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Sample records for notch signaling pathways

  1. The Notch signaling pathway as a mediator of tumor survival

    PubMed Central

    Pine, Sharon R.

    2013-01-01

    The Notch signaling pathway is evolutionarily conserved and responsible for cell fate determination in the developing embryo and mature tissue. At the molecular level, ligand binding activates Notch signaling by liberating the Notch intracellular domain, which then translocates into the nucleus and activates gene transcription. Despite the elegant simplicity of this pathway, which lacks secondary messengers or a signaling cascade, Notch regulates gene expression in a highly context- and cell-type-dependent manner. Notch signaling is frequently dysregulated, most commonly by overactivation, across many cancers and confers a survival advantage on tumors, leading to poorer outcomes for patients. Recent studies demonstrate how Notch signaling increases tumor cell proliferation and provide evidence that active Notch signaling maintains the cancer stem-cell pool, induces epithelial–mesenchymal transition and promotes chemoresistance. These studies imply that pharmacological inhibition of Notch signaling may refine control of cancer therapy and improve patient survival. Gamma secretase inhibitors (GSIs) are drugs that inhibit Notch signaling and may be successful in controlling cancer cell growth in conjunction with standard chemotherapy, but substantial side effects have hampered their widespread use. Recent efforts have been aimed at the development of antibodies against specific Notch receptors and ligands with the hope of limiting side effects while providing the same therapeutic benefit as GSIs. Together, studies characterizing Notch signaling and modulation have offered hope that refined methods targeting Notch may become powerful tools in anticancer therapeutics. PMID:23585460

  2. The Notch signaling pathway as a mediator of tumor survival.

    PubMed

    Capaccione, Kathleen M; Pine, Sharon R

    2013-07-01

    The Notch signaling pathway is evolutionarily conserved and responsible for cell fate determination in the developing embryo and mature tissue. At the molecular level, ligand binding activates Notch signaling by liberating the Notch intracellular domain, which then translocates into the nucleus and activates gene transcription. Despite the elegant simplicity of this pathway, which lacks secondary messengers or a signaling cascade, Notch regulates gene expression in a highly context- and cell-type-dependent manner. Notch signaling is frequently dysregulated, most commonly by overactivation, across many cancers and confers a survival advantage on tumors, leading to poorer outcomes for patients. Recent studies demonstrate how Notch signaling increases tumor cell proliferation and provide evidence that active Notch signaling maintains the cancer stem-cell pool, induces epithelial-mesenchymal transition and promotes chemoresistance. These studies imply that pharmacological inhibition of Notch signaling may refine control of cancer therapy and improve patient survival. Gamma secretase inhibitors (GSIs) are drugs that inhibit Notch signaling and may be successful in controlling cancer cell growth in conjunction with standard chemotherapy, but substantial side effects have hampered their widespread use. Recent efforts have been aimed at the development of antibodies against specific Notch receptors and ligands with the hope of limiting side effects while providing the same therapeutic benefit as GSIs. Together, studies characterizing Notch signaling and modulation have offered hope that refined methods targeting Notch may become powerful tools in anticancer therapeutics. PMID:23585460

  3. Targeting the Notch signaling pathway in cancer therapeutics

    PubMed Central

    Guo, Huajiao; Lu, Yi; Wang, Jianhua; Liu, Xia; Keller, Evan T; Liu, Qian; Zhou, Qinghua; Zhang, Jian

    2014-01-01

    Despite advances in surgery, imaging, chemotherapy, and radiotherapy, the poor overall cancer-related death rate remains unacceptable. Novel therapeutic strategies are desperately needed. Nowadays, targeted therapy has become the most promising therapy and a welcome asset to the cancer therapeutic arena. There is a large body of evidence demonstrating that the Notch signaling pathway is critically involved in the pathobiology of a variety of malignancies. In this review, we provide an overview of emerging data, highlight the mechanism of the Notch signaling pathway in the development of a wide range of cancers, and summarize recent progress in therapeutic targeting of the Notch signaling pathway. PMID:26767041

  4. Involvement of Notch1/Hes signaling pathway in ankylosing spondylitis

    PubMed Central

    Xu, Wei; Liang, Chao-Ge; Li, Yi-Fan; Ji, Yun-Han; Qiu, Wen-Jun; Tang, Xian-Zhong

    2015-01-01

    We aimed to investigate the role of Notch1/Hes signaling pathway in the pathogenesis of abnormal ossification of hip ligament in patients with ankylosing spondylitis (AS). 22 AS patients scheduled for artificial hip arthroplasty were randomly chosen as AS group. As controls, we used 4 patients diagnosed with transcervical fracture who underwent hip replacement surgery. Notch1 and Hes mRNA expressions were detected by real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR). Immunohistochemistry (IHC) was used to detect Notch1 and Hes protein expression. Correlation analyses of Notch-l and Hes with AS-related clinical factors were conducted with spearman’s correlation analysis and partial correlation analysis. RFQ-PCR results showed significant differences in Notch1 and Hes mRNA expressions between AS group and the control group (all P < 0.05). IHC analysis further indicated positive nuclear signals of Notch1 and Hes protein, indicating functional activation of the Notch1 and Hes pathways. Semi-quantitative IHC showed a higher Notch1 and Hes expression levels in AS group compared to the control group (all P < 0.05). Correlation analysis suggested that Hes protein expression was positively associated with the clinical course of the disease in AS patients. In conclusion, Notch1 and Hes overexpression was clearly detected in hip joint ligaments of AS patients, Hes protein expression was associated with the clinical course of AS. Taken together, we suggest that signaling pathways mediated by Notch1-Hes may contribute to ligament ossification of hip joints in AS patients. PMID:26045779

  5. SEPT4 is regulated by the Notch signaling pathway.

    PubMed

    Liu, Wenbin

    2012-04-01

    Notch receptor-mediated signaling is an evolutionarily conserved pathway that regulates diverse developmental processes and its dysregulation has been implicated in a variety of developmental disorders and cancers. Notch functions in these processes by activating expression of its target genes. Septin 4 (SEPT4) is a polymerizing GTP-binding protein that serves as scaffold for diverse molecules and is involved in cell proliferation and apoptosis. After activation of the Notch signal, the expression of SEPT4 is up-regulated and cell proliferation is inhibited. When the Notch signal is inhibited by the CSL (CBF1/Su(H)/Lag-1)-binding-domain-negative Mastermind-like protein 1, the expression of SEPT4 is down-regulated, proliferation and colony formation of cells are promoted, but cell adhesion ability is decreased. Nevertheless, the SEPT4 expression is not affected after knock-down of CSL. Meanwhile, if SEPT4 activity is inhibited through RNA interference, the protein level and activity of NOTCH1 remains unchanged, but cell proliferation is dysregulated. This indicates that SEPT4 is a Notch target gene. This relationship between Notch signaling pathway and SEPT4 offers a potential basis for further study of developmental control and carcinogenesis. PMID:21938432

  6. Activation of the Notch signaling pathway promotes neurovascular repair after traumatic brain injury

    PubMed Central

    Ran, Qi-shan; Yu, Yun-hu; Fu, Xiao-hong; Wen, Yuan-chao

    2015-01-01

    The Notch signaling pathway plays a key role in angiogenesis and endothelial cell formation, but it remains unclear whether it is involved in vascular repair by endothelial progenitor cells after traumatic brain injury. Therefore, in the present study, we controlled the Notch signaling pathway using overexpression and knockdown constructs. Activation of the Notch signaling pathway by Notch1 or Jagged1 overexpression enhanced the migration, invasiveness and angiogenic ability of endothelial progenitor cells. Suppression of the Notch signaling pathway with Notch1 or Jagged1 siRNAs reduced the migratory capacity, invasiveness and angiogenic ability of endothelial progenitor cells. Activation of the Notch signaling pathway in vivo in a rat model of mild traumatic brain injury promoted neurovascular repair. These findings suggest that the activation of the Notch signaling pathway promotes blood vessel formation and tissue repair after brain trauma. PMID:26487853

  7. Small molecules intercept Notch signaling and the early secretory pathway.

    PubMed

    Krämer, Andreas; Mentrup, Torben; Kleizen, Bertrand; Rivera-Milla, Eric; Reichenbach, Daniela; Enzensperger, Christoph; Nohl, Richard; Täuscher, Eric; Görls, Helmar; Ploubidou, Aspasia; Englert, Christoph; Werz, Oliver; Arndt, Hans-Dieter; Kaether, Christoph

    2013-11-01

    Notch signaling has a pivotal role in numerous cell-fate decisions, and its aberrant activity leads to developmental disorders and cancer. To identify molecules that influence Notch signaling, we screened nearly 17,000 compounds using automated microscopy to monitor the trafficking and processing of a ligand-independent Notch-enhanced GFP (eGFP) reporter. Characterization of hits in vitro by biochemical and cellular assays and in vivo using zebrafish led to five validated compounds, four of which induced accumulation of the reporter at the plasma membrane by inhibiting γ-secretase. One compound, the dihydropyridine FLI-06, disrupted the Golgi apparatus in a manner distinct from that of brefeldin A and golgicide A. FLI-06 inhibited general secretion at a step before exit from the endoplasmic reticulum (ER), which was accompanied by a tubule-to-sheet morphological transition of the ER, rendering FLI-06 the first small molecule acting at such an early stage in secretory traffic. These data highlight the power of phenotypic screening to enable investigations of central cellular signaling pathways. PMID:24077179

  8. Endosomal sorting of Notch receptors through COMMD9-dependent pathways modulates Notch signaling

    PubMed Central

    Li, Haiying; Koo, Yeon; Mao, Xicheng; Sifuentes-Dominguez, Luis; Morris, Lindsey L.; Jia, Da; Miyata, Naoteru; Faulkner, Rebecca A.; van Deursen, Jan M.; Vooijs, Marc; Billadeau, Daniel D.; van de Sluis, Bart; Cleaver, Ondine

    2015-01-01

    Notch family members are transmembrane receptors that mediate essential developmental programs. Upon ligand binding, a proteolytic event releases the intracellular domain of Notch, which translocates to the nucleus to regulate gene transcription. In addition, Notch trafficking across the endolysosomal system is critical in its regulation. In this study we report that Notch recycling to the cell surface is dependent on the COMMD–CCDC22–CCDC93 (CCC) complex, a recently identified regulator of endosomal trafficking. Disruption in this system leads to intracellular accumulation of Notch2 and concomitant reduction in Notch signaling. Interestingly, among the 10 copper metabolism MURR1 domain containing (COMMD) family members that can associate with the CCC complex, only COMMD9 and its binding partner, COMMD5, have substantial effects on Notch. Furthermore, Commd9 deletion in mice leads to embryonic lethality and complex cardiovascular alterations that bear hallmarks of Notch deficiency. Altogether, these studies highlight that the CCC complex controls Notch activation by modulating its intracellular trafficking and demonstrate cargo-specific effects for members of the COMMD protein family. PMID:26553930

  9. Cardioprotective actions of Notch1 against myocardial infarction via LKB1-dependent AMPK signaling pathway.

    PubMed

    Yang, Hui; Sun, Wanqing; Quan, Nanhu; Wang, Lin; Chu, Dongyang; Cates, Courtney; Liu, Quan; Zheng, Yang; Li, Ji

    2016-05-15

    AMP-activated protein kinase (AMPK) signaling pathway plays a pivotal role in intracellular adaptation to energy stress during myocardial ischemia. Notch1 signaling in the adult myocardium is also activated in response to ischemic stress. However, the relationship between Notch1 and AMPK signaling pathways during ischemia remains unclear. We hypothesize that Notch1 as an adaptive signaling pathway protects the heart from ischemic injury via modulating the cardioprotective AMPK signaling pathway. C57BL/6J mice were subjected to an in vivo ligation of left anterior descending coronary artery and the hearts from C57BL/6J mice were subjected to an ex vivo globe ischemia and reperfusion in the Langendorff perfusion system. The Notch1 signaling was activated during myocardial ischemia. A Notch1 γ-secretase inhibitor, dibenzazepine (DBZ), was intraperitoneally injected into mice to inhibit Notch1 signaling pathway by ischemia. The inhibition of Notch1 signaling by DBZ significantly augmented cardiac dysfunctions caused by myocardial infarction. Intriguingly, DBZ treatment also significantly blunted the activation of AMPK signaling pathway. The immunoprecipitation experiments demonstrated that an interaction between Notch1 and liver kinase beta1 (LKB1) modulated AMPK activation during myocardial ischemia. Furthermore, a ligand of Notch1 Jagged1 can significantly reduce cardiac damage caused by ischemia via activation of AMPK signaling pathway and modulation of glucose oxidation and fatty acid oxidation during ischemia and reperfusion. But Jagged1 did not have any cardioprotections on AMPK kinase dead transgenic hearts. Taken together, the results indicate that the cardioprotective effect of Notch1 against ischemic damage is mediated by AMPK signaling via an interaction with upstream LKB1.

