Notch signaling in blood vessels: from morphogenesis to homeostasis

Notch signaling is an evolutionarily conserved intercellular signaling pathway that plays numerous crucial roles in vascular development and physiology. Compelling evidence indicates that Notch signaling is vital for vascular morphogenesis including arterial and venous differentiation and endothelial tip and stalk cell specification during sprouting angiogenesis and also vessel maturation featured by mural cell differentiation and recruitment. Notch signaling is also required for vascular homeostasis in adults by keeping quiescent phalanx cells from re-entering cell cycle and by modulating the behavior of endothelial progenitor cells. We will summarize recent advances of Notch pathway in vascular biology with special emphasis on the underlying molecular mechanisms.     

Presenilin-based genetic screens in Drosophila melanogaster identify novel notch pathway modifiers

Presenilin is the enzymatic component of gamma-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimer's disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for gamma-tubulin in the pathway.     

Making sense out of missense mutations: Mechanistic dissection of Notch receptors through structure-function studies in Drosophila

Notch signaling is involved in the development of almost all organ systems and is required post-developmentally to modulate tissue homeostasis. Rare variants in Notch signaling pathway genes are found in patients with rare Mendelian disorders, while unique or recurrent somatic mutations in a similar set of genes are identified in cancer. The human genome contains four genes that encode Notch receptors, NOTCH1-4, all of which are linked to genetic diseases and cancer. Although some mutations have been classified as clear loss- or gain-of-function alleles based on cellular or rodent based assay systems, the functional consequence of many variants/mutations in human Notch receptors remain unknown. In this review, I will first provide an overview of the domain structure of Notch receptors and discuss how each module is known to regulate Notch signaling activity in vivo using the Drosophila Notch receptor as an example. Next, I will introduce some interesting mutant alleles that have been isolated in the fly Notch gene over the past > 100 years of research and discuss how studies of these mutations have facilitated the understanding of Notch biology. By identifying unique alleles of the fly Notch gene through forward genetic screens, mapping their molecular lesions and characterizing their phenotypes in depth, one can begin to unravel new mechanistic insights into how different domains of Notch fine-tune signaling output. Such information can be useful in deciphering the functional consequences of rare variants/mutations in human Notch receptors, which in turn can influence disease management and therapy.     

Notch3 Interactome Analysis Identified WWP2 as a Negative Regulator of Notch3 Signaling in Ovarian Cancer

The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD) interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2 increases mono-ubiquitination of the membrane-tethered Notch3 fragment, therefore attenuating Notch3 pathway activity in cancer cells and leading to cell cycle arrest. The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor. Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas. Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance. Taken together, our results provide evidence that WWP2 serves as a tumor suppressor by negatively regulating Notch3 signaling in ovarian cancer.     

Expression of Notch1 and Jagged2 in the enteric nervous system.

The Notch signaling pathway is a vitally important pathway in regulating brain development. To explore the involvement of the Notch pathway in neuronal cells of adult rat gut, we investigated the expression of Notch1 and Jagged2 by in situ hybridization (ISH) and immunohistochemistry (IHC). In the enteric nervous system, Notch1 and Jagged2 were expressed in ganglia of the submucosal and myenteric plexus. Notch1 was preferentially expressed in cholinergic neurons lacking calretinin or nitric oxide synthase (NOS), whereas Jagged2 was present in most neuron subtypes. We propose that Notch1 and Jagged2 have a continuing role in the maintenance and function of neuronal cells in the adult enteric nervous system.     

Notch and presenilins in vertebrates and invertebrates: implications for neuronal development and degeneration

Recent progress in elucidating the biology of Notch and presenilin has revealed a close functional relationship between these two proteins during cell fate determination in worms, flies and humans. Presenilins are required for the putatively intramembranous proteolysis of Notch to release its intracellular domain to the nucleus. This finding establishes a specific biochemical role for presenilins in Notch signaling and interfaces with emerging evidence about how frizzled, disheveled and numerous other genes regulate the highly complex Notch pathway. Advances in understanding Notch and presenilin functions in the differentiation of neurons and non-neural cells have important implications not only for development but also for late-life degenerative disorders such as Alzheimer's disease.     

Targeting Notch signaling cross-talk with estrogen receptor and ErbB-2 in breast cancer

The Notch signaling pathway is receiving considerable interest because of its pervasive importance in developmental biology and more recently, in the post-natal functions of the immune system and in cancer biology.Our observations, together with those of other laboratories, support a context-dependent role for Notch signaling in breast cancer.Targeting Notch signaling paves the way to new therapeutic strategy.     

