Conditional deletion of Notch1 and Notch2 genes in excitatory neurons of postnatal forebrain does not cause neurodegeneration or reduction of Notch mRNAs and proteins

Activation of Notch signaling requires intramembranous cleavage by γ-secretase to release the intracellular domain. We previously demonstrated that presenilin and nicastrin, components of the γ-secretase complex, are required for neuronal survival in the adult cerebral cortex. Here we investigate whether Notch1 and/or Notch2 are functional targets of presenilin/γ-secretase in promoting survival of excitatory neurons in the adult cerebral cortex by generating Notch1, Notch2, and Notch1/Notch2 conditional knock-out (cKO) mice. Unexpectedly, we did not detect any neuronal degeneration in the adult cerebral cortex of these Notch cKO mice up to ～2 years of age, whereas conditional inactivation of presenilin or nicastrin using the same αCaMKII-Cre transgenic mouse caused progressive, striking neuronal loss beginning at 4 months of age. More surprisingly, we failed to detect any reduction of Notch1 and Notch2 mRNAs and proteins in the cerebral cortex of Notch1 and Notch2 cKO mice, respectively, even though Cre-mediated genomic deletion of the floxed Notch1 and Notch2 exons clearly took place in the cerebral cortex of these cKO mice. Furthermore, introduction of Cre recombinase into primary cortical cultures prepared from postnatal floxed Notch1/Notch2 pups, where Notch1 and Notch2 are highly expressed, completely eliminated their expression, indicating that the floxed Notch1 and Notch2 alleles can be efficiently inactivated in the presence of Cre. Together, these results demonstrate that Notch1 and Notch2 are not involved in the age-related neurodegeneration caused by loss of presenilin or γ-secretase and suggest that there is no detectable expression of Notch1 and Notch2 in pyramidal neurons of the adult cerebral cortex.     

Notch2 and Notch3 function together to regulate vascular smooth muscle development

Notch signaling has been implicated in the regulation of smooth muscle differentiation, but the precise role of Notch receptors is ill defined. Although Notch3 receptor expression is high in smooth muscle, Notch3 mutant mice are viable and display only mild defects in vascular patterning and smooth muscle differentiation. Notch2 is also expressed in smooth muscle and Notch2 mutant mice show cardiovascular abnormalities indicative of smooth muscle defects. Together, these findings infer that Notch2 and Notch3 act together to govern vascular development and smooth muscle differentiation. To address this hypothesis, we characterized the phenotype of mice with a combined deficiency in Notch2 and Notch3. Our results show that when Notch2 and Notch3 genes are simultaneously disrupted, mice die in utero at mid-gestation due to severe vascular abnormalities. Assembly of the vascular network occurs normally as assessed by Pecam1 expression, however smooth muscle cells surrounding the vessels are grossly deficient leading to vascular collapse. In vitro analysis show that both Notch2 and Notch3 robustly activate smooth muscle differentiation genes, and Notch3, but not Notch2 is a target of Notch signaling. These data highlight the combined actions of the Notch receptors in the regulation of vascular development, and suggest that while these receptors exhibit compensatory roles in smooth muscle, their functions are not entirely overlapping.     