  10. Notch Signaling in Pancreatic Development

    PubMed Central

    Li, Xu-Yan; Zhai, Wen-Jun; Teng, Chun-Bo

    2015-01-01

    The Notch signaling pathway plays a significant role in embryonic cell fate determination and adult tissue homeostasis. Various studies have demonstrated the deep involvement of Notch signaling in the development of the pancreas and the lateral inhibition of Notch signaling in pancreatic progenitor differentiation and maintenance. The targeted inactivation of the Notch pathway components promotes premature differentiation of the endocrine pancreas. However, there is still the contrary opinion that Notch signaling specifies the endocrine lineage. Here, we review the current knowledge of the Notch signaling pathway in pancreatic development and its crosstalk with the Wingless and INT-1 (Wnt) and fibroblast growth factor (FGF) pathways. PMID:26729103

  11. Jagged1-selective notch signaling induces smooth muscle differentiation via a RBP-Jkappa-dependent pathway.

    PubMed

    Doi, Hiroshi; Iso, Tatsuya; Sato, Hiroko; Yamazaki, Miki; Matsui, Hiroki; Tanaka, Toru; Manabe, Ichiro; Arai, Masashi; Nagai, Ryozo; Kurabayashi, Masahiko

    2006-09-29

    The Notch signaling pathway plays a crucial role in specifying cellular fates by interaction between cellular neighbors; however, the molecular mechanism underlying smooth muscle cell (SMC) differentiation by Notch signaling has not been well characterized. Here we demonstrate that Jagged1-Notch signaling promotes SMC differentiation from mesenchymal cells. Overexpression of the Notch intracellular domain, an activated form of Notch, up-regulates the expression of multiple SMC marker genes including SMC-myosin heavy chain (Sm-mhc) in mesenchymal 10T1/2 cells, but not in non-mesenchymal cells. Physiological Notch stimulation by its ligand Jagged1, but not Dll4, directly induces Sm-mhc expression in 10T1/2 cells without de novo protein synthesis, indicative of a ligand-selective effect. Jagged1-induced expression of SM-MHC was blocked bygamma-secretase inhibitor, N-(N-(3,5-difluorophenyl)-l-alanyl)-S-phenylglycine t-butyl ester, which impedes Notch signaling. Using Rbp-jkappa-deficient cells and site-specific mutagenesis of the SM-MHC gene, we show that such an induction is independent of the myocardin-serum response factor-CArG complex, but absolutely dependent on RBP-Jkappa, a major mediator of Notch signaling, and its cognate binding sequence. Of importance, Notch signaling and myocardin synergistically activate SM-MHC gene expression. Taken together, these data suggest that the Jagged1-Notch pathway constitutes an instructive signal for SMC differentiation through an RBP-Jkappa-dependent mechanism and augments gene expression mediated by the myocardin-SRF-CArG complex. Given that Notch pathway components are expressed in vascular SMC during normal development and disease, Notch signaling is likely to play a pivotal role in such situations to modulate the vascular smooth muscle cell phenotype. PMID:16867989

  12. Targeting stem cell signaling pathways for drug discovery: advances in the Notch and Wnt pathways.

    PubMed

    An, Songzhu Michael; Ding, Qiang; Zhang, Jie; Xie, JingYi; Li, LingSong

    2014-06-01

    Signaling pathways transduce extracellular stimuli into cells through molecular cascades to regulate cellular functions. In stem cells, a small number of pathways, notably those of TGF-β/BMP, Hedgehog, Notch, and Wnt, are responsible for the regulation of pluripotency and differentiation. During embryonic development, these pathways govern cell fate specifications as well as the formation of tissues and organs. In adulthood, their normal functions are important for tissue homeostasis and regeneration, whereas aberrations result in diseases, such as cancer and degenerative disorders. In complex biological systems, stem cell signaling pathways work in concert as a network and exhibit crosstalk, such as the negative crosstalk between Wnt and Notch. Over the past decade, genetic and genomic studies have identified a number of potential drug targets that are involved in stem cell signaling pathways. Indeed, discovery of new targets and drugs for these pathways has become one of the most active areas in both the research community and pharmaceutical industry. Remarkable progress has been made and several promising drug candidates have entered into clinical trials. This review focuses on recent advances in the discovery of novel drugs which target the Notch and Wnt pathways.

  13. Activation of the Notch signaling pathway in response to pulp capping of rat molars.

    PubMed

    Løvschall, H; Tummers, M; Thesleff, I; Füchtbauer, E-M; Poulsen, K

    2005-08-01

    Notch signaling is an evolutionarily conserved pathway that controls the developmental choices made by individual cells. Cells communicate via Notch receptors and their ligands, which direct decisions on the fate of stem cells according to the states of their neighbors. In this study we explored Notch signaling after the pulp capping of adult first upper rat molars. The wound was capped with calcium hydroxide. In situ hybridization revealed an increased expression of Notch signaling genes on day 1, which showed a tendency to decrease on day 3. Notch1 increased in the subodontoblast zone and close to the lesion limited to a few cells. Notch2 increased in pulp stroma surrounded by coronal odontoblasts. Notch1 and, especially, Notch3 expression increased, corresponding to perivascular cell groups. A low increase of ligand expression was observed near the injury with Delta1 expression along the dentin wall and Jagged1 in the stroma. Expression of the downstream target, Hes1, was observed along the lesion and adjacent dentin walls. Hes5 expression was not observed. The results indicate that Notch signaling is activated in response to injury and associated with the differentiation of pulp cells into perivascular cells and odontoblasts. The findings are consistent with the concept that the Notch pathway controls stem cell fate during pulp regeneration. PMID:16048523

  14. Notch1 Regulates Hippocampal Plasticity Through Interaction with the Reelin Pathway, Glutamatergic Transmission and CREB Signaling

    PubMed Central

    Brai, Emanuele; Marathe, Swananda; Astori, Simone; Fredj, Naila Ben; Perry, Elisabeth; Lamy, Christophe; Scotti, Alessandra; Alberi, Lavinia

    2015-01-01

    Notch signaling plays a crucial role in adult brain function such as synaptic plasticity, memory and olfaction. Several reports suggest an involvement of this pathway in neurodegenerative dementia. Yet, to date, the mechanism underlying Notch activity in mature neurons remains unresolved. In this work, we investigate how Notch regulates synaptic potentiation and contributes to the establishment of memory in mice. We observe that Notch1 is a postsynaptic receptor with functional interactions with the Reelin receptor, apolipoprotein E receptor 2 (ApoER2) and the ionotropic receptor, N-methyl-D-aspartate receptor (NMDAR). Targeted loss of Notch1 in the hippocampal CA fields affects Reelin signaling by influencing Dab1 expression and impairs the synaptic potentiation achieved through Reelin stimulation. Further analysis indicates that loss of Notch1 affects the expression and composition of the NMDAR but not AMPAR. Glutamatergic signaling is further compromised through downregulation of CamKII and its secondary and tertiary messengers resulting in reduced cAMP response element-binding (CREB) signaling. Our results identify Notch1 as an important regulator of mechanisms involved in synaptic plasticity and memory formation. These findings emphasize the possible involvement of this signaling receptor in dementia. Highlights In this paper, we propose a mechanism for Notch1-dependent plasticity that likely underlies the function of Notch1 in memory formation: Notch1 interacts with another important developmental pathway, the Reelin cascade. Notch1 regulates both NMDAR expression and composition. Notch1 influences a cascade of cellular events culminating in CREB activation. PMID:26635527

  15. Notch signaling genes

    PubMed Central

    Terragni, Jolyon; Zhang, Guoqiang; Sun, Zhiyi; Pradhan, Sriharsa; Song, Lingyun; Crawford, Gregory E; Lacey, Michelle; Ehrlich, Melanie

    2014-01-01

    Notch intercellular signaling is critical for diverse developmental pathways and for homeostasis in various types of stem cells and progenitor cells. Because Notch gene products need to be precisely regulated spatially and temporally, epigenetics is likely to help control expression of Notch signaling genes. Reduced representation bisulfite sequencing (RRBS) indicated significant hypomethylation in myoblasts, myotubes, and skeletal muscle vs. many nonmuscle samples at intragenic or intergenic regions of the following Notch receptor or ligand genes: NOTCH1, NOTCH2, JAG2, and DLL1. An enzymatic assay of sites in or near these genes revealed unusually high enrichment of 5-hydroxymethylcytosine (up to 81%) in skeletal muscle, heart, and cerebellum. Epigenetics studies and gene expression profiles suggest that hypomethylation and/or hydroxymethylation help control expression of these genes in heart, brain, myoblasts, myotubes, and within skeletal muscle myofibers. Such regulation could promote cell renewal, cell maintenance, homeostasis, and a poised state for repair of tissue damage. PMID:24670287

  16. hCLP46 regulates U937 cell proliferation via Notch signaling pathway

    SciTech Connect

    Ma, Wenzhan; Du, Jie; Chu, Qiaoyun; Wang, Youxin; Liu, Lixin; Song, Manshu; Wang, Wei

    2011-04-29

    Highlights: {yields} Knock down of hCLP46 by RNAi impairs mammalian Notch signaling. {yields} hCLP46 affects neither cell surface Notch1 expression nor ligand-receptor binding. {yields} Knock down of hCLP46 inhibits U937 cell-growth by up-regulation of CDKN1B. -- Abstract: Human CAP10-like protein 46 kDa (hCLP46) is the homolog of Rumi, which is the first identified protein O-glucosyltransferase that modifies Notch receptor in Drosophila. Dysregulation of hCLP46 occurs in many hematologic diseases, but the role of hCLP46 remains unclear. Knockdown of hCLP46 by RNA interference resulted in decreased protein levels of endogenous Notch1, Notch intracellular domain (NICD) and Notch target gene Hes-1, suggesting the impairment of the Notch signaling. However, neither cell surface Notch expression nor ligand binding activities were affected. In addition, down-regulated expression of hCLP46 inhibited the proliferation of U937 cells, which was correlated with increased cyclin-dependent kinase inhibitor (CDKI) CDKN1B (p27) and decreased phosphorylation of retinoblastoma (RB) protein. We showed that lack of hCLP46 results in impaired ligand induced Notch activation in mammalian cell, and hCLP46 regulates the proliferation of U937 cell through CDKI-RB signaling pathway, which may be important for the pathogenesis of leukemia.

  17. Quinomycin A targets Notch signaling pathway in pancreatic cancer stem cells

    PubMed Central

    Ponnurangam, Sivapriya; Dandawate, Prasad R.; Dhar, Animesh; Tawfik, Ossama W.; Parab, Rajashri R.; Mishra, Prabhu Dutt; Ranadive, Prafull; Sharma, Rajiv; Mahajan, Girish; Umar, Shahid; Weir, Scott J.; Sugumar, Aravind; Jensen, Roy A.; Padhye, Subhash B.; Balakrishnan, Arun; Anant, Shrikant; Subramaniam, Dharmalingam

    2016-01-01

    Cancer stem cells (CSCs) appear to explain many aspects of the neoplastic evolution of tumors and likely account for enhanced therapeutic resistance following treatment. Dysregulated Notch signaling, which affects CSCs plays an important role in pancreatic cancer progression. We have determined the ability of Quinomycin to inhibit CSCs and the Notch signaling pathway. Quinomycin treatment resulted in significant inhibition of proliferation and colony formation in pancreatic cancer cell lines, but not in normal pancreatic epithelial cells. Moreover, Quinomycin affected pancreatosphere formation. The compound also decreased the expression of CSC marker proteins DCLK1, CD44, CD24 and EPCAM. In addition, flow cytometry studies demonstrated that Quinomycin reduced the number of DCLK1+ cells. Furthermore, levels of Notch 1–4 receptors, their ligands Jagged1, Jagged2, DLL1, DLL3, DLL4 and the downstream target protein Hes-1 were reduced. The γ-secretase complex proteins, Presenilin 1, Nicastrin, Pen2, and APH-1, required for Notch activation also exhibited decreased expression. Ectopic expression of the Notch Intracellular Domain (NICD) partially rescued the cells from Quinomycin mediated growth suppression. To determine the effect of Quinomycin on tumor growth in vivo, nude mice carrying tumor xenografts were administered Quinomycin intraperitoneally every day for 21 days. Treatment with the compound significantly inhibited tumor xenograft growth, coupled with significant reduction in the expression of CSC markers and Notch signaling proteins. Together, these data suggest that Quinomycin is a potent inhibitor of pancreatic cancer that targets the stem cells by inhibiting Notch signaling proteins. PMID:26673007

  18. Corilagin suppresses cholangiocarcinoma progression through Notch signaling pathway in vitro and in vivo.

    PubMed

    Gu, Yue; Xiao, Linfeng; Ming, Yanlin; Zheng, Zhizhong; Li, Wengang

    2016-05-01

    Corilagin is a natural plant polyphenol tannic acid with antitumor, anti-inflammatory, and anti-oxidative properties. However, the mechanisms of its actions are largely unknown. Our group reported that corilagin could induce cell inhibition in human breast cancer cell line MCF-7 and human liver hepatocellular carcinoma cell lines HepG2. We report here that corilagin inhibits cholangiocarcinoma (CCA) development through regulating Notch signaling pathway. We found that, in vitro, corilagin inhibited CCA cell proliferation, migration and invasion, promoted CCA cell apoptosis, and inhibited Notch1 and Notch signaling pathway protein expression. Co-immunoprecipitation was used to establish Notch intracellular domain (NICD) interaction with MAML1 and P300 in CCA. Importantly, corilagin reduced Hes1 mRNA level through inhibiting Hes1 promoter activity. In nude mice, corilagin inhibited CCA growth and repressed the expression of Notch1 and mTOR. These results indicate that corilagin may control CCA cell growth by downregulating the expression of Notch1. Therefore, our findings suggest that corilagin may have the potential to become a new therapeutic drug for human CCA. PMID:26935808

