Dynamic maternal and fetal Notch activity and expression in placentation
Heather LevinChantae S. Sullivan-PykeVirginia E. PapaioannouRonald J. WapnerJan KitajewskiCarrie J. ShawberNataki C. Douglas
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Placentation
JAG1
Trophoblast
Decidua
Notch 1
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Notch signalling is essential for blood vessel formation. During angiogenesis, the Notch ligand DLL4 on the leading tip cell activates Notch receptors on the adjacent stalk cells. DLL4-Notch signalling is impaired by the Notch ligand JAG1 in endothelial cells. The Delta/Serrate/Lag2 (DSL) domain of the Notch ligands binds to the EGF-like repeats 11–13 of the Notch receptor. This study aimed to elucidate how soluble proteins containing these short domains interfere with Notch signalling during angiogenesis. Adenoviral vectors were generated to express the DSL domains of DLL1, DLL4, JAG1, and the Notch1 EGF-like repeats 11–13 fused to immunoglobulin-G heavy chain. These soluble ligand peptides inhibited Notch signalling in endothelial cells and this caused hyperbranching in cellular angiogenesis assays and in the neonatal mouse retina. The soluble Notch receptor peptides bound stronger to JAG1 than DLL4 ligands, resulting in increased signalling activity. This led to impaired tip cell formation and less vessel sprouting in the retina. The minimal binding domains of Notch ligands are sufficient to interfere with Notch signalling. The corresponding soluble Notch1 EGF11-13 peptide binds stronger to inhibitory Notch ligands and thereby promotes Notch signalling in endothelial cells.
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It has been demonstrated that adult human circulating endothelial progenitor cells (EPC) can differentiate to a cardiomyogenic phenotype. Notch signaling promotes epithelial-to-mesenchymal transformation and plays a prominent role in heart and vessel development. Here, we investigated the role of Notch activation for cardiac differentiation of EPC in a co-culture system with neonatal rat cardiomyocyte (CM). EPC expressed the receptors Notch-1 and Notch-2, whereas CM expressed high level of Notch ligand Jagged-1. Therefore, we hypothesized that CM may activate Notch signaling within EPC. Indeed, after co-culture, Notch activation was detected in EPC by immunohistochemical detection of the intracellular cleavage fragment of Notch-1 (NICD), whereas NICD was only rarely detected in EPC before co-culture. Western blot analysis confirmed Notch cleavage after co-culture. RT-PCR directed against the human specific sequences of the Notch target genes Hey2 and Hes1 demonstrated a transient activation of the transcriptional activity of Notch in human EPC after co-culture with CM. Inhibition of γ-secretase significantly blocked Notch cleavages and NICD translocations. Furthermore, the expression of the cardiac marker protein γ-sarcomeric actinin and troponin T was significantly suppressed by γ-secretase inhibition (55.8 ± 8.1% and 54.0 ± 10.5% of control, respectively) or addition of soluble recombinant Jagged-1, indicating that Notch activation facilitates cardiac marker gene expression. Because non-canonical Wnts have previously been shown to promote cardiac differentiation, we additionally determined the influence of Notch activation on the expression of Wnt5a and. Wnt5a and Wnt11 expressions in the human cells was induced by the co-culture and was blocked by γ-secretase inhibitor. Likewise, stimulation of Notch signaling by immobilized Jagged-1 promoted NICD cleavage and Wnt5a expression in EPC. These data suggested that Notch is transiently activated upon co-culture of EPC with neonatal rat CM. γ-Secretase-dependent Notch activation is required for cardiac gene expression in human cells and induces the expression of non-canonical Wnt proteins, which may act in an paracrine manner to further amplify cardiac differentiation.
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Medium conditioned by decidual cells decreased the growth of cultured BeWo choriocarcinoma cells. The degree of inhibition was dependent on the concentration of the conditioned medium used, and suggested that maternal decidua might regulate the growth of the fetal placenta. Medium from BeWo cells and primary trophoblast had the opposing effect and increased the growth of cultured decidual cells for up to 120 h of culture. These results suggest that a regulatory loop to control placental and decidual growth exists at the materno—fetal interface, and this may be an important factor in the development of adequate placentation and the subsequent growth of the placenta during pregnancy.
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The glycosyltransferase EOGT transfers O-GlcNAc to a consensus site in epidermal growth factor-like (EGF) repeats of a limited number of secreted and membrane proteins, including Notch receptors. In EOGT-deficient cells, the binding of DLL1 and DLL4, but not JAG1, canonical Notch ligands was reduced, and ligand-induced Notch signaling was impaired. Mutagenesis of O-GlcNAc sites on NOTCH1 also resulted in decreased binding of DLL4. EOGT functions were investigated in retinal angiogenesis that depends on Notch signaling. Global or endothelial cell-specific deletion of Eogt resulted in defective retinal angiogenesis, with a mild phenotype similar to that caused by reduced Notch signaling in retina. Combined deficiency of different Notch1 mutant alleles exacerbated the abnormalities in Eogt−/− retina, and Notch target gene expression was decreased in Eogt−/−endothelial cells. Thus, O-GlcNAc on EGF repeats of Notch receptors mediates ligand-induced Notch signaling required in endothelial cells for optimal vascular development.
