated by the ligand-independent endocytosis and traffic of the Notch receptor. Our results show that Notch associates with Armadillo near the adherens junctions and that it is rapidly endocytosed promoting the traffic of an activated form of Armadillo into endosomal compartments, where it may be degraded. As Notch has the ability to interact with and downregulate activated forms of Armadillo, it is possible that in vivo Notch regulates the transcriptionally competent pool of Armadillo. These interactions reveal a previously unknown activity of Notch, which serves to buffer the function of activated Armadillo and might underlie some of its transcription-independent effects.
Author Summary
Establishment of the correct shape and pattern of tissues within an organism requires the integration of molecular information present in signalling and transcriptional networks and demands delicate exchanges and balances of their activities. A large body of experimental work has revealed close correlations in the activities of two pathways: Notch and Wnt, which suggest the existence of multiple links between them. Notch signalling relies in part upon the activity of the Notch protein, a membrane-bound receptor with a transcription factor domain that can be released from the membrane by proteolytic cleavage. On the other hand Wnt proteins are ligands that trigger changes in the activity of ß-catenin, which is called Armadillo in the fruit fly Drosophila melanogaster. In this study we uncover a previously unknown activity for Notch: endocytosis and trafficking of full length Notch, which targets Armadillo for degradation. This activity of Notch is independent of its ligands, Delta and Serrate, and of its downstream effector, the transcription factor Suppressor of Hairless. We further show that in the absence of Notch, which has been shown to act as a tumor suppressor in mammals, expression of an activated form of Armadillo causes tissue overgrowth and changes in the polarity of cells. Our results suggest that Drosophila Notch can promote the degradation of activated forms of Armadillo and may buffer cells against fluctuations in Wnt signalling activity.
doi:10.1371/journal.pbio.1000169
PMCID: PMC2716527  PMID: 19668359
14.  The Role of Notch in the Kidney, Development and Beyond 
The Journal of pathology  2011;226(2):394-403.
Notch is a highly conserved cell-cell communication mechanism that regulates development, tissue homeostasis and repair. Recent studies indicate that Notch plays a key role in kidney development by establishing proximal tubular epithelial cell fate and cell type specification in the renal collecting system. Notch signalling is markedly reduced in the adult kidney, however, increased Notch signalling has been noted in both acute and chronic kidney injury. Increased glomerular epithelial Notch signaling has been associated with albuminuria and glomerulosclerosis, while tubular epithelial Notch activation might cause altered repair and fibrosis development. Here, we review the role of Notch signalling in the kidney during development as well as in acute and chronic injury.
doi:10.1002/path.2967
PMCID: PMC3677191  PMID: 21952830
Notch signalling; development; repair; regeneration; kidney fibrosis; chronic kidney disease; renal cell cancer
15.  Glucocorticoid and Growth Factor Synergism Requirement for Notch4 Chromatin Domain Activation▿  
Molecular and Cellular Biology  2007;27(6):2411-2422.
The Notch signaling pathway modulates cell fate in diverse contexts, including vascular development. Notch4 is selectively expressed in vascular endothelium and regulates vascular remodeling. The signal-dependent transcription factor activator protein 1 (AP-1) activates Notch4 transcription in endothelial cells, but other factors/signals that regulate Notch4 are largely unknown. We demonstrate that, unlike the established transrepression mechanism in which the glucocorticoid receptor (GR) antagonizes AP-1, AP-1 and GR synergistically activated Notch4 transcription in endothelial cells. Fibroblast growth factor 2 (FGF-2) and cortisol induced AP-1 and GR occupancy, respectively, at a Notch4 promoter composite response element consisting of an imperfect half-glucocorticoid response element and an AP-1 motif, which mediated signal-dependent activation. Analysis of Notch4 promoter complex assembly provided evidence that GR and AP-1 independently occupy the composite response element, but AP-1 stabilizes GR occupancy. In multipotent 10T1/2 cells, FGF-2 and cortisol induced a histone modification pattern at the Notch4 locus mimicking that present in endothelial cells and reprogrammed Notch4 from a repressed to an active state. These results establish the molecular basis for a novel AP-1/GR-Notch4 axis in vascular endothelium.
doi:10.1128/MCB.02152-06
PMCID: PMC1820485  PMID: 17220278
16.  Physiological Notch Signaling Maintains Bone Homeostasis via RBPjk and Hey Upstream of NFATc1 
PLoS Genetics  2012;8(3):e1002577.
Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo.
Author Summary
Osteoporosis is a disease caused by disruption of the balance between bone formation and resorption resulting in a net loss of bone mass. Although anti-resorptive agents are the current mainstay of osteoporosis therapy, novel strategies to promote bone formation are critically needed for more effective prevention and treatment of the disease. Notch signaling, an evolutionally conserved mechanism among multi-cellular organisms, was recently shown to control bone formation and therefore represents a potential target pathway for novel bone-promoting therapeutics. In this study we elucidate the intracellular signaling mechanism through which Notch controls bone formation, providing a molecular framework that may guide future drug development.
doi:10.1371/journal.pgen.1002577
PMCID: PMC3310726  PMID: 22457635
17.  Activated Notch1 Target Genes during Embryonic Cell Differentiation Depend on the Cellular Context and Include Lineage Determinants and Inhibitors 
PLoS ONE  2010;5(7):e11481.
Background
Notch receptor signaling controls developmental cell fates in a cell-context dependent manner. Although Notch signaling directly regulates transcription via the RBP-J/CSL DNA binding protein, little is known about the target genes that are directly activated by Notch in the respective tissues.
Methodology/Principal Findings
To analyze how Notch signaling mediates its context dependent function(s), we utilized a Tamoxifen-inducible system to activate Notch1 in murine embryonic stem cells at different stages of mesodermal differentiation and performed global transcriptional analyses. We find that the majority of genes regulated by Notch1 are unique for the cell type and vary widely dependent on other signals. We further show that Notch1 signaling regulates expression of genes playing key roles in cell differentiation, cell cycle control and apoptosis in a context dependent manner. In addition to the known Notch1 targets of the Hes and Hey families of transcriptional repressors, Notch1 activates the expression of regulatory transcription factors such as Sox9, Pax6, Runx1, Myf5 and Id proteins that are critically involved in lineage decisions in the absence of protein synthesis.
Conclusion/Significance
We suggest that Notch signaling determines lineage decisions and expansion of stem cells by directly activating both key lineage specific transcription factors and their repressors (Id and Hes/Hey proteins) and propose a model by which Notch signaling regulates cell fate commitment and self renewal in dependence of the intrinsic and extrinsic cellular context.
doi:10.1371/journal.pone.0011481
PMCID: PMC2900208  PMID: 20628604
18.  Novel insights into the differential functions of Notch ligands in vascular formation 
The Notch signaling pathway is a critical component of vascular formation and morphogenesis in both development and disease. Compelling evidence indicates that Notch signaling is required for the induction of arterial-cell fate during development and for the selection of endothelial tip and stalk cells during sprouting angiogenesis. In mammals, two of the four Notch receptors (Notch1 and Notch4) and three of the five Notch ligands (Jagged1, Dll1, and Dll4) are predominantly expressed in vascular endothelial cells and are important for many aspects of vascular biology. During arterial cell-fate selection and angiogenesis, the roles of Notch1 and Notch4 are thought to be similar, and the function of Dll4 is well-characterized. However, the molecular mechanisms that determine the functional similarities and differences of Notch ligands in vascular endothelial cells remain largely unknown; consequently, additional research is needed to elucidate the ligand-specific functions and mechanisms associated with Notch activation in the vascular endothelium. Results from recent studies indicate that Dll1 and Dll4 have distinct roles in the specification and maintenance of arterial cell identity, while Dll4 and Jagged1 have opposing functions in tip- and stalk-cell selection during sprouting angiogenesis. This review will focus on the newly discovered, distinct functions of several Notch ligands in the regulation of blood vessel formation and will provide perspectives for future research in the field.
doi:10.1186/2040-2384-1-8
PMCID: PMC2794854  PMID: 20016694
19.  Notch Signaling and Hes Labeling in the Normal and Drug-Damaged Organ of Corti 
Hearing research  2009;249(1-2):15-22.