  19. Ginsenoside Rb1 inhibits matrix metalloproteinase 13 through down-regulating Notch signaling pathway in osteoarthritis

    PubMed Central

    Wang, Wei; Zeng, Li; Wang, Ze-ming; Zhang, Sihan; Rong, Xiao-Feng

    2015-01-01

    Mounting evidence suggests that an excess of matrix metalloproteinase-13 (MMP-13) plays an important role in the breakdown of extracellular matrix in osteoarthritis (OA). Here, the effects of ginsenoside Rb1 (GRb1) on the expression of MMP-13 in IL-1β-induced SW 1353 chondrosarcoma cells and an experimental rat model of OA induced by anterior cruciate ligament transection (ACLT) were investigated. SW1353 chondrosarcoma cells were pretreated with or without GRb1 and Notch signaling pathway inhibitor, DAPT, then were stimulated with IL-1β. In rats, experimental OA was induced by ACLT. These rats then received intra-articular injections of vehicle, an inhibitor of γ-secretase, DAPT, and/or GRb1. Expression of MMP-13, collagen type II (CII), Notch1, and jagged 1 (JAG1) were verified by western blotting and immunohistochemistry. In addition, levels of MMP-13 mRNA were detected using quantitative real-time PCR. In histological analyses, treatment with DAPT reduced the number of cartilage lesions present and the expressions of MMP-13, CII, Notch1, and JAG1. In addition, treatment with GRb1 was associated with lower levels of Notch1 and JAG1 in both IL-1β-induced SW1353 chondrosarcoma cells and in the rat OA model. Furthermore, the suppressive effect of GRb1 on MMP-13 was greater than that exhibited by the signaling pathway inhibitor. In conclusion, GRb1 inhibits MMP-13 through down-regulating Notch signaling pathway in OA. PMID:26062798

  20. Notch signaling pathway and Cdx2 expression in the development of Barrett's esophagus.

    PubMed

    Tamagawa, Yuji; Ishimura, Norihisa; Uno, Goichi; Yuki, Takafumi; Kazumori, Hideaki; Ishihara, Shunji; Amano, Yuji; Kinoshita, Yoshikazu

    2012-06-01

    Cdx2 expression in esophageal stem cells induced by reflux bile acids may be an important factor for development of Barrett's esophagus, whereas Notch signaling is a molecular signaling pathway that plays an important role in the determination of cell differentiation. ATOH1 (a factor associated with Notch signaling) plays an important role in differentiation of stem cells into goblet cells. However, the relationship between the Notch signaling pathway and Cdx2 expression in the development of Barrett's esophagus has not been explored. The aim of this study was to investigate the interrelationship between Notch signaling and Cdx2 in esophageal epithelial cells. The expressions of Cdx2, MUC2, and intracellular signaling molecules related to Notch signaling (Notch1, Hes1, and ATOH1) were examined using real-time polymerase chain reaction (PCR) and immunohistochemical staining with biopsy specimens obtained from esophageal intestinal metaplasia (IM) with goblet cells (IM⁺) and columnar epithelium not accompanied by goblet cells (IM⁻). For in vitro experiments, we employed human esophageal epithelial cell lines (OE33, OE19, and Het-1A). After forced Cdx2 expression by applying a Cdx2 expression vector to the cells, changes in the expressions of Notch1, Hes1, ATOH1, Cdx2, and MUC2 were analyzed by real-time PCR and western blot analysis. Changes in expressions of Notch1, Hes1, ATOH1, Cdx2, and MUC2 in cells were analyzed following stimulation with bile acids in the presence or absence of Cdx2 blocking with Cdx2-siRNA. Suppressed Hes1 and enhanced ATOH1 and MUC2 expressions were identified in IM⁺ specimens. Forced expression of Cdx2 in cells suppressed Hes1, and enhanced ATOH1 and MUC2 expressions, whereas bile acids suppressed Hes1, and enhanced ATOH1, Cdx2, and MUC2 expressions. On the other hand, these effects were blocked by siRNA-based Cdx2 downregulation. Enhanced expression of Cdx2 by stimulation with bile acids may induce intestinal differentiation of

  1. Notch signalling pathway as an oncogenic factor involved in cancer development

    PubMed Central

    Piecuch, Adam; Dittfeld, Anna; Mielańczyk, Łukasz; Michalski, Marek; Wyrobiec, Grzegorz; Harabin-Słowińska, Marzena; Kurek, Józef; Wojnicz, Romuald

    2016-01-01

    Notch signalling is an evolutionarily conserved signalling pathway, which plays a significant role in a wide array of cellular processes including proliferation, differentiation, and apoptosis. Nevertheless, it must be noted that Notch is a binary cell fate determinant, and its overexpression has been described as oncogenic in a broad range of human malignancies. This finding led to interest in therapeutically targeting this pathway especially by the use of GSIs, which block the cleavage of Notch at the cell membrane and inhibit release of the transcriptionally active NotchIC subunit. Preclinical cancer models have clearly demonstrated that GSIs suppress the growth of such malignancies as pancreatic, breast, and lung cancer; however, GSI treatment in vivo is associated with side effects, especially those within the gastrointestinal tract. Although intensive studies are associated with the role of γ-secretase in pathological states, it should be pointed out that this complex impacts on proteolytic cleavages of around 55 membrane proteins. Therefore, it is clear that GSIs are highly non-specific and additional drugs must be designed, which will more specifically target components of the Notch signalling.

  2. Notch signalling pathway as an oncogenic factor involved in cancer development.

    PubMed

    Brzozowa-Zasada, Marlena; Piecuch, Adam; Dittfeld, Anna; Mielańczyk, Łukasz; Michalski, Marek; Wyrobiec, Grzegorz; Harabin-Słowińska, Marzena; Kurek, Józef; Wojnicz, Romuald

    2016-01-01

    Notch signalling is an evolutionarily conserved signalling pathway, which plays a significant role in a wide array of cellular processes including proliferation, differentiation, and apoptosis. Nevertheless, it must be noted that Notch is a binary cell fate determinant, and its overexpression has been described as oncogenic in a broad range of human malignancies. This finding led to interest in therapeutically targeting this pathway especially by the use of GSIs, which block the cleavage of Notch at the cell membrane and inhibit release of the transcriptionally active NotchIC subunit. Preclinical cancer models have clearly demonstrated that GSIs suppress the growth of such malignancies as pancreatic, breast, and lung cancer; however, GSI treatment in vivo is associated with side effects, especially those within the gastrointestinal tract. Although intensive studies are associated with the role of γ-secretase in pathological states, it should be pointed out that this complex impacts on proteolytic cleavages of around 55 membrane proteins. Therefore, it is clear that GSIs are highly non-specific and additional drugs must be designed, which will more specifically target components of the Notch signalling. PMID:27688721

  3. Notch signalling pathway as an oncogenic factor involved in cancer development

    PubMed Central

    Piecuch, Adam; Dittfeld, Anna; Mielańczyk, Łukasz; Michalski, Marek; Wyrobiec, Grzegorz; Harabin-Słowińska, Marzena; Kurek, Józef; Wojnicz, Romuald

    2016-01-01

    Notch signalling is an evolutionarily conserved signalling pathway, which plays a significant role in a wide array of cellular processes including proliferation, differentiation, and apoptosis. Nevertheless, it must be noted that Notch is a binary cell fate determinant, and its overexpression has been described as oncogenic in a broad range of human malignancies. This finding led to interest in therapeutically targeting this pathway especially by the use of GSIs, which block the cleavage of Notch at the cell membrane and inhibit release of the transcriptionally active NotchIC subunit. Preclinical cancer models have clearly demonstrated that GSIs suppress the growth of such malignancies as pancreatic, breast, and lung cancer; however, GSI treatment in vivo is associated with side effects, especially those within the gastrointestinal tract. Although intensive studies are associated with the role of γ-secretase in pathological states, it should be pointed out that this complex impacts on proteolytic cleavages of around 55 membrane proteins. Therefore, it is clear that GSIs are highly non-specific and additional drugs must be designed, which will more specifically target components of the Notch signalling. PMID:27688721

  4. Constitutively active Notch1 induces growth arrest of HPV-positive cervical cancer cells via separate signaling pathways

    SciTech Connect

    Talora, Claudio; Cialfi, Samantha; Segatto, Oreste; Morrone, Stefania; Kim Choi, John; Frati, Luigi; Paolo Dotto, Gian; Gulino, Alberto; Screpanti, Isabella . E-mail: isabella.screpanti@uniroma1.it

    2005-05-01

    Notch signaling plays a key role in cell-fate determination and differentiation in different organisms and cell types. Several reports suggest that Notch signaling may be involved in neoplastic transformation. However, in primary keratinocytes, Notch1 can function as a tumor suppressor. Similarly, in HPV-positive cervical cancer cells, constitutively active Notch1 signaling was found to cause growth suppression. Activated Notch1 in these cells represses viral E6/E7 expression through AP-1 down-modulation, resulting in increased p53 expression and a block of pRb hyperphosphorylation. Here we show that in cervical cancer cell lines in which Notch1 ability to repress AP-1 activity is impaired, Notch1-enforced expression elicits an alternative pathway leading to growth arrest. Indeed, activated Notch1 signaling suppresses activity of the helix-loop-helix transcription factor E47, via ERK1/2 activation, resulting in inhibition of cell cycle progression. Moreover, we found that RBP-J{kappa}-dependent Notch signaling is specifically repressed in cervical cancer cells and this repression could provide one such mechanism that needs to be activated for cervical carcinogenesis. Finally, we show that inhibition of endogenous Notch1 signaling, although results in a proliferative advantage, sensitizes cervical cancer cell lines to drug-induced apoptosis. Together, our results provide novel molecular insights into Notch1-dependent growth inhibitory effects, counteracting the transforming potential of HPV.

  5. Notch and Wnt/β-catenin signaling pathway play important roles in activating liver cancer stem cells

    PubMed Central

    Wang, Ronghua; Sun, Qian; Wang, Peng; Liu, Man; Xiong, Si; Luo, Jing; Huang, Hai; Du, Qiang; Geller, David A.; Cheng, Bin

    2016-01-01

    Human hepatocellular carcinoma (HCC) is driven and maintained by liver cancer stem cells (LCSCs) that display stem cell properties. These LCSCs are promoted by the intersecting of Notch and Wnt/β-Catenin signaling pathways. In this study, we demonstrate that LCSCs with markers CD90, CD24, CD13, and CD133 possess stem properties of self-renewal and tumorigenicity in NOD/SCID mice. The increased expression of these markers was correlated with advanced disease stage, larger tumors, and worse overall survival in 61 HCC cases. We also found that both Notch and Wnt/β-catenin signaling pathways played important roles in increasing the stem-ness characteristics of LCSCs. Our data suggested that Notch1 was downstream of Wnt/β-catenin. The active form of Notch1 intracellular domain (NICD) expression depended on Wnt/β-catenin pathway activation. Moreover, Notch1 negatively contributed to Wnt/β-catenin signaling modulation. Knock down of Notch1 with lentivirus N1ShRNA up-regulated the active form of β-catenin. Ectopic expression of NICD with LV-Notch1 in LCSCs attenuated β-catenin/TCF dependent luciferase activity significantly. In addition, there was a non-proteasome mediated feedback loop between Notch1 and Wnt/β-catenin signaling in LCSCs. The central role of Notch and the Wnt/β-catenin signaling pathway in LCSCs may provide an attractive therapeutic strategy against HCC. PMID:26735577

  6. Epidermal stem cells (ESCs) accelerate diabetic wound healing via the Notch signalling pathway

    PubMed Central

    Yang, Rong-Hua; Qi, Shao-Hai; Shu, Bin; Ruan, Shu-Bin; Lin, Ze-Peng; Lin, Yan; Shen, Rui; Zhang, Feng-Gang; Chen, Xiao-Dong; Xie, Ju-Lin

    2016-01-01

    Chronic, non-healing wounds are a major complication of diabetes. Recently, various cell therapies have been reported for promotion of diabetic wound healing. Epidermal stem cells (ESCs) are considered a powerful tool for tissue therapy. However, the effect and the mechanism of the therapeutic properties of ESCs in the diabetic wound healing are unclear. Herein, to determine the ability of ESCs to diabetic wound healing, a dorsal skin defect in a streptozotocin (STZ)-induced diabetes mellitus (DM) mouse model was used. ESCs were isolated from mouse skin. We found that both the mRNA and protein levels of a Notch ligand Jagged1 (Jag1), Notch1 and Notch target gene Hairy Enhancer of Split-1 (Hes1) were significantly increased at the wound margins. In addition, we observed that Jag1 was high expressed in ESCs. Overexpression of Jag1 promotes ESCs migration, whereas knockdown Jag1 resulted in a significant reduction in ESCs migration in vitro. Importantly, Jag1 overexpression improves diabetic wound healing in vivo. These results provide evidence that ESCs accelerate diabetic wound healing via the Notch signalling pathway, and provide a promising potential for activation of the Notch pathway for the treatment of diabetic wound. PMID:27129289