Notch 家族四个蛋白质在人胰腺癌细胞 HPAC 中对 YY1 和 EGFR水平的影响

Notch信号通路是肿瘤形成过程中一种重要的信号通路,其中心分子为Notch受体. Notch受体为细胞膜上的单次跨膜蛋白,介导细胞间信号转导,哺乳动物细胞内包括Notch1、Notch2、Notch3和Notch4的4个成员. Notch家族4个蛋白质在结构和功能上存在差异.前期研究显示,Notch1信号通路与转录因子YY1（YING-YANG 1）、表皮生长因子受体（EGFR）间存在调控作用. 本研究在人胰腺癌细胞HPAC中,采用RNA干扰技术,分别降低细胞中Notch家族4个蛋白质的表达,检测YY1和EGFR在mRNA和蛋白质水平上的表达;并构建相应的激活形式的Notch受体--Notch胞内结构域（Notch intracellular domain,NICD）真核表达质粒,在HPAC细胞中分别过表达4种NICD,检测其对YY1和EGFR表达水平的影响. 结果显示,降低细胞中Notch1或Notch3的表达,均使HPAC细胞中EGFR mRNA和蛋白质水平升高（P<0.05）,而降低Notch2和Notch4后,EGFR mRNA和蛋白质水平没有改变（P>0.05）.分别降低4个Notch的表达,对YY1的蛋白质和mRNA表达水平均没有改变（P>0.05）. 在HPAC细胞中过表达N1ICD和N3ICD后,YY1和EGFR的蛋白质水平降低（P<0.05）,而过表达N2ICD和N4ICD后,YY1和EGFR蛋白质水平没有改变（P>0.05）.分别过表达4种NICD均没有改变YY1和EGFR的mRNA表达水平（P>0.05）.初步得出结论是,在HPAC细胞中,Notch信号通路经Notch1和Notch3影响EGFR的表达. Notch1和Notch3对EGFR的表达可能具有负调控作用. 在Notch1和Notch3过度激活时,这种调控作用通过YY1介导. 本文可为深入研究Notch信号通路对胰腺癌发生发展的作用机制提供有意义的参考.     

Regulation of Notch signaling by glycosylation

Notch receptors are approximately 300 kDa cell surface glycoproteins whose activation by Notch ligands regulates cell fate decisions in the metazoa. The extracellular domain of Notch receptors has many epidermal growth factor like repeats that are glycosylated with O-fucose and O-glucose glycans as well as N-glycans. Disruption of O-fucose glycan synthesis leads to severe Notch signaling defects in Drosophila and mammals. Removal or addition of O-fucose glycan consensus sites on Notch receptors also leads to Notch signaling defects. Ligand binding and ligand-induced Notch signaling assays have provided insights into how changes in the O-fucose glycans of Notch receptors alter Notch signaling.     

Notch2, but not Notch1, is required for proximal fate acquisition in the mammalian nephron

The Notch pathway regulates cell fate determination in numerous developmental processes. Here we report that Notch2 acts non-redundantly to control the processes of nephron segmentation through an Rbp-J-dependent process. Notch1 and Notch2 are detected in the early renal vesicle. Genetic analysis reveals that only Notch2 is required for the differentiation of proximal nephron structures (podocytes and proximal convoluted tubules) despite the presence of activated Notch1 in the nuclei of putative proximal progenitors. The inability of endogenous Notch1 to compensate for Notch2 deficiency may reflect sub-threshold Notch1 levels in the nucleus. In line with this view, forced expression of a gamma-secretase-independent form of Notch1 intracellular domain drives the specification of proximal fates where all endogenous, ligand-dependent Notch signaling is blocked by a gamma-secretase inhibitor. These results establish distinct (non-redundant), instructive roles for Notch receptors in nephron segmentation.     

SEL-10 is an inhibitor of notch signaling that targets notch for ubiquitin-mediated protein degradation

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.     

Low Density Lipoprotein Receptor-related Protein-1 (LRP1) Regulates Thrombospondin-2 (TSP2) Enhancement of Notch3 Signaling

Intracellular trafficking of Notch and Notch ligands modulates signaling, suggesting that choreography of ligand and receptor translocation is essential for optimal Notch activity. Indeed, a major model for Notch signaling posits that Notch trans-endocytosis into the ligand-expressing (signal sending) cell is a key driving force for Notch signal transduction. The extracellular protein thrombospondin-2 (TSP2) enhances Notch signaling and binds to both Jagged1 and Notch3 ectodomains, potentially bridging two essential extracellular components of Notch signaling. We investigated the role of low density lipoprotein receptor-related protein-1 (LRP1), a TSP2 receptor, in the regulation of Notch3 signaling. TSP2 potentiation of Notch is blocked by the receptor-associated protein (an inhibitor of low density lipoprotein receptor-related protein function) and requires LRP1 expression in the signal-sending cell. TSP2 stimulates Notch3 endocytosis into wild type fibroblasts but not LRP1-deficient fibroblasts. Finally, recombinant Notch3 and Jagged1 interact with the LRP1 85-kDa B-chain, a subunit that lacks known ligand binding function. Our data suggest that LRP1 and TSP2 stimulate Notch activity by driving trans-endocytosis of the Notch ectodomain into the signal-sending cell and demonstrate a novel, non-cell autonomous function of LRP1 in cell-cell signaling.     

