Ligand-induced Notch Signaling Research Articles

A role for actomyosin contractility in Notch signaling

BackgroundNotch-Delta signaling functions across a wide array of animal systems to break symmetry in a sheet of undifferentiated cells and generate cells with different fates, a process known as lateral inhibition. Unlike many other signaling systems, however, since both the ligand and receptor are transmembrane proteins, the activation of Notch by Delta depends strictly on cell-cell contact. Furthermore, the binding of the ligand to the receptor may not be sufficient to induce signaling, since recent work in cell culture suggests that ligand-induced Notch signaling also requires a mechanical pulling force. This tension exposes a cleavage site in Notch that, when cut, activates signaling. Although it is not known if mechanical tension contributes to signaling in vivo, others have suggested that this is how endocytosis of the receptor-ligand complex contributes to the cleavage and activation of Notch. In a similar way, since Notch-mediated lateral inhibition at a distance in the dorsal thorax of the pupal fly is mediated via actin-rich protrusions, it is possible that cytoskeletal forces generated by networks of filamentous actin and non-muscle myosin during cycles of protrusion extension and retraction also contribute to Notch signaling.ResultsTo test this hypothesis, we carried out a detailed analysis of the role of myosin II-dependent tension in Notch signaling in the developing fly and in cell culture. Using dynamic fluorescence-based reporters of Notch, we found that myosin II is important for signaling in signal sending and receiving cells in both systems—as expected if myosin II-dependent tension across the Notch-Delta complex contributes to Notch activation. While myosin II was found to contribute most to signaling at a distance, it was also required for maximal signaling between adjacent cells that share lateral contacts and for signaling between cells in culture.ConclusionsTogether these results reveal a previously unappreciated role for non-muscle myosin II contractility in Notch signaling, providing further support for the idea that force contributes to the cleavage and activation of Notch in the context of ligand-dependent signaling, and a new paradigm for actomyosin-based mechanosensation.

Notch signaling: A sweet strategy.

Glycosylation of Notch receptors regulates ligand-induced Notch signaling, which is essential for normal development in animals. Fucose analogs targeting Notch glycosylation serve as ligand-specific Notch inhibitors and facilitate the understanding of how O-glycan regulates Notch–ligand interactions.

New insights into the tetraspanin Tspan5 using novel monoclonal antibodies

Tspan5 is a member of a subgroup of tetraspanins referred to as TspanC8. These tetraspanins directly interact with the metalloprotease ADAM10, regulate its exit from the endoplasmic reticulum and subsequent trafficking, and differentially regulate its ability to cleave various substrates and activate Notch signaling. The study of Tspan5 has been limited by the lack of good antibodies. This study provides new insights into Tspan5 using new monoclonal antibodies (mAbs), including two mAbs recognizing both Tspan5 and the highly similar tetraspanin Tspan17. Using these mAbs, we show that endogenous Tspan5 associates with ADAM10 in human cell lines and in mouse tissues where it is the most abundant, such as the brain, the lung, the kidney, or the intestine. We also uncover two TspanC8-specific motifs in the large extracellular domain of Tspan5 that are important for ADAM10 interaction and exit from the endoplasmic reticulum. One of the anti-Tspan5 mAbs does not recognize Tspan5 associated with ADAM10, providing a convenient way to measure the fraction of Tspan5 not associated with ADAM10. This fraction is minor in the cell lines tested, and it increases upon transfection of cells with TspanC8 tetraspanins such as Tspan15 or Tspan33 that inhibit Notch signaling. Finally, two antibodies inhibit ligand-induced Notch signaling, and this effect is stronger in cells depleted of the TspanC8 tetraspanin Tspan14, further indicating that Tspan5 and Tspan14 can compensate for each other in Notch signaling.

O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals.

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.