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Notch signaling provides an important cue in mammalian developmental. It is a key player in T cell development and function. Notch ligands, such as Delta-like ligands (DLL) 1, 3, 4 and Jagged 1, 2, can impact Notch signaling positively or negatively, by trans-activation or cis-inhibition. Trans and cis interactions are receptor-ligand interaction on two adjacent cells and interaction on the same cell respectively. The former sends an activation signal and the latter, a signal for inhibition of Notch. However, earlier reports suggested that Notch is activated in the absence of Notch ligand-expressing APCs in a purified population of CD4 T cells. Thus the role of ligands in Notch activation, in a purified population of CD4 T cells, remains obscure. In this study we demonstrate that mature CD4 T cells are capable of expressing Notch ligands on their surface very early upon activation with soluble antibodies against CD3 and CD28. Moreover, signaling solely through CD28 induces Notch ligand expression and CD3 signaling inhibits ligand expression, in contrast to Notch which is induced by CD3 signaling. Additionally, by using decoys mimicking the Notch extracellular domain, we demonstrated that DLL1, DLL4, and JAG1, expressed on the T cells, can cis-interact with the Notch receptor and inhibit activation of Notch. Thus, our data indicate a novel mechanism of the regulation of Notch ligand expression on CD4 T cells and its impact on activated Notch.
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The Notch signaling pathway is an important therapeutic target for the treatment of inflammatory diseases and cancer. We previously created ligand-specific inhibitors of Notch signaling comprised of Fc fusions to specific EGF-like repeats of the Notch1 extracellular domain, called Notch decoys, which bound ligands, blocked Notch signaling, and showed anti-tumor activity with low toxicity. However, the study of their function depended on virally mediated expression, which precluded dosage control and limited clinical applicability. We have refined the decoy design to create peptibody-based Notch inhibitors comprising the core binding domains, EGF-like repeats 10-14, of either Notch1 or Notch4. These Notch peptibodies showed high secretion properties and production yields that were improved by nearly 100-fold compared to previous Notch decoys. Using surface plasmon resonance spectroscopy coupled with co-immunoprecipitation assays, we observed that Notch1 and Notch4 peptibodies demonstrate strong but distinct binding properties to Notch ligands DLL4 and JAG1. Both Notch1 and Notch4 peptibodies interfere with Notch signaling in endothelial cells and reduce expression of canonical Notch targets after treatment. While prior DLL4 inhibitors cause hyper-sprouting, the Notch1 peptibody reduced angiogenesis in a 3-dimensional in vitro sprouting assay. Administration of Notch1 peptibodies to neonate mice resulted in reduced radial outgrowth of retinal vasculature, confirming anti-angiogenic properties. We conclude that purified Notch peptibodies comprising EGF-like repeats 10-14 bind to both DLL4 and JAG1 ligands and exhibit anti-angiogenic properties. Based on their secretion profile, unique Notch inhibitory activities, and anti-angiogenic properties, Notch peptibodies present new opportunities for therapeutic Notch inhibition.
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Notch is a critical signaling pathway that controls cell fate and tissue homeostasis, but the functional characterization of Notch ligand domains that activate Notch receptors remains incomplete. Here, we established a method for immobilizing Notch ligand proteins onto beads to measure time-dependent Notch activity after the addition of Notch ligand-coated beads. A comparison between activities by the Notch ligand found on the cell surface to that of the ligand immobilized on beads showed that immobilized Notch ligand protein produces comparable signal activity during the first 10 h. Follow-up truncation studies showed that the N-terminal epidermal growth factor (EGF) repeat three region of delta like canonical Notch ligand 4 (DLL4) or jagged 1 (JAG1) is the minimum region for activating Notch signaling, and the DLL4 EGF repeat three domain may have a role in activation through a mechanism other than by increasing binding affinity. In addition, we found that reconstruction of the DLL4 delta and OSM-11 (DOS) motif (N257P) resulted in an increase in both binding affinity and signaling activity, which suggests that the role of the DOS motif is conserved among Notch ligands. Furthermore, active DLL4 protein on beads promoted T cell differentiation or inhibited B cell differentiation in vitro, whereas JAG1 proteins on beads did not have any effect. Taken together, our findings provide unambiguous evidence for the role of different Notch ligands and their domains in Notch signal activation, and may be potential tools for controlling Notch signaling activation. J. Cell. Biochem. 118: 785-796, 2017. © 2016 Wiley Periodicals, Inc.
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