The microRNA pathway regulates the temporal pattern of Notch signaling in Drosophila follicle cells

Multicellular development requires the correct spatial and temporal regulation of cell division and differentiation. These processes are frequently coordinated by the activities of various signaling pathways such as Notch signaling. From a screen for modifiers of Notch signaling in Drosophila we have identified the RNA helicase Belle, a recently described component of the RNA interference pathway, as an important regulator of the timing of Notch activity in follicle cells. We found that loss of Belle delays activation of Notch signaling, which results in delayed follicle cell differentiation and defects in the cell cycle. Because mutations in well-characterized microRNA components phenocopied the Notch defects observed in belle mutants, Belle might be functioning in the microRNA pathway in follicle cells. The effect of loss of microRNAs on Notch signaling occurs upstream of Notch cleavage, as expression of the constitutively active intracellular domain of Notch in microRNA-defective cells restored proper activation of Notch. Furthermore, we present evidence that the Notch ligand Delta is an important target of microRNA regulation in follicle cells and regulates the timing of Notch activation through cis inhibition of Notch. Here we have uncovered a complex regulatory process in which the microRNA pathway promotes Notch activation by repressing Delta-mediated inhibition of Notch in follicle cells.     

Physical interaction of Delta1, Jagged1, and Jagged2 with Notch1 and Notch3 receptors

The Delta/Serrate/LAG-2 (DSL) domain-containing proteins, Delta1, Jagged1, and Jagged2, are considered to be ligands for Notch receptors. However, the physical interaction between the three DSL proteins and respective Notch receptors remained largely unknown. In this study, we investigated this issue through the targeting of Notch1 and Notch3 in two experimental systems using fusion proteins comprising their extracellular portions. Cell-binding assays showed that soluble forms of Notch1 and Notch3 proteins physically bound to the three DSL proteins on the cell surface. In solid-phase binding assays using immobilized soluble Notch1 and Notch3 proteins, it was revealed that each DSL protein directly bound to the soluble Notch proteins with different affinities. All interactions between the DSL proteins and soluble Notch proteins were dependent on Ca(2+). Taken together, these results suggest that Delta1, Jagged1, and Jagged2 are ligands for Notch1 and Notch3 receptors.     

The O-fucosyltransferase O-fut1 is an extracellular component that is essential for the constitutive endocytic trafficking of Notch in Drosophila

Notch is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell-fate decisions. Endocytic trafficking of Notch plays important roles in the activation and downregulation of this receptor. A Drosophila O-FucT-1 homolog, encoded by O-fut1, catalyzes the O-fucosylation of Notch, a modification essential for Notch signaling and ligand binding. It was recently proposed that O-fut1 acts as a chaperon for Notch in the endoplasmic reticulum and is required for Notch to exit the endoplasmic reticulum. Here, we report that O-fut1 has additional functions in the endocytic transportation of Notch. O-fut1 was indispensable for the constitutive transportation of Notch from the plasma membrane to the early endosome, which we show was independent of the O-fucosyltransferase activity of O-fut1. We also found that O-fut1 promoted the turnover of Notch, which consequently downregulated Notch signaling. O-fut1 formed a stable complex with the extracellular domain of Notch. In addition, O-fut1 protein added to conditioned medium and endocytosed was sufficient to rescue normal Notch transportation to the early endosome in O-fut1 knockdown cells. Thus, an extracellular interaction between Notch and O-fut1 is essential for the normal endocytic transportation of Notch. We propose that O-fut1 is the first example, except for ligands, of a molecule that is required extracellularly for receptor transportation by endocytosis.     