  7. Epidermal stem cells (ESCs) accelerate diabetic wound healing via the Notch signalling pathway.

    PubMed

    Yang, Rong-Hua; Qi, Shao-Hai; Shu, Bin; Ruan, Shu-Bin; Lin, Ze-Peng; Lin, Yan; Shen, Rui; Zhang, Feng-Gang; Chen, Xiao-Dong; Xie, Ju-Lin

    2016-08-01

    Chronic, non-healing wounds are a major complication of diabetes. Recently, various cell therapies have been reported for promotion of diabetic wound healing. Epidermal stem cells (ESCs) are considered a powerful tool for tissue therapy. However, the effect and the mechanism of the therapeutic properties of ESCs in the diabetic wound healing are unclear. Herein, to determine the ability of ESCs to diabetic wound healing, a dorsal skin defect in a streptozotocin (STZ)-induced diabetes mellitus (DM) mouse model was used. ESCs were isolated from mouse skin. We found that both the mRNA and protein levels of a Notch ligand Jagged1 (Jag1), Notch1 and Notch target gene Hairy Enhancer of Split-1 (Hes1) were significantly increased at the wound margins. In addition, we observed that Jag1 was high expressed in ESCs. Overexpression of Jag1 promotes ESCs migration, whereas knockdown Jag1 resulted in a significant reduction in ESCs migration in vitro Importantly, Jag1 overexpression improves diabetic wound healing in vivo These results provide evidence that ESCs accelerate diabetic wound healing via the Notch signalling pathway, and provide a promising potential for activation of the Notch pathway for the treatment of diabetic wound. PMID:27129289

  8. Epidermal growth factor promotes proliferation of dermal papilla cells via Notch signaling pathway.

    PubMed

    Zhang, Haihua; Nan, Weixiao; Wang, Shiyong; Zhang, Tietao; Si, Huazhe; Wang, Datao; Yang, Fuhe; Li, Guangyu

    2016-08-01

    The effect of epidermal growth factor (EGF) on the development and growth of hair follicle is controversial. In the present study, 2-20 ng/ml EGF promoted the growth of mink hair follicles in vitro, whereas 200 ng/ml EGF inhibited follicle growth. Further, dermal papilla (DP) cells, a group of mesenchymal cells that govern hair follicle development and growth, were isolated and cultured in vitro. Treatment with or forced expression of EGF accelerated proliferation and induced G1/S transition in DP cells. Moreover, EGF upregulated the expression of DP mesenchymal genes, such as alkaline phosphatase (ALP) and insulin-like growth factor (IGF-1), as well as the Notch pathway molecules including Notch1, Jagged1, Hes1 and Hes5. In addition, inhibition of Notch signaling pathway by DAPT significantly reduced the basal and EGF-enhanced proliferation rate, and also suppressed cell cycle progression. We also show that the expression of several follicle-regulatory genes, such as Survivin and Msx2, were upregulated by EGF, and was inhibited by DAPT. In summary, our study demonstrates that the concentration of EGF is critical for the switch between hair follicle growth and inhibition, and EGF promotes DP cell proliferation via Notch signaling pathway.

  9. mTORC1 Prevents Preosteoblast Differentiation through the Notch Signaling Pathway

    PubMed Central

    Huang, Bin; Wang, Yongkui; Wang, Wenhao; Chen, Juan; Lai, Pinglin; Liu, Zhongyu; Yan, Bo; Xu, Song; Zhang, Zhongmin; Zeng, Chun; Rong, Limin; Liu, Bin; Cai, Daozhang; Jin, Dadi; Bai, Xiaochun

    2015-01-01

    The mechanistic target of rapamycin (mTOR) integrates both intracellular and extracellular signals to regulate cell growth and metabolism. However, the role of mTOR signaling in osteoblast differentiation and bone formation is undefined, and the underlying mechanisms have not been elucidated. Here, we report that activation of mTOR complex 1 (mTORC1) is required for preosteoblast proliferation; however, inactivation of mTORC1 is essential for their differentiation and maturation. Inhibition of mTORC1 prevented preosteoblast proliferation, but enhanced their differentiation in vitro and in mice. Activation of mTORC1 by deletion of tuberous sclerosis 1 (Tsc1) in preosteoblasts produced immature woven bone in mice due to excess proliferation but impaired differentiation and maturation of the cells. The mTORC1-specific inhibitor, rapamycin, restored these in vitro and in vivo phenotypic changes. Mechanistically, mTORC1 prevented osteoblast maturation through activation of the STAT3/p63/Jagged/Notch pathway and downregulation of Runx2. Preosteoblasts with hyperactive mTORC1 reacquired the capacity to fully differentiate and maturate when subjected to inhibition of the Notch pathway. Together, these findings identified the role of mTORC1 in osteoblast formation and established that mTORC1 prevents preosteoblast differentiation and maturation through activation of the Notch pathway. PMID:26241748

  10. Protective effect of curcumin on acute airway inflammation of allergic asthma in mice through Notch1-GATA3 signaling pathway.

    PubMed

    Chong, Lei; Zhang, Weixi; Nie, Ying; Yu, Gang; Liu, Liu; Lin, Li; Wen, Shunhang; Zhu, Lili; Li, Changchong

    2014-10-01

    Curcumin, a natural product derived from the plant Curcuma longa, has been found to have anti-inflammatory, antineoplastic and antifibrosis effects. It has been reported that curcumin attenuates allergic airway inflammation in mice through inhibiting NF-κB and its downstream transcription factor GATA3. It also has been proved the antineoplastic effect of curcumin through down-regulating Notch1 receptor and its downstream nuclear transcription factor NF-κB levels. In this study, we aimed to investigate the anti-inflammatory effect of curcumin on acute allergic asthma and its underlying mechanisms. 36 male BALB/c mice were randomly divided into four groups (normal, asthma, asthma+budesonide and asthma+curcumin groups). BALF (bronchoalveolar lavage fluid) and lung tissues were analyzed for airway inflammation and the expression of Notch1, Notch2, Notch3, Notch4 and the downstream transcription factor GATA3. Our findings showed that the levels of Notch1 and Notch2 receptors were up-regulated in asthma group, accompanied by the increased expression of GATA3. But the expression of Notch2 receptor was lower than Notch1 receptor. Curcumin pretreatment improved the airway inflammatory cells infiltration and reversed the increasing levels of Notch1/2 receptors and GATA3. Notch3 receptor was not expressed in all of the four groups. Notch4 receptor protein and mRNA expression level in the four groups had no significant differences. The results of the present study suggested that Notch1 and Notch2 receptor, major Notch1 receptor, played an important role in the development of allergic airway inflammation and the inhibition of Notch1-GATA3 signaling pathway by curcumin can prevent the development and deterioration of the allergic airway inflammation. This may be a possible therapeutic option of allergic asthma.

  11. Notch signaling in gastrointestinal tract (review).

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2007-01-01

    Notch signaling is one of key pathways constituting the stem cell signaling network. DLL1, DLL3, DLL4, JAG1 and JAG2 with DSL domain are typical Notch ligands, while DNER, F3/Contactin and NB-3 without DSL domain are atypical Notch ligands. Notch-ligand binding to NOTCH1, NOTCH2, NOTCH3 or NOTCH4 receptor induces the receptor proteolysis by metalloprotease and gamma-secretase to release Notch intracellular domain (NICD). Typical Notch ligands transduce signals to the CSL-NICD-Mastermind complex for the maintenance of stem or progenitor (transit-amplifying) cells through transcriptional activation of HES1, HES5, HES7, HEY1, HEY2 and HEYL genes, and also to the NF-kappaB-NICD complex for the augmentation of NF-kappaB signaling. Atypical Notch ligands transduce signals to the CSL-NICD-Deltex complex for the differentiation of progenitor cells through MAG transcriptional activation. Notch signals are transduced to the canonical pathway (CSL-NICD-Mastermind signaling cascade) or the non-canonical pathway (NF-kappaB-NICD and CSL-NICD-Deltex signaling cascades) based on the expression profile of Notch ligands, Notch receptors, and Notch signaling modifiers. Canonical Notch signaling is activated in the stem or progenitor domain of gastrointestinal epithelium, such as basal layer in esophagus and lower part of the crypt in colon. Notch signaling to inhibit secretory cell differentiation is oncogenic in gastric cancer and colorectal cancer, while Notch signaling to promote keratinocyte differentiation is anti-oncogenic in esophageal squamous cell carcinoma (SCC). Single nucleotide polymorphism (SNP), epigenetic change, and genetic alteration of genes encoding Notch signaling-associated molecules will be utilized as biomarkers for gastrointestinal cancer. gamma-Secretase inhibitors, functioning as Notch signaling inhibitors, will be applied as anti-cancer drugs for gastric cancer and colorectal cancer.

  12. Dominant Enhancers of Egfr in Drosophila Melanogaster: Genetic Links between the Notch and Egfr Signaling Pathways

    PubMed Central

    Price, J. V.; Savenye, E. D.; Lum, D.; Breitkreutz, A.

    1997-01-01

    The Drosophila epidermal growth factor receptor (EGFR) is a key component of a complex signaling pathway that participates in multiple developmental processes. We have performed an F(1) screen for mutations that cause dominant enhancement of wing vein phenotypes associated with mutations in Egfr. With this screen, we have recovered mutations in Hairless (H), vein, groucho (gro), and three apparently novel loci. All of the E(Egfr)s we have identified show dominant interactions in transheterozygous combinations with each other and with alleles of N or Su(H), suggesting that they are involved in cross-talk between the N and EGFR signaling pathways. Further examination of the phenotypic interactions between Egfr, H, and gro revealed that reductions in Egfr activity enhanced both the bristle loss associated with H mutations, and the bristle hyperplasia and ocellar hypertrophy associated with gro mutations. Double mutant combinations of Egfr and gro hypomorphic alleles led to the formation of ectopic compound eyes in a dosage sensitive manner. Our findings suggest that these E(Egfr)s represent links between the Egfr and Notch signaling pathways, and that Egfr activity can either promote or suppress Notch signaling, depending on its developmental context. PMID:9383058

  13. Notch Signaling Components

    PubMed Central

    Liu, Zhi-Yan; Wu, Tao; Li, Qing; Wang, Min-Cong; Jing, Li; Ruan, Zhi-Ping; Yao, Yu; Nan, Ke-Jun; Guo, Hui

    2016-01-01

    Abstract Non-small-cell lung cancer (NSCLC) is a lethal and aggressive malignancy. Currently, the identities of prognostic and predictive makers of NSCLC have not been fully established. Dysregulated Notch signaling has been implicated in many human malignancies, including NSCLC. However, the prognostic value of measuring Notch signaling and the utility of developing Notch-targeted therapies in NSCLC remain inconclusive. The present study investigated the association of individual Notch receptor and ligand levels with lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) prognosis using the Kaplan-Meier plotte database. This online database encompasses 2437 lung cancer samples. Hazard ratios with 95% confidence intervals were calculated. The results showed that higher Notch1, Notch2, JAG1, and DLL1 mRNA expression predicted better overall survival (OS) in lung ADC, but showed no significance in SCC patients. Elevated Notch3, JAG2, and DLL3 mRNA expression was associated with poor OS of ADC patients, but not in SCC patients. There was no association between Notch4 and OS in either lung ADC or SCC patients. In conclusion, the set of Notch1, Notch2, JAG1, DLL1 and that of Notch3, JAG2, DLL3 played opposing prognostic roles in lung ADC patients. Neither set of Notch receptors and ligands was indicative of lung SCC prognosis. Notch signaling could serve as promising marker to predict outcomes in lung ADC patients. The distinct features of lung cancer subtypes and Notch components should be considered when developing future Notch-targeted therapies. PMID:27196489

  14. Monomeric C-reactive protein and Notch-3 co-operatively increase angiogenesis through PI3K signalling pathway.

    PubMed

    Boras, Emhamed; Slevin, Mark; Alexander, M Yvonne; Aljohi, Ali; Gilmore, William; Ashworth, Jason; Krupinski, Jerzy; Potempa, Lawrence A; Al Abdulkareem, Ibrahim; Elobeid, Adila; Matou-Nasri, Sabine