Notch signaling regulates antigen sensitivity of naive CD4+ T cells by tuning co-stimulation (LYM7P.613)

Abstract Adaptive immune responses begin when naïve CD4+ T cells engage peptide+major histocompatibility complex class II and costimulatory molecules on antigen-presenting cells (APC). Notch signaling can influence effector functions in differentiated CD4+ T helper and T regulatory cells. Whether and how ligand-induced Notch signaling influences the initial priming of CD4+ T cells has not been addressed. We have found that Delta Like Ligand 4 (DLL4)-induced Notch signaling potentiates phosphatidyl inositol 3-OH kinase (PI3K)-dependent signaling downstream of the T cell receptor+CD28, allowing naïve CD4+ T cells to respond to lower doses of antigen. In vitro, DLL4-deficient APC were less efficient stimulators of CD4+ T cell activation, metabolism, proliferation, and cytokine secretion. With deletion of DLL4 from CD11c+ APC in vivo, these deficits translated to an impaired ability to mount effective CD4+ T cell-dependent immune responses. These data implicate Notch signaling as an important regulator of adaptive immune responses.

Notch Signaling Regulates Antigen Sensitivity of Naive CD4+ T Cells by Tuning Co-stimulation

Adaptive immune responses begin when naive CD4(+) T cells engage peptide+major histocompatibility complex class II and co-stimulatory molecules on antigen-presenting cells (APCs). Notch signaling can influence effector functions in differentiated CD4(+) T helper and T regulatory cells. Whether and how ligand-induced Notch signaling influences the initial priming of CD4(+) T cells has not been addressed. We have found that Delta Like Ligand 4 (DLL4)-induced Notch signaling potentiates phosphatidylinositol 3-OH kinase (PI3K)-dependent signaling downstream of the T cell receptor+CD28, allowing naive CD4(+) T cells to respond to lower doses of antigen. In vitro, DLL4-deficient APCs were less efficient stimulators of CD4(+) T cell activation, metabolism, proliferation, and cytokine secretion. With deletion of DLL4 from CD11c(+) APCs in vivo, these deficits translated to an impaired ability to mount an effective CD4(+)-dependent anti-tumor response. These data implicate Notch signaling as an important regulator of adaptive immune responses.

The notch ligand JAGGED1 as a target for anti-tumor therapy.

The Notch pathway is increasingly attracting attention as a source of therapeutic targets for cancer. Ligand-induced Notch signaling has been implicated in various aspects of cancer biology; as a consequence, pan-Notch inhibitors and therapeutic antibodies targeting one or more of the Notch receptors have been investigated for cancer therapy. Alternatively, Notch ligands provide attractive options for therapy in cancer treatment due to their more restricted expression and better-defined functions, as well as their low rate of mutations in cancer. One of the Notch ligands, Jagged1 (JAG1), is overexpressed in many cancer types, and plays an important role in several aspects of tumor biology. In fact, JAG1-stimulated Notch activation is directly implicated in tumor growth through maintaining cancer stem cell populations, promoting cell survival, inhibiting apoptosis, and driving cell proliferation and metastasis. In addition, JAG1 can indirectly affect cancer by influencing tumor microenvironment components such as tumor vasculature and immune cell infiltration. This article gives an overview of JAG1 and its role in tumor biology, and its potential as a therapeutic target.

Activation of Notch Signaling Mediates Ex Vivo Expansion of Multilineage, Engrafting Murine Hematopoietic Progenitors From Embryonic Sources.

Abstract 2321An important goal in the application of pluripotent stem cells (PSC) for therapeutic purposes is the derivation of hematopoietic stem and progenitor cells (HSPC) capable of efficient engraftment in vivo. Fundamental to achieving this goal is improved understanding of key signal pathways required to establish, maintain and expand HSPCs from embryonic sources. Ex vivo activation of Notch signaling in mouse bone marrow and human cord blood-derived HSC can facilitate expansion of rapidly engrafting multilineage progenitors, which has recently been translated for therapeutic purposes. In contrast, similar expansion of engrafting progenitors has not been successful from PSC. This prompted us to evaluate whether embryonic-derived HSPC have capacity to respond to ligand-induced Notch signaling ex vivo, and whether Notch activation could promote expansion of engrafting progenitors from these embryonic sources. We have examined the effects of ex vivo activation of Notch receptors by immobilized, exogenous Notch ligands on highly enriched populations of embryonic HSC and HSC precursors (pre-HSC) at various developmental stages. We find that activation of Notch by the ligand Delta1 within HSC/pre-HSC isolated from embryonic aorta-gonad-mesonephros (AGM) promotes expansion of progenitors with erythromyeloid colony forming potential and T/B-lymphoid potential in vitro, with concurrent expression of surface phenotypes resembling fetal liver-stage HSC. Furthermore, Notch activation in embryonic HSPC also mediates expansion of progenitors with rapidly engrafting myeloid and lymphoid capacity in irradiated mouse models. Our results demonstrate that embryonic stage HSPC have capacity to expand in response to Notch activation, and thus further studies comparing AGM- and PSC-derived hematopoietic precursors are needed to elucidate differences that may account for failure to expand HSPC from PSC. Disclosures:Bernstein:Seattle Genetics, Inc.: Consultancy.