Notch signaling in cancer

The evolutionarily conserved developmental pathway driven by Notch receptors and ligands has acquired multiple post-natal homeostatic functions in vertebrates. Potential roles in human physiology and pathology are being studied by an increasingly large number of investigators. While the canonical Notch signaling pathway is deceptively simple, the consequences of Notch activation on cell fate are complex and context-dependent. The manner in which other signaling pathways cross-talk with Notch signaling appears to be extraordinarily complex. Recent observations have demonstrated the importance of endocytosis, multiple ubiquitin ligases, non-visual beta-arrestins and hypoxia in modulating Notch signaling. Structural biology is shedding light on the molecular mechanisms whereby Notch interacts with its nuclear partners. Genomics is slowly unraveling the puzzle of Notch target genes in several systems. At the same time, interest in modulating Notch signaling for medical purposes has dramatically increased. Over the last few years we have learned much about Notch signaling in cancer, immune disorders, neurological disorders and most recently, stroke. The role of Notch signaling in normal and transformed stem cells is under intense investigation. Some Notch-modulating drugs are already in clinical trials, and others at various stages of development. This review will focus on the most recent findings on Notch signaling in cancer and discuss their potential clinical implications.     

Mammary development and breast cancer: the role of stem cells

The mammary gland is a highly regenerative organ that can undergo multiple cycles of proliferation, lactation and involution, a process controlled by stem cells. The last decade much progress has been made in the identification of signaling pathways that function in these stem cells to control self-renewal, lineage commitment and epithelial differentiation in the normal mammary gland. The same signaling pathways that control physiological mammary development and homeostasis are also often found deregulated in breast cancer. Here we provide an overview on the functional and molecular identification of mammary stem cells in the context of both normal breast development and breast cancer. We discuss the contribution of some key signaling pathways with an emphasis on Notch receptor signaling, a cell fate determination pathway often deregulated in breast cancer. A further understanding of the biological roles of the Notch pathway in mammary stem cell behavior and carcinogenesis might be relevant for the development of future therapies.     

Scalloped and strawberry notch are target genes of Notch signaling in the context of wing margin formation in Drosophila.

The Notch pathway regulates the differentiation of many cell types throughout development of higher metazoa. Different cellular responses are elicited through specific activation of distinct Notch target genes. In the Drosophila wing, for example, the cut gene is activated by Notch signaling along the dorso-ventral boundary but, as we show here, not in other cell types. We identify additional regulatory components, scalloped and strawberry notch, that are targets of the Notch pathway specifically within the wing anlagen. As suggested by physical interactions, these proteins could be co-factors of the cut trans-regulator Vestigial. Additional regulatory input comes from the Wingless pathway. Our data support a model, whereby context specific involvement of distinct co-regulators modulates Notch target gene activation.     

Notch信号与心脏干细胞关系的研究进展

Notch信号是广泛存在于各种动物细胞中高度保守的信号途径,在干细胞生物学功能中发挥重要作用。心脏干细胞（cardiac stem cells,CSCs）是存在于心脏特殊微环境下的多潜能干细胞,其表面存在Notch受体,而与其相邻的细胞可表达Notch配体,提示心脏干细胞中的Notch信号在某些条件下可被活化。该文从Notch信号通路的组成和激活、CSCs的界定与来源、CSCs主要类型的一般生物学特征及Notch信号通路与CSCs形成、分化和增殖的关系等方面进行综述,并展望了基于CSCs在心肌再生相关转化医学研究中的前景。     

Endosomal entry regulates Notch receptor activation in Drosophila melanogaster

Signaling through the transmembrane receptor Notch is widely used throughout animal development and is a major regulator of cell proliferation and differentiation. During canonical Notch signaling, internalization and recycling of Notch ligands controls signaling activity, but the involvement of endocytosis in activation of Notch itself is not well understood. To address this question, we systematically assessed Notch localization, processing, and signaling in a comprehensive set of Drosophila melanogaster mutants that block access of cargo to different endocytic compartments. We find that gamma-secretase cleavage and signaling of endogenous Notch is reduced in mutants that impair entry into the early endosome but is enhanced in mutants that increase endosomal retention. In mutants that block endosomal entry, we also uncover an alternative, low-efficiency Notch trafficking route that can contribute to signaling. Our data show that endosomal access of the Notch receptor is critical to achieve physiological levels of signaling and further suggest that altered residence in distinct endocytic compartments could underlie pathologies involving aberrant Notch pathway activation.     

Epithelial Mesenchymal Transition and Pancreatic Tumor Initiating CD44+/EpCAM+ Cells Are Inhibited by γ-Secretase Inhibitor IX

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a high rate of metastasis. Recent studies have indicated that the Notch signalling pathway is important in PDAC initiation and maintenance, although the specific cell biological roles of the pathway remain to be established. Here we sought to examine this question in established pancreatic cancer cell lines using the γ-secretase inhibitor IX (GSI IX) to inactivate Notch. Based on the known roles of Notch in development and stem cell biology, we focused on effects on epithelial mesenchymal transition (EMT) and on pancreatic tumor initiating CD44+/EpCAM+ cells. We analyzed the effect of the GSI IX on growth and epithelial plasticity of human pancreatic cancer cell lines, and on the tumorigenicity of pancreatic tumor initiating CD44+/EpCAM+ cells. Notably, apoptosis was induced after GSI IX treatment and EMT markers were selectively targeted. Furthermore, under GSI IX treatment, decline in the growth of pancreatic tumor initiating CD44+/EpCAM+ cells was observed in vitro and in a xenograft mouse model. This study demonstrates a central role of Notch signalling pathway in pancreatic cancer pathogenesis and identifies an effective approach to inhibit selectively EMT and suppress tumorigenesis by eliminating pancreatic tumor initiating CD44+/EpCAM+ cells.     