    2014-10-01

    C-reactive protein (CRP) is the most acute-phase reactant serum protein of inflammation and a strong predictor of cardiovascular disease. Its expression is associated with atherosclerotic plaque instability and the formation of immature micro-vessels. We have previously shown that CRP upregulates endothelial-derived Notch-3, a key receptor involved in vascular development, remodelling and maturation. In this study, we investigated the links between the bioactive monomeric CRP (mCRP) and Notch-3 signalling in angiogenesis. We used in vitro (cell counting, wound-healing and tubulogenesis assays) and in vivo (chorioallantoic membrane) angiogenic assays and Western blotting to study the angiogenic signalling pathways induced by mCRP and Notch-3 activator chimera protein (Notch-3/Fc). Our results showed an additive effect on angiogenesis of mCRP stimulatory effect combined with Notch-3/Fc promoting bovine aortic endothelial cell (BAEC) proliferation, migration, tube formation in Matrigel(TM) with up-regulation of phospho-Akt expression. The pharmacological blockade of PI3K/Akt survival pathway by LY294002 fully inhibited in vitro and in vivo angiogenesis induced by mCRP/Notch-3/Fc combination while blocking Notch signalling by gamma-secretase inhibitor (DAPT) partially inhibited mCRP/Notch-3/Fc-induced angiogenesis. Using a BAEC vascular smooth muscle cell co-culture sprouting angiogenesis assay and transmission electron microscopy, we showed that activation of both mCRP and Notch-3 signalling induced the formation of thicker sprouts which were shown later by Western blotting to be associated with an up-regulation of N-cadherin expression and a down-regulation of VE-cadherin expression. Thus, mCRP combined with Notch-3 activator promote angiogenesis through the PI3K/Akt pathway and their therapeutic combination has potential to promote and stabilize vessel formation whilst reducing the risk of haemorrhage from unstable plaques. PMID:24972386

  15. Protein O-fucosyltransferase 1 expression impacts myogenic C2C12 cell commitment via the Notch signaling pathway.

    PubMed

    Der Vartanian, Audrey; Audfray, Aymeric; Al Jaam, Bilal; Janot, Mathilde; Legardinier, Sébastien; Maftah, Abderrahman; Germot, Agnès

    2015-01-01

    The Notch signaling pathway plays a crucial role in skeletal muscle regeneration in mammals by controlling the transition of satellite cells from quiescence to an activated state, their proliferation, and their commitment toward myotubes or self-renewal. O-fucosylation on Notch receptor epidermal growth factor (EGF)-like repeats is catalyzed by the protein O-fucosyltransferase 1 (Pofut1) and primarily controls Notch interaction with its ligands. To approach the role of O-fucosylation in myogenesis, we analyzed a murine myoblastic C2C12 cell line downregulated for Pofut1 expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown of Pofut1 affected the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7(+)/MyoD(-) cells and earlier myogenic program entrance were observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue of Pofut1 expression in knockdown cells restored Notch signaling activation and a normal course in C2C12 differentiation. Our results establish the critical role of Pofut1 on Notch pathway activation during myogenic differentiation.

  16. Protein O-Fucosyltransferase 1 Expression Impacts Myogenic C2C12 Cell Commitment via the Notch Signaling Pathway

    PubMed Central

    Der Vartanian, Audrey; Audfray, Aymeric; Al Jaam, Bilal; Janot, Mathilde; Legardinier, Sébastien; Maftah, Abderrahman

    2014-01-01

    The Notch signaling pathway plays a crucial role in skeletal muscle regeneration in mammals by controlling the transition of satellite cells from quiescence to an activated state, their proliferation, and their commitment toward myotubes or self-renewal. O-fucosylation on Notch receptor epidermal growth factor (EGF)-like repeats is catalyzed by the protein O-fucosyltransferase 1 (Pofut1) and primarily controls Notch interaction with its ligands. To approach the role of O-fucosylation in myogenesis, we analyzed a murine myoblastic C2C12 cell line downregulated for Pofut1 expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown of Pofut1 affected the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7+/MyoD− cells and earlier myogenic program entrance were observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue of Pofut1 expression in knockdown cells restored Notch signaling activation and a normal course in C2C12 differentiation. Our results establish the critical role of Pofut1 on Notch pathway activation during myogenic differentiation. PMID:25384974

  17. Notch Signaling in Neuroendocrine Tumors

    PubMed Central

    Crabtree, Judy S.; Singleton, Ciera S.; Miele, Lucio

    2016-01-01

    Carcinoids and neuroendocrine tumors (NETs) are a heterogeneous group of tumors that arise from the neuroendocrine cells of the GI tract, endocrine pancreas, and the respiratory system. NETs remain significantly understudied with respect to molecular mechanisms of pathogenesis, particularly the role of cell fate signaling systems such as Notch. The abundance of literature on the Notch pathway is a testament to its complexity in different cellular environments. Notch receptors can function as oncogenes in some contexts and tumor suppressors in others. The genetic heterogeneity of NETs suggests that to fully understand the roles and the potential therapeutic implications of Notch signaling in NETs, a comprehensive analysis of Notch expression patterns and potential roles across all NET subtypes is required. PMID:27148486

  18. The Notch and TGF-β Signaling Pathways Contribute to the Aggressiveness of Clear Cell Renal Cell Carcinoma

    PubMed Central

    Sjölund, Jonas; Manna, Sugata; Moustakas, Aristidis; Ljungberg, Börje; Johansson, Martin; Fredlund, Erik; Axelson, Håkan

    2011-01-01

    Background Despite recent progress, therapy for metastatic clear cell renal cell carcinoma (CCRCC) is still inadequate. Dysregulated Notch signaling in CCRCC contributes to tumor growth, but the full spectrum of downstream processes regulated by Notch in this tumor form is unknown. Methodology/Principal Findings We show that inhibition of endogenous Notch signaling modulates TGF-β dependent gene regulation in CCRCC cells. Analysis of gene expression data representing 176 CCRCCs showed that elevated TGF-β pathway activity correlated significantly with shortened disease specific survival (log-rank test, p = 0.006) and patients with metastatic disease showed a significantly elevated TGF-β signaling activity (two-sided Student's t-test, p = 0.044). Inhibition of Notch signaling led to attenuation of both basal and TGF-β1 induced TGF-β signaling in CCRCC cells, including an extensive set of genes known to be involved in migration and invasion. Functional analyses revealed that Notch inhibition decreased the migratory and invasive capacity of CCRCC cells. Conclusion An extensive cross-talk between the Notch and TGF-β signaling cascades is present in CCRCC and the functional properties of these two pathways are associated with the aggressiveness of this disease. PMID:21826227

  19. Dystroglycan is involved in skin morphogenesis downstream of the Notch signaling pathway.

    PubMed

    Sirour, Cathy; Hidalgo, Magdalena; Bello, Valérie; Buisson, Nicolas; Darribère, Thierry; Moreau, Nicole

    2011-08-15

    Dystroglycan (Dg) is a transmembrane protein involved both in the assembly and maintenance of basement membrane structures essential for tissue morphogenesis, and the transmission of signals across the plasma membrane. We used a morpholino knockdown approach to investigate the function of Dg during Xenopus laevis skin morphogenesis. The loss of Dg disrupts epidermal differentiation by affecting the intercalation of multiciliated cells, deposition of laminin, and organization of fibronectin in the extracellular matrix (ECM). Depletion of Dg also affects cell-cell adhesion, as shown by the reduction of E-cadherin expression at the intercellular contacts, without affecting the distribution of β(1) integrins. This was associated with a decrease of cell proliferation, a disruption of multiciliated-cell intercalation, and the down-regulation of the transcription factor P63, a marker of differentiated epidermis. In addition, we demonstrated that inhibition or activation of the Notch pathway prevents and promotes transcription of X-dg. Our study showed for the first time in vivo that Dg, in addition to organizing laminin in the ECM, also acts as a key signaling component in the Notch pathway. PMID:21680717

  20. Notch signaling in cerebrovascular diseases (Review)

    PubMed Central

    Cai, Zhiyou; Zhao, Bin; Deng, Yanqing; Shangguan, Shouqin; Zhou, Faming; Zhou, Wenqing; Li, Xiaoli; Li, Yanfeng; Chen, Guanghui

    2016-01-01

    The Notch signaling pathway is a crucial regulator of numerous fundamental cellular processes. Increasing evidence suggests that Notch signaling is involved in inflammation and oxidative stress, and thus in the progress of cerebrovascular diseases. In addition, Notch signaling in cerebrovascular diseases is associated with apoptosis, angiogenesis and the function of blood-brain barrier. Despite the contradictory results obtained to date as to whether Notch signaling is harmful or beneficial, the regulation of Notch signaling may provide a novel strategy for the treatment of cerebrovascular diseases. PMID:27574001

  1. The PDZ Protein Canoe/AF-6 Links Ras-MAPK, Notch and Wingless/Wnt Signaling Pathways by Directly Interacting with Ras, Notch and Dishevelled

    PubMed Central

    Carmena, Ana; Speicher, Stephan; Baylies, Mary

    2006-01-01

    Over the past few years, it has become increasingly apparent that signal transduction pathways are not merely linear cascades; they are organized into complex signaling networks that require high levels of regulation to generate precise and unique cell responses. However, the underlying regulatory mechanisms by which signaling pathways cross-communicate remain poorly understood. Here we show that the Ras-binding protein Canoe (Cno)/AF-6, a PDZ protein normally associated with cellular junctions, is a key modulator of Wingless (Wg)/Wnt, Ras-Mitogen Activated Protein Kinase (MAPK) and Notch (N) signaling pathways cross-communication. Our data show a repressive effect of Cno/AF-6 on these three signaling pathways through physical interactions with Ras, N and the cytoplasmic protein Dishevelled (Dsh), a key Wg effector. We propose a model in which Cno, through those interactions, actively coordinates, at the membrane level, Ras-MAPK, N and Wg signaling pathways during progenitor specification. PMID:17183697

  2. Inhibition of notch signaling pathway prevents cholestatic liver fibrosis by decreasing the differentiation of hepatic progenitor cells into cholangiocytes.

    PubMed

    Zhang, Xiao; Du, Guangli; Xu, Ying; Li, Xuewei; Fan, Weiwei; Chen, Jiamei; Liu, Cheng; Chen, Gaofeng; Liu, Chenghai; Zern, Mark A; Mu, Yongping; Liu, Ping

    2016-03-01

    Although hepatic progenitor cells (HPCs) are known to contribute to cholestatic liver fibrosis (CLF), how Notch signaling modulates the differentiation of HPCs to cholangiocytes in CLF is unknown. Thus, using a rat model of CLF that is induced by bile duct ligation, we inhibited Notch signaling with DAPT. In vivo, CK19, OV6, Sox9, and EpCAM expression was increased significantly. Notch signaling increased after bile duct ligation, and DAPT treatment reduced the expression of CK19, OV6, Sox9, and EpCAM and blocked cholangiocyte proliferation and CLF. In vitro, treatment of a WB-F344 cell line with sodium butyrate resulted in increased mRNA and protein expression of CK19, Sox9, and EpCAM, but Notch signaling was activated. Both of these processes were inhibited by DAPT. This study reveals that Notch signaling activation is required for HPC differentiation into cholangiocytes in CLF, and inhibition of the Notch signaling pathway may offer a therapeutic approach for treating CLF.

  3. WNT antagonist, DKK2, is a Notch signaling target in intestinal stem cells: augmentation of a negative regulation system for canonical WNT signaling pathway by the Notch-DKK2 signaling loop in primates.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2007-01-01

    Notch and WNT signaling pathways are key components of the stem cell signaling network. Canonical WNT signaling to intestinal progenitor cells leads to transcriptional activation of the JAG1 gene, encoding Serrate-type Notch ligand. JAG1 then binds to the Notch receptor on adjacent stem cells to induce Notch receptor proteolyses for the release of Notch intracellular domain (NICD). NICD is associated with CSL/RBPSUH and Mastermind (MAML1, MAML2, or MAML3) to activate Notch target genes, such as HES1 and HES5. Although WNT-dependent Notch signaling activation in intestinal stem cells is clarified, the effects of Notch signaling activation on WNT signaling in progenitor cells remain unclear. We searched for Notch-response element (NRE) in the promoter region of genes encoding secreted WNT signaling inhibitors, including DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1. Double NREs were identified within human DKK2 promoter by bioinformatics and human intelligence (Humint). The human DKK2 gene was characterized as Notch signaling target in intestinal stem cells. Because DKK2 is a key player in the stem cell signaling network, the DKK2 gene at human chromosome 4q25 is a candidate tumor suppressor gene inactivated due to epigenetic silencing and/or deletion. The chimpanzee DKK2 gene was identified within the NW_105990.1 genome sequence, while the cow Dkk2 gene was identified within the AC156664.2 and AC158038.2 genome sequences. Chimpanzee DKK2 and cow Dkk2 showed 98.5% and 95.8% total-amino-acid identity with human DKK2, respectively. Double NREs in human DKK2 promoter were conserved in chimpanzee DKK2 promoter, partially in rat Dkk2 promoter, but not in cow and mouse Dkk2 promoters. The Notch-DKK2 signaling loop, created or potentiated in primates, was complementary to WNT-DKK1 and BMP-IHH-SFRP1 signaling loops for negative regulation of canonical WNT signaling pathway. Together, these facts indicate that DKK2 promoter evolution resulted in the

  4. Naringin Stimulates Osteogenic Differentiation of Rat Bone Marrow Stromal Cells via Activation of the Notch Signaling Pathway