The effects of methylmercury on Notch signaling during embryonic neural development in Drosophila melanogaster

Methylmercury (MeHg) is a ubiquitous toxicant that targets the developing fetal nervous system. MeHg interacts with the Notch signaling pathway, a highly-conserved intercellular signaling mechanism required for normal development. Notch signaling is conveyed by activation of the genes in the enhancer of split (E(spl)) locus in Drosophila. We have previously shown that acute high doses of MeHg upregulate several E(spl) genes in Drosophila neural-derived C6 cells. Furthermore, MeHg induction of E(spl) can occur independent of the Notch receptor itself. We now show that MeHg, unlike inorganic mercury (HgCl2), preferentially upregulates E(spl)mδ and E(spl)mγ in Drosophila C6 cells. This is distinct from Delta ligand-induced Notch signaling in which no induction of E(spl)mδ is seen. MeHg is also seen to specifically upregulate E(spl)mδ in Drosophila embryos where HgCl2 showed no such effect. Additionally, treatment of embryos with MeHg caused a consistent failure in axonal outgrowth of the intersegmental nerve (ISN). This ISN phenotype was partially replicated by genetic activation of the Notch pathway, but was not replicated by increasing expression of E(spl)mδ. These data suggest a role for Notch signaling and the E(spl)mδ target gene in MeHg toxicity, however, the site of action for E(spl)mδ in this system remains to be elucidated.

Feed-forward Signaling by Membrane-bound Ligand Receptor Circuit: THE CASE OF NOTCH DELTA-LIKE 4 LIGAND IN ENDOTHELIAL CELLS

The DELTA like-4 ligand (DLL4) belongs to the highly conserved NOTCH family and is specifically expressed in the endothelium. DLL4 regulates crucial processes in vascular growth, including endothelial cell (EC) sprouting and arterial specification. Its expression is increased by VEGF-A. In the present study, we show that VEGF-induced DLL4 expression depends on NOTCH activation. VEGF-induced DLL4 expression was prevented by the blockage of NOTCH signaling with γ-secretase or ADAM inhibitors in human cardiac microvascular ECs. Similar to VEGF-A, recombinant DLL4 itself stimulated NOTCH signaling and resulted in up-regulation of DLL4, suggesting a positive feed-forward mechanism. These effects were abrogated by NOTCH inhibitors but not by inhibition of VEGF signaling. NOTCH activation alone suffices to induce DLL4 expression as illustrated by the positive effect of NOTCH intracellular domain (NICD)-1 or -4 overexpression. To discriminate between NICD/RBP-Jκ and FOXC2-regulated DLL4 expression, DLL4 promoter activity was assessed in promoter deletion experiments. NICD induced promoter activity was dependent on RBP-Jκ site but independent of the FOXC2 binding site. Accordingly, constitutively active FOXC2 did not affect DLL4 expression. The notion that the positive feed-forward mechanism might propagate NOTCH activation to neighboring ECs was supported by our observation that DLL4-eGFP-transfected ECs induced DLL4 expression in nontransfected cells in their vicinity. In summary, our data provide evidence for a mechanism by which VEGF or ligand-induced NOTCH signaling up-regulates DLL4 through a positive feed-forward mechanism. By this mechanism, DLL4 could propagate its own expression and enable synchronization of NOTCH expression and signaling between ECs.