The origin of the ankyrin repeat region in Notch intracellular domains is critical for regulation of HES promoter activity

Notch signal transduction is mediated by proteolysis of the receptor and translocation of the intracellular domain (IC) into the nucleus, where it functions as a regulator of HES gene expression after binding to the DNA-binding protein RBP-J kappa. The mammalian Notch receptors are structurally very similar, but have distinct functions. Most notably, Notch 1 IC is a potent activator of the HES promoter, while Notch 3 IC is a much weaker activator and can repress Notch 1 IC-mediated HES activation in certain contexts. In this report we explore the molecular basis for this functional difference between Notch 1 and Notch 3 IC. We find that Notch 3 IC, like Notch 1 IC, can bind the SKIP and PCAF proteins. Furthermore, both Notch 1 and Notch 3 ICs displace the co-repressor SMRT from the DNA-binding protein RBP-J kappa on the HES promoter. The latter observation suggests that both Notch 3 IC and Notch 1 IC can access RBP-J kappa in vivo, and that the difference in activation capacity instead stems from structural differences in the two ICs when positioned on RBP-J kappa. We show that two distinct regions in the Notch IC are critical for the difference between the Notch 1 and Notch 3 IC. First, the origin of the ankyrin repeat region is important, i.e. only chimeric ICs containing a Notch 1-derived ankyrin repeat region are potent activators. Second, we identify a novel important region in the Notch IC. This region, named the RE/AC region (for repression/activation), is located immediately C-terminal to the ankyrin repeat region, and is required for Notch 1 IC's ability to activate and for Notch 3 IC's ability to repress a HES promoter. The interplay between the RE/AC region and the ankyrin repeat region provides a basis to understand the difference in HES activation between structurally similar Notch receptors.     

Roles of Pofut1 and O-fucose in mammalian Notch signaling

Mammalian Notch receptors contain 29-36 epidermal growth factor (EGF)-like repeats that may be modified by protein O-fucosyltransferase 1 (Pofut1), an essential component of the canonical Notch signaling pathway. The Drosophila orthologue Ofut1 is proposed to function as both a chaperone required for stable cell surface expression of Notch and a protein O-fucosyltransferase. Here we investigate these dual roles of Pofut1 in relation to endogenous Notch receptors of Chinese hamster ovary and murine embryonic stem (ES) cells. We show that fucosylation-deficient Lec13 Chinese hamster ovary cells have wild type levels of Pofut1 and cell surface Notch receptors. Nevertheless, they have reduced binding of Notch ligands and low levels of Delta1- and Jagged1-induced Notch signaling. Exogenous fucose but not galactose rescues both ligand binding and Notch signaling. Murine ES cells lacking Pofut1 also have wild type levels of cell surface Notch receptors. However, Pofut1-/- ES cells do not bind Notch ligands or exhibit Notch signaling. Although overexpression of fucosyltransferase-defective Pofut1 R245A in Pofut1-/- cells partially rescues ligand binding and Notch signaling, this effect is not specific. The same rescue is achieved by an unrelated, inactive, endoplasmic reticulum glucosidase. Therefore, mammalian Notch receptors require Pofut1 for the generation of optimally functional Notch receptors, but, in contrast to Drosophila, Pofut1 is not required for stable cell surface expression of Notch. Importantly, we also show that, under certain circumstances, mammalian Notch receptors are capable of signaling in the absence of Pofut1 and O-fucose.     

Notch signaling controls proliferation through cell-autonomous and non-autonomous mechanisms in the Drosophila eye

During Drosophila eye development, localized Notch signaling at the dorsal ventral (DV)-midline promotes growth of the entire eye field. This long-range action of Notch signaling may be mediated through the diffusible ligand of the Jak/STAT pathway, Unpaired (Upd), which was recently identified as a downstream target of Notch. However, Notch activity has not been shown to be cell-autonomously required for Upd expression and therefore yet another diffusible signal may be required for Notch activation of Upd. Our results clarify the Notch requirement, demonstrating that Notch activity at the DV-midline leads to cell-autonomous expression of Upd as monitored in loss and gain-of-function Notch clones. In addition, mutations in the Jak/STAT pathway interact genetically with the Notch pathway by suppressing Notch mediated overgrowth. N(act) clones show non-autonomous effects on the cell cycle anterior to the furrow, indicating function of the Jak/STAT pathway. However, cell-autonomous effects of Notch within and posterior to the furrow are independent of Upd. Here, Notch autonomously maintains cells in a proliferative state and blocks photoreceptor differentiation.