    PubMed Central

    Yu, Guo-yong; Zheng, Gui-zhou; Chang, Bo; Hu, Qin-xiao; Lin, Fei-xiang; Liu, De-zhong; Wu, Chu-cheng; Du, Shi-xin

    2016-01-01

    Naringin is a major flavonoid found in grapefruit and is an active compound extracted from the Chinese herbal medicine Rhizoma Drynariae. Naringin is a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of naringin involves the Notch signaling pathway. Rat bone marrow stromal cells (BMSCs) were cultured in osteogenic medium containing-naringin, with or without DAPT (an inhibitor of Notch signaling), the effects on ALP activity, calcium deposits, osteogenic genes (ALP, BSP, and cbfa1), adipogenic maker gene PPARγ2 levels, and Notch expression were examined. We found that naringin dose-dependently increased ALP activity and Alizarin red S staining, and treatment at the optimal concentration (50 μg/mL) increased mRNA levels of osteogenic genes and Notch1 expression, while decreasing PPARγ2 mRNA levels. Furthermore, treatment with DAPT partly reversed effects of naringin on BMSCs, as judged by decreases in naringin-induced ALP activity, calcium deposits, and osteogenic genes expression, as well as upregulation of PPARγ2 mRNA levels. These results suggest that the osteogenic effect of naringin partly involves the Notch signaling pathway. PMID:27069482

  5. Metastasis-associated lung adenocarcinoma transcript 1 promotes the proliferation of chondrosarcoma cell via activating Notch-1 signaling pathway

    PubMed Central

    Xu, Fengqin; Zhang, Zhi-qiang; Fang, Yong-chao; Li, Xiao-lei; Sun, Yu; Xiong, Chuan-zhi; Yan, Lian-qi; Wang, Qiang

    2016-01-01

    Background Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) is identified to be overexpressed in several cancers. However, the role of MALAT-1 in chondrosarcoma is poorly understood. Methods The expression of MALAT-1 and Notch-1 signaling pathway was detected in chondrosarcoma tissues and chondrosarcoma cells by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was performed to examine the cell viability of chondrosarcoma cells transfected with si-MALAT-1 or pcDNA-MALAT-1. Then the expression of Notch-1 signaling pathway was detected when MALAT-1 was upregulated or downregulated in chondrosarcoma cells. A subcutaneous chondrosarcoma cells xenograft model was used to confirm the effect of MALAT-1 on tumor growth in vivo. Results We found the increased expression of MALAT-1 and Notch-1 signaling pathway in chondrosarcoma tissue and cells. MALAT-1 promoted the proliferation of chondrosarcoma cells. In addition, MALAT-1 activated the Notch-1 signaling pathway at posttranscriptional level in chondrosarcoma cells. Meanwhile, overexpression of Notch-1 reversed the effect of si-MALAT-1 on the proliferation of chondrosarcoma cells. Finally, we found that MALAT-1 promoted the tumor growth in a subcutaneous chondrosarcoma cells xenograft model, which confirmed the promoted effect of MALAT-1 on the tumor growth in vivo. Conclusion Taken together, our study demonstrated that MALAT-1 promoted the proliferation of chondrosarcoma cell via activating Notch-1 signaling pathway. PMID:27110130

  6. An activated Notch1 signaling pathway inhibits cell proliferation and induces apoptosis in human esophageal squamous cell carcinoma cell line EC9706.

    PubMed

    Lu, Zhaoming; Liu, Hongtao; Xue, Lexun; Xu, Peirong; Gong, Tianxiao; Hou, Guiqin

    2008-03-01

    Previous studies have demonstrated that Notch1 signaling pathway plays a major role in maintaining the balance of cell proliferation, differentiation and apoptosis, and is closely associated with tumorigenesis. However, roles of Notch1 signaling pathway in esophageal squamous cell carcinoma (ESCC), which is a common cause of mortality in China, remain poorly understood. Therefore, a novel strategy for seeking a rational molecular therapeutic target for ESCC is urgently needed. The purpose of this study is to examine the effect of the active Notch1 signaling pathway on the proliferation and apoptosis of ESCC cells and to investigate the underlying molecular mechanisms in carcinogenesis of the esophagus. The results revealed that a constitutively activated Notch1 signaling pathway was observed in ESCC cell line EC9706, through a pcNICD vector mediated expression system. Clearly, the activated Notch1 signaling pathway gave rise to proliferation suppression of the cells, accompanied with a cell cycle inhibition at the G0/G1 phase and apoptosis. In contrast to the expression of CDK2, cyclin D1 and cyclin E observed in EC9706 cells untreated and transfected with pcDNA3.1, there was a markedly decrease in the cells stably expressing Notch1 NICD. Up- and down-regulations of GSK3 beta and beta-catenin, respectively, indicated that Notch1 inhibited proliferation and induced apoptosis of EC9706 cells through Wnt-mediated signaling pathway. These findings suggest that Notch1 signaling pathway may participate in carcinogenesis of the esophagus.

  7. Role of Notch-1 signaling pathway in PC12 cell apoptosis induced by amyloid beta-peptide (25–35)

    PubMed Central

    Liang, Huimin; Zhang, Yaozhou; Shi, Xiaoyan; Wei, Tianxiang; Lou, Jiyu

    2014-01-01

    Recent studies have demonstrated that Notch-1 expression is increased in the hippocampus of Alzheimer's disease patients. We speculate that Notch-1 signaling may be involved in PC12 cell apoptosis induced by amyloid beta-peptide (25–35) (Aβ25–35). In the present study, PC12 cells were cultured with different doses (0, 0.1, 1.0, 10 and 100 nmol/L) of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, a Notch-1 signaling pathway inhibitor, for 30 minutes. Then cultured cells were induced with Aβ25–35 for 48 hours. Pretreatment of PC12 cells with high doses of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (> 10 nmol/L) prolonged the survival of PC12 cells after Aβ25–35 induction, decreased the expression of apoptosis-related proteins caspase-3, -8, -9, increased the activity of oxidative stress-related superoxide dismutase and catalase, inhibited the production of active oxygen, and reduced nuclear factor kappa B expression. This study indicates that the Notch-1 signaling pathway plays a pivotal role in Aβ25–35-induced PC12 apoptosis. PMID:25221582

  8. Adropin is a brain membrane-bound protein regulating physical activity via the NB-3/Notch signaling pathway in mice.

    PubMed

    Wong, Chi-Ming; Wang, Yudong; Lee, Jimmy Tsz Hang; Huang, Zhe; Wu, Donghai; Xu, Aimin; Lam, Karen Siu Ling

    2014-09-12

    Adropin is a highly conserved polypeptide that has been suggested to act as an endocrine factor that plays important roles in metabolic regulation, insulin sensitivity, and endothelial functions. However, in this study, we provide evidence demonstrating that adropin is a plasma membrane protein expressed abundantly in the brain. Using a yeast two-hybrid screening approach, we identified NB-3/Contactin 6, a brain-specific, non-canonical, membrane-tethered Notch1 ligand, as an interaction partner of adropin. Furthermore, this interaction promotes NB3-induced activation of Notch signaling and the expression of Notch target genes. We also generated and characterized adropin knockout mice to explore the role of adropin in vivo. Adropin knockout mice exhibited decreased locomotor activity and impaired motor coordination coupled with defective synapse formation, a phenotype similar to NB-3 knockout mice. Taken together, our data suggest that adropin is a membrane-bound protein that interacts with the brain-specific Notch1 ligand NB3. It regulates physical activity and motor coordination via the NB-3/Notch signaling pathway and plays an important role in cerebellum development in mice.

  9. The Parkinson’s Disease-Associated Protein Kinase LRRK2 Modulates Notch Signaling through the Endosomal Pathway

    PubMed Central

    Imai, Yuzuru; Kobayashi, Yoshito; Inoshita, Tsuyoshi; Meng, Hongrui; Arano, Taku; Uemura, Kengo; Asano, Takeshi; Yoshimi, Kenji; Zhang, Chang-Liang; Matsumoto, Gen; Ohtsuka, Toshiyuki; Kageyama, Ryoichiro; Kiyonari, Hiroshi; Shioi, Go; Nukina, Nobuyuki; Hattori, Nobutaka; Takahashi, Ryosuke

    2015-01-01

    Leucine-rich repeat kinase 2 (LRRK2) is a key molecule in the pathogenesis of familial and idiopathic Parkinson’s disease (PD). We have identified two novel LRRK2-associated proteins, a HECT-type ubiquitin ligase, HERC2, and an adaptor-like protein with six repeated Neuralized domains, NEURL4. LRRK2 binds to NEURL4 and HERC2 via the LRRK2 Ras of complex proteins (ROC) domain and NEURL4, respectively. HERC2 and NEURL4 link LRRK2 to the cellular vesicle transport pathway and Notch signaling, through which the LRRK2 complex promotes the recycling of the Notch ligand Delta-like 1 (Dll1)/Delta (Dl) through the modulation of endosomal trafficking. This process negatively regulates Notch signaling through cis-inhibition by stabilizing Dll1/Dl, which accelerates neural stem cell differentiation and modulates the function and survival of differentiated dopaminergic neurons. These effects are strengthened by the R1441G ROC domain-mutant of LRRK2. These findings suggest that the alteration of Notch signaling in mature neurons is a component of PD etiology linked to LRRK2. PMID:26355680

  10. The thyroid hormone nuclear receptor TRα1 controls the Notch signaling pathway and cell fate in murine intestine.

    PubMed

    Sirakov, Maria; Boussouar, Amina; Kress, Elsa; Frau, Carla; Lone, Imtiaz Nisar; Nadjar, Julien; Angelov, Dimitar; Plateroti, Michelina

    2015-08-15

    Thyroid hormones control various aspects of gut development and homeostasis. The best-known example is in gastrointestinal tract remodeling during amphibian metamorphosis. It is well documented that these hormones act via the TR nuclear receptors, which are hormone-modulated transcription factors. Several studies have shown that thyroid hormones regulate the expression of several genes in the Notch signaling pathway, indicating a possible means by which they participate in the control of gut physiology. However, the mechanisms and biological significance of this control have remained unexplored. Using multiple in vivo and in vitro approaches, we show that thyroid hormones positively regulate Notch activity through the TRα1 receptor. From a molecular point of view, TRα1 indirectly controls Notch1, Dll1, Dll4 and Hes1 expression but acts as a direct transcriptional regulator of the Jag1 gene by binding to a responsive element in the Jag1 promoter. Our findings show that the TRα1 nuclear receptor plays a key role in intestinal crypt progenitor/stem cell biology by controlling the Notch pathway and hence the balance between cell proliferation and cell differentiation.

  11. Co-regulation of the Notch and Wnt signaling pathways promotes supporting cell proliferation and hair cell regeneration in mouse utricles

    PubMed Central

    Wu, Jingfang; Li, Wenyan; Lin, Chen; Chen, Yan; Cheng, Cheng; Sun, Shan; Tang, Mingliang; Chai, Renjie; Li, Huawei

    2016-01-01

    This work sought to determine the crosstalk between the Notch and Wnt signaling pathways in regulating supporting cell (SC) proliferation and hair cell (HC) regeneration in mouse utricles. We cultured postnatal day (P)3 and P60 mouse utricles, damaged the HCs with gentamicin, and treated the utricles with the γ-secretase inhibitor DAPT to inhibit the Notch pathway and with the Wnt agonist QS11 to active the Wnt pathway. We also used Sox2-CreER, Notch1-flox (exon 1), and Catnb-flox (exon 3) transgenic mice to knock out the Notch pathway and activate the Wnt pathway in Sox2+ SCs. Notch inhibition alone increased SC proliferation and HC number in both undamaged and damaged utricles. Wnt activation alone promoted SC proliferation, but the HC number was not significantly increased. Here we demonstrated the cumulative effects of Notch inhibition and Wnt activation in regulating SC proliferation and HC regeneration. Simultaneously inhibiting Notch and overexpressing Wnt led to significantly greater SC proliferation and greater numbers of HCs than manipulating either pathway alone. Similar results were observed in the transgenic mice. This study suggests that the combination of Notch inhibition and Wnt activation can significantly promote SC proliferation and increase the number of regenerated HCs in mouse utricle. PMID:27435629

  12. Integration of Orthogonal Signaling by the Notch and Dpp Pathways in Drosophila

    PubMed Central

    Stroebele, Elizabeth; Erives, Albert

    2016-01-01

    The transcription factor Suppressor of Hairless and its coactivator, the Notch intracellular domain, are polyglutamine (pQ)-rich factors that target enhancer elements and interact with other locally bound pQ-rich factors. To understand the functional repertoire of such enhancers, we identify conserved regulatory belts with binding sites for the pQ-rich effectors of both Notch and BMP/Dpp signaling, and the pQ-deficient tissue selectors Apterous (Ap), Scalloped (Sd), and Vestigial (Vg). We find that the densest such binding site cluster in the genome is located in the BMP-inducible nab locus, a homolog of the vertebrate transcriptional cofactors NAB1/NAB2. We report three major findings. First, we find that this nab regulatory belt is a novel enhancer driving dorsal wing margin expression in regions of peak phosphorylated Mad in wing imaginal discs. Second, we show that Ap is developmentally required to license the nab dorsal wing margin enhancer (DWME) to read out Notch and Dpp signaling in the dorsal compartment. Third, we find that the nab DWME is embedded in a complex of intronic enhancers, including a wing quadrant enhancer, a proximal wing disc enhancer, and a larval brain enhancer. This enhancer complex coordinates global nab expression via both tissue-specific activation and interenhancer silencing. We suggest that DWME integration of BMP signaling maintains nab expression in proliferating margin descendants that have divided away from Notch–Delta boundary signaling. As such, uniform expression of genes like nab and vestigial in proliferating compartments would typically require both boundary and nonboundary lineage-specific enhancers. PMID:26975664