Epsin potentiates Notch pathway activity in Drosophila and C. elegans

Endocytosis and trafficking within the endocytosis pathway are known to modulate the activity of different signaling pathways. Epsins promote endocytosis and are postulated to target specific proteins for regulated endocytosis. Here, we present a functional link between the Notch pathway and epsins. We identify the Drosophila ortholog of epsin, liquid facets (lqf), as an inhibitor of cardioblast development in a genetic screen for mutants that affect heart development. We find that lqf inhibits cardioblast development and promotes the development of fusion-competent myoblasts, suggesting a model in which lqf acts on or in fusion-competent myoblasts to prevent their acquisition of the cardioblast fate. lqf and Notch exhibit essentially identical heart phenotypes, and lqf genetically interacts with the Notch pathway during multiple Notch-dependent events in Drosophila. We extended the link between the Notch pathway and epsin function to C. elegans, where the C. elegans lqf ortholog acts in the signaling cell to promote the glp-1/Notch pathway activity during germline development. Our results suggest that epsins play a specific, evolutionarily conserved role to promote Notch signaling during animal development and support the idea that they do so by targeting ligands of the Notch pathway for endocytosis.     

Notch信号通路与血液系统和白血病形成

Notch基因编码着一类进化上高度保守的跨膜受体蛋白家族,其信号通路是由Notch受体、Notch配体、CSL DNA结合蛋白组成。该信号通路能够调节淋巴细胞的发育和分化过程,介导心血管系统的形成,参与肿瘤的发生和发展。近年来有研究表明,Notch信号通路在造血作用及白血病的发生过程中起关键作用。本文综述了Notch信号通路在淋巴细胞及造血干细胞的发育和分化中的作用,进一步探讨了其对造血作用和白血病发生发展的调节,以期能够对临床造血疾病的治疗提供帮助。     

Downregulation of microRNA-592 protects mice from hypoplastic heart and congenital heart disease by inhibition of the Notch signaling pathway through upregulating KCTD10

Evidence has demonstrated that the microRNA (miR) may play a significant role in the development of congenital heart disease (CHD). Here, we explore the mechanism of microRNA-592 (miR-592) in heart development and CHD with the involvement of KCTD10 and Notch signaling pathway in a CHD mouse model. Cardiac tissues were extracted from CHD and normal mice. Immunohistochemistry staining was performed to detect positive expression rate of KCTD10. A series of inhibitor, activators, and siRNAs was introduced to verified regulatory functions for miR-592 governing KCTD10 in CHD. Furthermore, the effect of miR-592 on cell proliferation and apoptosis was also investigated. Downregulated positive rate of KCTD10 was observed in CHD mice. Downregulation of miR-592 would upregulate expression of KCTD10 and inhibit the activation of Notch signaling pathway, thus promote cell proliferation. This study demonstrates that downregulation of miR-592 prevents CHD and hypoplastic heart by inhibition of the Notch signaling pathway via negatively binding to KCTD10.     

Decoding the PTM-switchboard of Notch

The developmentally indispensable Notch pathway exhibits a high grade of pleiotropism in its biological output. Emerging evidence supports the notion of post-translational modifications (PTMs) as a modus operandi controlling dynamic fine-tuning of Notch activity. Although, the intricacy of Notch post-translational regulation, as well as how these modifications lead to multiples of divergent Notch phenotypes is still largely unknown, numerous studies show a correlation between the site of modification and the output. These include glycosylation of the extracellular domain of Notch modulating ligand binding, and phosphorylation of the PEST domain controlling half-life of the intracellular domain of Notch. Furthermore, several reports show that multiple PTMs can act in concert, or compete for the same sites to drive opposite outputs. However, further investigation of the complex PTM crosstalk is required for a complete understanding of the PTM-mediated Notch switchboard. In this review, we aim to provide a consistent and up-to-date summary of the currently known PTMs acting on the Notch signaling pathway, their functions in different contexts, as well as explore their implications in physiology and disease. Furthermore, we give an overview of the present state of PTM research methodology, and allude to a future with PTM-targeted Notch therapeutics.     

Inducible RNA interference uncovers the Drosophila protein Bx42 as an essential nuclear cofactor involved in Notch signal transduction

We used the UAS/GAL4 two component system to induce mRNA interference (mRNAi) during Drosophila development. In the adult eye the expression from white transgenes or the resident white locus is significantly repressed by the induction of UAS-wRNAi using different GAL4 expressing strains. By induced RNAi we demonstrate that the conserved nuclear protein Bx42 is essential for the development of many tissues. Phenotypically the effects of Bx42 RNAi resemble those obtained for certain classes of Notch mutants, pointing to an involvement of Bx42 in the Notch signal transduction pathway. The wing phenotype following overexpression of Suppressor of Hairless is strongly enhanced by simultaneous Bx42 RNAi induction in the same tissue. Target genes of Notch signaling like cut and Enhancer of split m8 were suppressed by induction of Bx42 RNAi. Our results demonstrate that inducible RNAi is a powerful tool to study the role of essential genes throughout development.