NOTCH1 missense alleles associated with left ventricular outflow tract defects exhibit impaired receptor processing and defective EMT

Notch signaling is essential for proper cardiac development. We recently identified missense variants in the NOTCH1 receptor in patients with diverse left ventricular outflow tract (LVOT) malformations (NOTCH1 G661S and NOTCH1 A683T) that reduce ligand-induced Notch signaling. Here, we examine the molecular mechanisms that contribute to reduced signaling and perturbed development. We find that NOTCH1 A683T exhibits reduced S1 cleavage due to impaired trafficking through the endoplasmic reticulum (ER). This observation is consistent with improper localization of the variant receptor to the ER and decreased presentation at the cell surface. In contrast, the nearby mutation NOTCH1 G661S exhibits reduced cell-surface presentation in the absence of overt folding or trafficking defects. To examine the implications of these variants in disease pathogenesis, we investigated their effect on epithelial-to-mesenchymal transition (EMT), a critical process for development of the outflow tract. We find that these LVOT-associated NOTCH1 alleles can contribute to defective EMT in endothelial cell lines through impaired induction of Snail and Hes family members. These data represent the first description of a molecular mechanism underlying NOTCH1 mutations in individuals with LVOT malformations, and have important implications regarding the functional contribution of these alleles to a complex set of developmental defects.

Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1

Nodal and Notch signaling pathways play essential roles in vertebrate development. Through a yeast two-hybrid screening, we identified Notch3 as a candidate binding partner of the Nodal coreceptor Cripto-1. Coimmunoprecipitation analysis confirmed the binding of Cripto-1 with all four mammalian Notch receptors. Deletion analyses revealed that the binding of Cripto-1 and Notch1 is mediated by the Cripto-1/FRL-1/Cryptic domain of Cripto-1 and the C-terminal region of epidermal growth factor-like repeats of Notch1. Binding of Cripto-1 to Notch1 occurred mainly in the endoplasmic reticulum-Golgi network. Cripto-1 expression resulted in the recruitment of Notch1 protein into lipid raft microdomains and enhancement of the furin-like protein convertase-mediated proteolytic maturation of Notch1 (S1 cleavage). Enhanced S1 cleavage resulted in the sensitization to ligand-induced activation of Notch signaling. In addition, knockdown of Cripto-1 expression in human and mouse embryonal carcinoma cells desensitized the ligand-induced Notch signaling activation. These results suggest a novel role of Cripto-1 in facilitating the posttranslational maturation of Notch receptors.

Jagged1-mediated Notch activation induces epithelial-to-mesenchymal transition through Slug-induced repression of E-cadherin

Aberrant expression of Jagged1 and Notch1 are associated with poor outcome in breast cancer. However, the reason that Jagged1 and/or Notch overexpression portends a poor prognosis is unknown. We identify Slug, a transcriptional repressor, as a novel Notch target and show that elevated levels of Slug correlate with increased expression of Jagged1 in various human cancers. Slug was essential for Notch-mediated repression of E-cadherin, which resulted in β-catenin activation and resistance to anoikis. Inhibition of ligand-induced Notch signaling in xenografted Slug-positive/E-cadherin–negative breast tumors promoted apoptosis and inhibited tumor growth and metastasis. This response was associated with down-regulated Slug expression, reexpression of E-cadherin, and suppression of active β-catenin. Our findings suggest that ligand-induced Notch activation, through the induction of Slug, promotes tumor growth and metastasis characterized by epithelial-to-mesenchymal transition and inhibition of anoikis.

C-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma

Human acute T-cell lymphoblastic leukemias and lymphomas (T-ALL) are commonly associated with gain-of-function mutations in Notch1 that contribute to T-ALL induction and maintenance. Starting from an expression-profiling screen, we identified c-myc as a direct target of Notch1 in Notch-dependent T-ALL cell lines, in which Notch accounts for the majority of c-myc expression. In functional assays, inhibitors of c-myc interfere with the progrowth effects of activated Notch1, and enforced expression of c-myc rescues multiple Notch1-dependent T-ALL cell lines from Notch withdrawal. The existence of a Notch1-c-myc signaling axis was bolstered further by experiments using c-myc-dependent murine T-ALL cells, which are rescued from withdrawal of c-myc by retroviral transduction of activated Notch1. This Notch1-mediated rescue is associated with the up-regulation of endogenous murine c-myc and its downstream transcriptional targets, and the acquisition of sensitivity to Notch pathway inhibitors. Additionally, we show that primary murine thymocytes at the DN3 stage of development depend on ligand-induced Notch signaling to maintain c-myc expression. Together, these data implicate c-myc as a developmentally regulated direct downstream target of Notch1 that contributes to the growth of T-ALL cells.