  13. Medicarpin, a Natural Pterocarpan, Heals Cortical Bone Defect by Activation of Notch and Wnt Canonical Signaling Pathways

    PubMed Central

    Gupta, Chandra Prakash; Kureel, Jyoti; Mansoori, Mohd Nizam; Shukla, Priyanka; John, Aijaz A.; Singh, Kavita; Purohit, Dipak; Awasthi, Pallavi; Singh, Divya; Goel, Atul

    2015-01-01

    We evaluated the bone regeneration and healing effect of Medicarpin (med) in cortical bone defect model that heals by intramembranous ossification. For the study, female Sprague–Dawley rats were ovariectomized and rendered osteopenic. A drill hole injury was generated in mid femoral bones of all the animals. Med treatment was commenced the day after and continued for 15 days. PTH was taken as a reference standard. Fifteen days post-treatment, animals were sacrificed. Bones were collected for histomorphometry studies at the injury site by micro-computed tomography (μCT) and confocal microscopy. RNA and protein was harvested from newly generated bone. For immunohistochemistry, 5μm sections of decalcified femur bone adjoining the drill hole site were cut. By μCT analysis and calcein labeling of newly generated bone it was found that med promotes bone healing and new bone formation at the injury site and was comparable to PTH in many aspects. Med treatment led to increase in the Runx-2 and osteocalcin signals indicating expansion of osteoprogenitors at the injury site as evaluated by qPCR and immunohistochemical localization. It was observed that med promoted bone regeneration by activating canonical Wnt and notch signaling pathway. This was evident by increased transcript and protein levels of Wnt and notch signaling components in the defect region. Finally, we confirmed that med treatment leads to elevated bone healing in pre-osteoblasts by co localization of beta catenin with osteoblast marker alkaline phosphatase. In conclusion, med treatment promotes new bone regeneration and healing at the injury site by activating Wnt/canonical and notch signaling pathways. This study also forms a strong case for evaluation of med in delayed union and non-union fracture cases. PMID:26657206

  14. Notch signaling in the developing cardiovascular system.

    PubMed

    Niessen, Kyle; Karsan, Aly

    2007-07-01

    The Notch proteins encompass a family of transmembrane receptors that have been highly conserved through evolution as mediators of cell fate. Recent findings have demonstrated a critical role of Notch in the developing cardiovascular system. Notch signaling has been implicated in the endothelial-to-mesenchymal transition during development of the heart valves, in arterial-venous differentiation, and in remodeling of the primitive vascular plexus. Mutations of Notch pathway components in humans are associated with congenital defects of the cardiovascular system such as Alagille syndrome, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), and bicuspid aortic valves. This article focuses on the role of the Notch pathway in the developing cardiovascular system and congenital human cardiovascular diseases.

  15. Estrogen improves the proliferation and differentiation of hBMSCs derived from postmenopausal osteoporosis through notch signaling pathway.

    PubMed

    Fan, Jin-Zhu; Yang, Liu; Meng, Guo-Lin; Lin, Yan-shui; Wei, Bo-Yuan; Fan, Jing; Hu, Hui-Min; Liu, Yan-Wu; Chen, Shi; Zhang, Jin-Kang; He, Qi-Zhen; Luo, Zhuo-Jing; Liu, Jian

    2014-07-01

    Estrogen deficiency is the main reason of bone loss, leading to postmenopausal osteoporosis, and estrogen replacement therapy (ERT) has been demonstrated to protect bone loss efficiently. Notch signaling controls proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Moreover, imperfect estrogen-responsive elements (EREs) were found in the 5'-untranslated region of Notch1 and Jagged1. Thus, we examined the molecular and biological links between estrogen and the Notch signaling in postmenopausal osteoporosis in vitro. hBMSCs were obtained from healthy women and patients with postmenopausal osteoporosis. Notch signaling molecules were quantified using real-time polymerase chain reaction (real-time PCR) and Western Blot. Luciferase reporter constructs with putative EREs were transfected into hBMSCs and analyzed. hBMSCs were transduced with lentiviral vectors containing human Notch1 intracellular domain (NICD1). We also used N-[N-(3, 5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester, a γ-secretase inhibitor, to suppress the Notch signaling. We found that estrogen enhanced the Notch signaling in hBMSCs by promoting the expression of Jagged1. hBMSCs cultured with estrogen resulted in the up-regulation of Notch signaling and increased proliferation and differentiation. Enhanced Notch signaling could enhance the proliferation and differentiation of hBMSCs from patients with postmenopausal osteoporosis (OP-hBMSCs). Our results demonstrated that estrogen preserved bone mass partly by activating the Notch signaling. Because long-term ERT has been associated with several side effects, the Notch signaling could be a potential target for treating postmenopausal osteoporosis.

  16. Activation of the NOTCH pathway in head and neck cancer.

    PubMed

    Sun, Wenyue; Gaykalova, Daria A; Ochs, Michael F; Mambo, Elizabeth; Arnaoutakis, Demetri; Liu, Yan; Loyo, Myriam; Agrawal, Nishant; Howard, Jason; Li, Ryan; Ahn, Sun; Fertig, Elana; Sidransky, David; Houghton, Jeffery; Buddavarapu, Kalyan; Sanford, Tiffany; Choudhary, Ashish; Darden, Will; Adai, Alex; Latham, Gary; Bishop, Justin; Sharma, Rajni; Westra, William H; Hennessey, Patrick; Chung, Christine H; Califano, Joseph A

    2014-02-15

    NOTCH1 mutations have been reported to occur in 10% to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation, and mutation analyses. Copy number increases were identified in NOTCH pathway genes, including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4 of the 37 tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptor mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently.

  17. Activation of the NOTCH pathway in Head and Neck Cancer

    PubMed Central

    Sun, Wenyue; Gaykalova, Daria A.; Ochs, Michael F.; Mambo, Elizabeth; Arnaoutakis, Demetri; Liu, Yan; Loyo, Myriam; Agrawal, Nishant; Howard, Jason; Li, Ryan; Ahn, Sun; Fertig, Elana; Sidransky, David; Houghton, Jeffery; Buddavarapu, Kalyan; Sanford, Tiffany; Choudhary, Ashish; Darden, Will; Adai, Alex; Latham, Gary; Bishop, Justin; Sharma, Rajni; Westra, William H.; Hennessey, Patrick; Chung, Christine H.; Califano, Joseph A.

    2014-01-01

    NOTCH1 mutations have been reported to occur in 10 to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation and mutation analyses. Copy number increases were identified in NOTCH pathway genes including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4/37 of the tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptors mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently. PMID:24351288

  18. Kinase active Misshapen regulates Notch signaling in Drosophila melanogaster.

    PubMed

    Mishra, Abhinava K; Sachan, Nalani; Mutsuddi, Mousumi; Mukherjee, Ashim

    2015-11-15

    Notch signaling pathway represents a principal cellular communication system that plays a pivotal role during development of metazoans. Drosophila misshapen (msn) encodes a protein kinase, which is related to the budding yeast Ste20p (sterile 20 protein) kinase. In a genetic screen, using candidate gene approach to identify novel kinases involved in Notch signaling, we identified msn as a novel regulator of Notch signaling. Data presented here suggest that overexpression of kinase active form of Msn exhibits phenotypes similar to Notch loss-of-function condition and msn genetically interacts with components of Notch signaling pathway. Kinase active form of Msn associates with Notch receptor and regulate its signaling activity. We further show that kinase active Misshapen leads to accumulation of membrane-tethered form of Notch. Moreover, activated Msn also depletes Armadillo and DE-Cadherin from adherens junctions. Thus, this study provides a yet unknown mode of regulation of Notch signaling by Misshapen. PMID:26431585

  19. Asperosaponin VI promotes progesterone receptor expression in decidual cells via the notch signaling pathway.

    PubMed

    Gao, Jie; Zhou, Chun; Li, Yadi; Gao, Feixia; Wu, Haiwang; Yang, Lilin; Qiu, Weiyu; Zhu, Lin; Du, Xin; Lin, Weixian; Huang, Dandan; Liu, Haibin; Liang, Chun; Luo, Songping

    2016-09-01

    Recurrent spontaneous abortion (RSA) is a common clinical condition, but its reasons remain unknown in 37-79% of the affected women. The steroid hormone progesterone (P4) is an integral mediator of early pregnancy events, exerting its effects via the progesterone receptor (PR). Dipsaci Radix (DR) has long been used for treating gynecological diseases in Chinese medicine, while its molecular mechanisms and active ingredients are still unclear. We report here the progesterone-like effects of the alcohol extraction and Asperosaponin VI from DR in primary decidual cells and HeLa cell line. We first determined the safe concentration of Asperosaponin VI in the cells with MTT assay and then found by using dual luciferase reporter and Western blotting that Asperosaponin VI significantly increased PR expression. Moreover, we explored the mechanisms of action of the DR extracts and Asperosaponin VI, and the results showed that they could activate Notch signaling, suggesting that they may function by promoting decidualization. PMID:27370099

  20. Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway

    PubMed Central

    Zhou, Yuning; Wang, Qingding; Weiss, Heidi L.; Evers, B. Mark

    2014-01-01

    The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis that is regulated by multiple signaling pathways. Previously, we have shown that the nuclear factor of activated T-cells 5 (NFAT5) is involved in the regulation of intestinal enterocyte differentiation. Here we show that treatment with sodium chloride (NaCl), which activates NFAT5 signaling, increased mTORC1 repressor regulated in development and DNA damage response 1 (REDD1) protein expression and inhibited mTOR signaling; these alterations were attenuated by knockdown of NFAT5. Knockdown of NFAT5 activated mammalian target of rapamycin (mTOR) signaling and significantly inhibited REDD1 mRNA expression and protein expression. Consistently, overexpression of NFAT5 increased REDD1 expression. In addition, knockdown of REDD1 activated mTOR and Notch signaling, whereas treatment with mTOR inhibitor rapamycin repressed Notch signaling and increased the expression of the goblet cell differentiation marker mucin 2 (MUC2). Moreover, knockdown of NFAT5 activated Notch signaling and decreased MUC2 expression, while overexpression of NFAT5 inhibited Notch signaling and increased MUC2 expression. Our results demonstrate a role for NFAT5 in the regulation of mTOR signaling in intestinal cells. Importantly, these data suggest that NFAT5 participates in the regulation of intestinal homeostasis via the suppression of mTORC1/Notch signaling pathway. PMID:25057011

  1. Growth hormone treatment of premature ovarian failure in a mouse model via stimulation of the Notch-1 signaling pathway

    PubMed Central

    LIU, TE; WANG, SUWEI; ZHANG, LINA; GUO, LIHE; YU, ZHIHUA; CHEN, CHUAN; ZHENG, JIN

    2016-01-01

    Premature ovarian failure (POF) is a condition affecting 1% of women in the general population, causing amenorrhea, hypergonadotropism and hypoestrogenism before the age of 40. Currently, POF cannot be reversed and, although treatments are available, there is an urgent need for improved treatment strategies. Growth hormone (GH) is a pleiotropic hormone that affects a broad spectrum of physiological functions, from carbohydrate and lipid metabolism to the immune response. GH has previously been used to treat POF in non-transgenic preclinical trials, but the biochemical mechanism underlying these effects are unclear. In the present study, a mouse model of POF was generated using cyclophosphamide. Treatment of POF mice with recombinant mouse growth hormone (rmGH) was revealed to markedly reduce POF histopathology in ovarian tissue, relieve ovarian granulosa cell injury, reduce the number of atretic follicles and significantly increase the number of mature oocytes. Furthermore, an enzyme-linked immunosorbent assay revealed that plasma estradiol levels increased and plasma follicle stimulating hormone levels decreased with time in a group of mice treated with a medium dose of rmGH (0.8 mg/kg) when compared with the POF model group (P<0.05). In addition, reverse transcription-quantitative polymerase chain reaction and immunohistochemical analysis demonstrated elevated levels of Notch-1 signaling pathway factors (Notch1, CBF1, and HES1) in wild-type mice and those treated with medium and high doses of rmGH, but not in those treated with low doses of rmGH. In conclusion, GH may promote ovarian tissue repair, estrogen release and oocyte maturation via activation of the Notch-1 signaling pathway in ovarian tissue. PMID:27347041