During the development of the inner ear, the Notch cell signaling pathway is responsible for the specification of the pro-sensory domain and influences cell fate decisions. It is assumed that Notch signaling ends during maturity and cannot be reinitiated to alter the fate of new or existing cells in the organ of Corti. This is in contrast to non-mammalian species which reinitiate Delta1-Notch1 signaling in response to trauma in the auditory epithelium, resulting in hair cell regeneration through transdifferentiation and/or mitosis. We report immunohistochemical data and Western protein analysis showing that in the aminoglycoside-damaged guinea pig organ of Corti, there is an increase in proteins involved in Notch activation occurring within 24 hours of a chemical hair cell lesion. The signaling response is characterized by the increased presence of Jagged1 ligand in pillar and Deiters cells, Notch1 signal in surviving supporting cell nuclei, and the absence of Jagged2 and Delta-like1. The pro-sensory bHLH protein Atoh1 was absent at all time points following an ototoxic lesion, while the repressor bHLH transcription factors Hes1 and Hes5 were detected in surviving supporting cell nuclei in the former inner and outer hair cell areas, respectively. Notch pathway proteins peaked at 2 weeks, decreased at 1 month, and nearly disappeared by 2 months. These results indicate that the mammalian auditory epithelium retains the ability to regulate Notch signaling and Notch-dependent Hes activity in response to cellular trauma and that the signaling is transient. Additionally, since Hes activity antagonizes the transcription of prosensory Atoh1, the presence of Hes after a lesion may prohibit the occurrence of transdifferentiation in the surviving supporting cells.
doi:10.1016/j.heares.2008.12.008
PMCID: PMC2796274  PMID: 19185606
20.  Molecular Determinants of NOTCH4 Transcription in Vascular Endothelium 
Molecular and Cellular Biology  2005;25(4):1458-1474.
The process whereby the primitive vascular network develops into the mature vasculature, known as angiogenic vascular remodeling, is controlled by the Notch signaling pathway. Of the two mammalian Notch receptors expressed in vascular endothelium, Notch1 is broadly expressed in diverse cell types, whereas Notch4 is preferentially expressed in endothelial cells. As mechanisms that confer Notch4 expression were unknown, we investigated how NOTCH4 transcription is regulated in human endothelial cells and in transgenic mice. The NOTCH4 promoter and the 5′ portion of NOTCH4 assembled into an endothelial cell-specific histone modification pattern. Analysis of NOTCH4 primary transcripts in human umbilical vein endothelial cells by RNA fluorescence in situ hybridization revealed that 36% of the cells transcribed one or both NOTCH4 alleles. The NOTCH4 promoter was sufficient to confer endothelial cell-specific transcription in transfection assays, but intron 1 or upstream sequences were required for expression in the vasculature of transgenic mouse embryos. Cell-type-specific activator protein 1 (AP-1) complexes occupied NOTCH4 chromatin and conferred endothelial cell-specific transcription. Vascular angiogenic factors activated AP-1 and reprogrammed the endogenous NOTCH4 gene in HeLa cells from a repressed to a transcriptionally active state. These results reveal an AP-1-Notch4 pathway, which we propose to be crucial for transducing angiogenic signals and to be deregulated upon aberrant signal transduction in cancer.
doi:10.1128/MCB.25.4.1458-1474.2005
PMCID: PMC548019  PMID: 15684396
21.  The Intracellular Form of Notch Blocks Transforming Growth Factor β-Mediated Growth Arrest in Mv1Lu Epithelial Cells 
Molecular and Cellular Biology  2003;23(18):6694-6701.
Notch signaling influences a variety of cell fate decisions during development, and constitutive activation of the pathway can provoke unbridled cell growth and cancer. The mechanisms by which Notch affects cell growth are not well established. We describe here a novel link between Notch and cell cycle control. We found that Mv1Lu epithelial cells harboring an oncogenic form of Notch (NICD) are resistant to the cell cycle-inhibitory effects of transforming growth factor β (TGF-β). NICD did not affect TGF-β signaling per se but blocked induction of the Cdk inhibitor p15INK4B. c-Myc, whose down-regulation by TGF-β is required for p15INK4B induction, remained elevated in the NICD-expressing cells. c-Myc expression was also maintained in low serum, indicating that Notch's effects on c-Myc are not specific to TGF-β. Our results are consistent with a model in which a strong Notch signal indirectly deregulates c-Myc expression and thereby renders Mv1Lu epithelial cells resistant to growth-inhibitory signals.
doi:10.1128/MCB.23.18.6694-6701.2003
PMCID: PMC193715  PMID: 12944493
22.  Myocardial Notch Signaling Reprograms Cardiomyocytes to a Conduction-Like Phenotype 
Circulation  2012;126(9):1058-1066.