Jagged1-induced Notch signaling drives proliferation of multiple myeloma cells

AbstractNotch receptors expressed on hematopoietic stem cells interact with their ligands on bone marrow stromal cells and thereby control cell fate decisions and survival. We recently demonstrated that Notch signaling is involved in proliferation and survival of B cell-derived tumor cells of classic Hodgkin disease and described a novel mechanism for the oncogenic capacity of Notch. In this study we investigated whether Notch signaling is involved in the tight interactions between neoplastic plasma cells and their bone marrow microenvironment, which are essential for tumor cell growth in multiple myeloma (MM). Here we demonstrate that Notch receptors and their ligand Jagged1 are highly expressed in cultured and primary MM cells, whereas nonneoplastic counterparts show low to undetectable levels of Notch. Functional data indicate that ligand-induced Notch signaling is a growth factor for MM cells and suggest that these interactions contribute to myelomagenesis in vivo. (Blood. 2004;103:3511-3515)

Notch signaling imposes two distinct blocks in the differentiation of C2C12 myoblasts.

Notch signal transduction regulates expression of downstream genes through the activation of the DNA-binding protein Su(H)/CBF1. In Drosophila most of Notch signaling requires Su(H); however, some Notch-dependent processes occur in the absence of Su(H) suggesting that Notch signaling does not always involve activation of this factor. Using constitutively active forms of Notch lacking CBF1-interacting sequences we identified a Notch signaling pathway that inhibits myogenic differentiation of C2C12 myoblasts in the absence of CBF1 activation. Here we show that ligand-induced Notch signaling suppresses myogenesis in C2C12 myoblasts that express a dominant negative form of CBF1, providing additional evidence for CBF1-independent Notch signal transduction. Surprisingly mutant forms of Notch deficient in CBF1 activation are unable to antagonize MyoD activity, despite the fact that they inhibit myogenesis. Moreover, Notch-induced antagonism of MyoD requires CBF1 suggesting that the CBF1-dependent pathway mediates a cell-type-specific block in the myogenic program. However, Notch signaling in the absence of CBF1 activation blocks both myogenesis and osteogenesis, indicative of a general block in cellular differentiation. Taken together our data provide evidence for two distinct Notch signaling pathways that function to block differentiation at separate steps during the process of myogenesis in C2C12 myoblasts.

Delta-induced Notch Signaling Mediated by RBP-J Inhibits MyoD Expression and Myogenesis

Signaling induced by interaction between the receptor Notch and its ligand Delta plays an important role in cell fate determination in vertebrates as well as invertebrates. Vertebrate Notch signaling has been investigated using its constitutively active form, i.e. the truncated intracellular region which is believed to mimic Notch-Delta signaling by interaction with a DNA-binding protein RBP-J. However, the molecular mechanism for Notch signaling triggered by ligand binding, which leads to inhibition of differentiation, is not clear. We have established a myeloma cell line expressing mouse Delta1 on its cell surface which can block muscle differentiation by co-culture with C2C12 muscle progenitor cells. We showed that Delta-induced Notch signaling stimulated transcriptional activation of RBP-J binding motif, containing promoters including the HES1 promoter. Furthermore, ligand-induced Notch signaling up-regulated HES1 mRNA expression within 1 h and subsequently reduced expression of MyoD mRNA. Since cycloheximide treatment did not inhibit induction of HES1 mRNA, the HES1 promoter appears to be a primary target of activated Notch. In addition, a transcriptionally active form of RBP-J, i.e. VP16-RBP-J, inhibited muscle differentiation of C2C12 cells by blocking the expression of MyoD protein. These results suggest that HES1 induction by the Delta1/Notch signaling is mediated by RBP-J and blocks myogenic differentiation of C2C12 cells by subsequent inhibition of MyoD expression.