Background
Notch signaling has previously been shown to play an essential role in regulating cell fate decisions and differentiation during cardiogenesis in many systems including Drosophila, Xenopus and mammals. We hypothesized that Notch may also be involved in directing the progressive lineage restriction of cardiomyocytes into specialized conduction cells.
Methods and Results
In hearts where Notch signaling is activated within the myocardium from early development onwards, Notch promotes a conduction-like phenotype based on ectopic expression of conduction system-specific genes and cell autonomous changes in electrophysiology. Using an in vitro assay to activate Notch in newborn cardiomyocytes, we observed global changes in the transcriptome as well as in action potential characteristics consistent with reprogramming to a conduction-like phenotype.
Conclusions
Notch can instruct the differentiation of chamber cardiac progenitors into specialized conduction-like cells. Plasticity remains in late-stage cardiomyocytes, which has potential implications for engineering of specialized cardiovascular tissues.
doi:10.1161/CIRCULATIONAHA.112.103390
PMCID: PMC3607542  PMID: 22837163
action potentials; conduction; pacemakers; Purkinje; reprogramming
23.  Cross-talk between the Notch and TGF-β signaling pathways mediated by interaction of the Notch intracellular domain with Smad3 
The Journal of Cell Biology  2003;163(4):723-728.
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.
doi:10.1083/jcb.200305112
PMCID: PMC2173673  PMID: 14638857
Hes-1; C2C12; CSL; Smad4; neural stem cell
24.  Involvement of Notch Signaling in Wound Healing 
PLoS ONE  2007;2(11):e1167.
The Notch signaling pathway is critically involved in cell fate decisions during development of many tissues and organs. In the present study we employed in vivo and cell culture models to elucidate the role of Notch signaling in wound healing. The healing of full-thickness dermal wounds was significantly delayed in Notch antisense transgenic mice and in normal mice treated with γ-secretase inhibitors that block proteolytic cleavage and activation of Notch. In contrast, mice treated with a Notch ligand Jagged peptide showed significantly enhanced wound healing compared to controls. Activation or inhibition of Notch signaling altered the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in a scratch wound healing model in ways consistent with roles for Notch signaling in wound healing functions all three cell types. These results suggest that Notch signaling plays important roles in wound healing and tissue repair, and that targeting the Notch pathway might provide a novel strategy for treatment of wounds and for modulation of angiogenesis in other pathological conditions.
doi:10.1371/journal.pone.0001167
PMCID: PMC2048753  PMID: 18000539
25.  Characterization of transgenic mice expressing cancer-associated variants of human NOTCH1 
Genesis (New York, N.Y. : 2000)  2012;50(2):112-118.
The Notch1 receptor plays a critical role in cell fate decisions during development. Activation of Notch signaling has been implicated in several types of cancer, particularly T-cell acute lymphoblastic leukemia (T-ALL). Consequently, several transgenic mouse strains have been made to study the role of Notch1 in T-ALL. However, the existing Notch1 transgenic lines mimic a translocation event found in only ~1% of T-ALL cases. Here we describe three novel NOTCH1 transgenic mouse strains that have Cre-inducible expression of the entire human NOTCH1 locus, each possessing a common mutation found in T-ALL. Unlike existing Notch1 transgenic strains, these NOTCH1 transgenic strains express full-length receptors from an endogenous human promoter that should be susceptible to a number of Notch antagonists that have recently been developed. These strains will allow researchers to modulate Notch signaling to study both normal development and cancer biology.
doi:10.1002/dvg.20798
PMCID: PMC3682665  PMID: 21898766
mouse models of cancer; BAC recombineering; BAC transgenesis

Results 1-25 (955992)