  2. Notch signaling drives multiple myeloma induced osteoclastogenesis

    PubMed Central

    Colombo, Michela; Thümmler, Katja; Mirandola, Leonardo; Garavelli, Silvia; Todoerti, Katia; Apicella, Luana; Lazzari, Elisa; Lancellotti, Marialuigia; Platonova, Natalia; Akbar, Moeed; Chiriva-Internati, Maurizio; Soutar, Richard; Neri, Antonino; Goodyear, Carl S.; Chiaramonte, Raffaella

    2014-01-01

    Multiple myeloma (MM) is closely associated with bone destruction. Once migrated to the bone marrow, MM cells unbalance bone formation and resorption via the recruitment and maturation of osteoclast precursors. The Notch pathway plays a key role in different types of cancer and drives several biological processes relevant in MM, including cell localization within the bone marrow, proliferation, survival and pharmacological resistance. Here we present evidences that MM can efficiently drive osteoclastogenesis by contemporaneously activating Notch signaling on tumor cells and osteoclasts through the aberrant expression of Notch ligands belonging to the Jagged family. Active Notch signaling in MM cells induces the secretion of the key osteoclastogenic factor, RANKL, which can be boosted in the presence of stromal cells. In turn, MM cells-derived RANKL causes the upregulation of its receptor, RANK, and Notch2 in pre-osteoclasts. Notch2 stimulates osteoclast differentiation by promoting autocrine RANKL signaling. Finally, MM cells through Jagged ligands expression can also activate Notch signaling in pre-osteoclast by direct contact. Such synergism between tumor cells and pre-osteoclasts in MM-induced osteoclastogenesis can be disrupted by silencing tumor-derived Jagged1 and 2. These results make the Jagged ligands new promising therapeutic targets in MM to contrast bone disease and the associated co-morbidities. PMID:25257302

  3. LFA-1/ICAM-1 Ligation in Human T Cells Promotes Th1 Polarization through a GSK3β Signaling-Dependent Notch Pathway.

    PubMed

    Verma, Navin K; Fazil, M H U Turabe; Ong, Seow Theng; Chalasani, Madhavi Latha S; Low, Jian Hui; Kottaiswamy, Amuthavalli; P, Praseetha; Kizhakeyil, Atish; Kumar, Sunil; Panda, Aditya K; Freeley, Michael; Smith, Sinead M; Boehm, Bernhard O; Kelleher, Dermot

    2016-07-01

    In this study, we report that the integrin LFA-1 cross-linking with its ligand ICAM-1 in human PBMCs or CD4(+) T cells promotes Th1 polarization by upregulating IFN-γ secretion and T-bet expression. LFA-1 stimulation in PBMCs, CD4(+) T cells, or the T cell line HuT78 activates the Notch pathway by nuclear translocation of cleaved Notch1 intracellular domain (NICD) and upregulation of target molecules Hey1 and Hes1. Blocking LFA-1 by a neutralizing Ab or specific inhibition of Notch1 by a γ-secretase inhibitor substantially inhibits LFA-1/ICAM-1-mediated activation of Notch signaling. We further demonstrate that the Notch pathway activation is dependent on LFA-1/ICAM-1-induced inactivation of glycogen synthase kinase 3β (GSK3β), which is mediated via Akt and ERK. Furthermore, in silico analysis in combination with coimmunoprecipitation assays show an interaction between NICD and GSK3β. Thus, there exists a molecular cross-talk between LFA-1 and Notch1 through the Akt/ERK-GSK3β signaling axis that ultimately enhances T cell differentiation toward Th1. Although clinical use of LFA-1 antagonists is limited by toxicity related to immunosuppression, these findings support the concept that Notch inhibitors could be attractive for prevention or treatment of Th1-related immunologic disorders and have implications at the level of local inflammatory responses. PMID:27206767

  4. miR-216a regulates snx5, a novel notch signaling pathway component, during zebrafish retinal development.

    PubMed

    Olena, Abigail F; Rao, Mahesh B; Thatcher, Elizabeth J; Wu, Shu-Yu; Patton, James G

    2015-04-01

    Precise regulation of Notch signaling is essential for normal vertebrate development. Mind bomb (Mib) is a ubiquitin ligase that is required for activation of Notch by Notch׳s ligand, Delta. Sorting Nexin 5 (SNX5) co-localizes with Mib and Delta complexes and has been shown to directly bind to Mib. We show that microRNA-216a (miR-216a) is expressed in the retina during early development and regulates snx5 to precisely regulate Notch signaling. miR-216a and snx5 have complementary expression patterns. Knocking down miR-216a and/or overexpression of snx5 resulted in increased Notch activation. Conversely, knocking down snx5 and/or miR-216a overexpression caused a decrease in Notch activation. We propose a model in which SNX5, precisely controlled by miR-216a, is a vital partner of Mib in promoting endocytosis of Delta and subsequent activation of Notch signaling.

  5. Hydrogen Sulfide Prevents Synaptic Plasticity from VD-Induced Damage via Akt/GSK-3β Pathway and Notch Signaling Pathway in Rats.

    PubMed

    Liu, Chunhua; Xu, Xiaxia; Gao, Jing; Zhang, Tao; Yang, Zhuo

    2016-08-01

    Our previous study has demonstrated that hydrogen sulfide (H2S) attenuates neuronal injury induced by vascular dementia (VD) in rats, but the mechanism is still poorly understood. In this study, we aimed to investigate whether the neuroprotection of H2S was associated with synaptic plasticity and try to interpret the potential underlying mechanisms. Adult male Wistar rats were suffered the ligation of bilateral common carotid arteries. At 24 h after surgery, rats were administered intraperitoneally with sodium hydrosulfide (NaHS, 5.6 mg·kg(-1)·day(-1)), a H2S donor, for 3 weeks in the VD+NaHS group and treated intraperitoneally with saline in the VD group respectively. Our results demonstrated that NaHS significantly decreased the level of glutamate. It obviously ameliorated cognitive flexibility as well as the spatial learning and memory abilities by Morris water maze. Moreover, NaHS significantly improved the long-term depression (LTD), and was able to elevate the expression of N-methyl-D-aspartate receptor subunit 2A, which plays a pivotal role in synaptic plasticity. Interestingly, NaHS decreased the phosphorylation of Akt, and it could maintain the activity of glycogen synthase kinase-3β (GSK-3β). Surprisingly, NaHS triggered the canonical Notch pathway by increasing expressions of Jagged-1 and Hes-1. These findings suggest that NaHS prevents synaptic plasticity from VD-induced damage partly via Akt/GSK-3β pathway and Notch signaling pathway.Hydrogen sulfide modulated the ratio of NMDAR 2A/2B and improved the synaptic plasticity via Akt/GSK-3β pathway and Notch signaling pathway in VD rats.

  6. Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signaling pathway

    PubMed Central

    Li, Hongda; Zhong, Xiaofen; Chau, Kevin Fongching; Santistevan, Nicholas J.; Guo, Weixiang; Kong, Guangyao; Li, Xuekun; Kadakia, Mitul; Masliah, Jamie; Chi, Jingyi; Jin, Peng; Zhang, Jing; Zhao, Xinyu; Chang, Qiang

    2014-01-01

    Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons. The precise control of the phosphorylation status of MeCP2 in neurons is critical for the normal development and function of the mammalian brain. However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts. Here we show that, in neural progenitor cells isolated from the adult mouse hippocampus, cell cycle-linked phosphorylation at serine 421 on MeCP2 is directly regulated by aurora kinase B, and modulates the balance between proliferation and neural differentiation through the Notch signaling pathway. Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signaling pathways for regulating diverse biological functions in different cell types. PMID:25420914

  7. HES6 promotes prostate cancer aggressiveness independently of Notch signalling

    PubMed Central

    Carvalho, Filipe L F; Marchionni, Luigi; Gupta, Anuj; Kummangal, Basheer A; Schaeffer, Edward M; Ross, Ashley E; Berman, David M

    2015-01-01

    Notch signalling is implicated in the pathogenesis of a variety of cancers, but its role in prostate cancer is poorly understood. However, selected Notch pathway members are overrepresented in high-grade prostate cancers. We comprehensively profiled Notch pathway components in prostate cells and found prostate cancer-specific up-regulation of NOTCH3 and HES6. Their expression was particularly high in androgen responsive lines. Up- and down-regulating Notch in these cells modulated expression of canonical Notch targets, HES1 and HEY1, which could also be induced by androgen. Surprisingly, androgen treatment also suppressed Notch receptor expression, suggesting that androgens can activate Notch target genes in a receptor-independent manner. Using a Notch-sensitive Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) reporter assay, we found that basal levels of Notch signalling were significantly lower in prostate cancer cells compared to benign cells. Accordingly pharmacological Notch pathway blockade did not inhibit cancer cell growth or viability. In contrast to canonical Notch targets, HES6, a HES family member known to antagonize Notch signalling, was not regulated by Notch signalling, but relied instead on androgen levels, both in cultured cells and in human cancer tissues. When engineered into prostate cancer cells, reduced levels of HES6 resulted in reduced cancer cell invasion and clonogenic growth. By molecular profiling, we identified potential roles for HES6 in regulating hedgehog signalling, apoptosis and cell migration. Our results did not reveal any cell-autonomous roles for canonical Notch signalling in prostate cancer. However, the results do implicate HES6 as a promoter of prostate cancer progression. PMID:25864518

  8. Notch signaling in the brain: in good and bad times.

    PubMed

    Alberi, Lavinia; Hoey, Sarah E; Brai, Emanuele; Scotti, Alessandra L; Marathe, Swananda

    2013-06-01

    Notch signaling is an evolutionarily conserved pathway, which is fundamental for neuronal development and specification. In the last decade, increasing evidence has pointed out an important role of this pathway beyond embryonic development, indicating that Notch also displays a critical function in the mature brain of vertebrates and invertebrates. This pathway appears to be involved in neural progenitor regulation, neuronal connectivity, synaptic plasticity and learning/memory. In addition, Notch appears to be aberrantly regulated in neurodegenerative diseases, including Alzheimer's disease and ischemic injury. The molecular mechanisms by which Notch displays these functions in the mature brain are not fully understood, but are currently the subject of intense research. In this review, we will discuss old and novel Notch targets and molecular mediators that contribute to Notch function in the mature brain and will summarize recent findings that explore the two facets of Notch signaling in brain physiology and pathology.

  9. Notch Signaling: A Potential Therapeutic Target for Hematologic Malignancies.

    PubMed

    Gao, Lingbao; Yuan, Keyu; Ding, Wei; Lin, Mei

    2016-01-01

    Notch signaling is a well-conserved cell-fate determining factor in embryo development, and the dyregulation of this signaling is frequently observed in many types of cancers, including hematological malignancies. In this review, we briefly describe the Notch signaling pathway, and we primarily focus on the relationship between Notch and hematological malignancies. We also discuss the clinical development of promising agents including γ-secretase inhibitors (GSIs) and monoclonal antibodies (mAbs). Complete response has been observed among patients with T-cell acute lymphoblastic leukemia (T-ALL) when treated with GSIs. Furthermore, a recent study has suggested that targeting Zmiz1, a direct, selective cofactor of Notch1, rather than targeting Notch directly, maybe helpful to reduce the current target-related toxicities. Taken together, we summarize the role of Notch signaling in hematological malignancies and discuss the treatment strategies for these diseases through targeting Notch signaling. PMID:27650987

  10. Rare Variants in the Notch Signaling Pathway Describe a Novel Type of Autosomal Recessive Klippel–Feil Syndrome

    PubMed Central

    Karaca, Ender; Yuregir, Ozge O.; Bozdogan, Sevcan T.; Aslan, Huseyin; Pehlivan, Davut; Jhangiani, Shalini N.; Akdemir, Zeynep C.; Gambin, Tomasz; Bayram, Yavuz; Atik, Mehmed M.; Erdin, Serkan; Muzny, Donna; Gibbs, Richard A.; Lupski, James R.

    2016-01-01

    Klippel–Feil syndrome is a rare disorder represented by a subgroup of segmentation defects of the vertebrae and characterized by fusion of the cervical vertebrae, low posterior hairline, and short neck with limited motion. Both autosomal dominant and recessive inheritance patterns were reported in families with Klippel–Feil. Mutated genes for both dominant (GDF6 and GDF3) and recessive (MEOX1) forms of Klippel–Feil syndrome have been shown to be involved in somite development via transcription regulation and signaling pathways. Heterotaxy arises from defects in proteins that function in the development of left–right asymmetry of the developing embryo. We describe a consanguineous family with a male proband who presents with classical Klippel–Feil syndrome together with heterotaxy (situs inversus totalis). The present patient also had Sprengel’s deformity, deformity of the sternum, and a solitary kidney. Using exome sequencing, we identified a homozygous frameshift mutation (c.299delT; p.L100fs) in RIPPLY2, a gene shown to play a crucial role in somitogenesis and participate in the Notch signaling pathway via negatively regulating Tbx6. Our data confirm RIPPLY2 as a novel gene for autosomal recessive Klippel–Feil syndrome, and in addition—from a mechanistic standpoint—suggest the possibility that mutations in RIPPLY2 could also lead to heterotaxy. PMID:26238661

  11. Rare variants in the notch signaling pathway describe a novel type of autosomal recessive Klippel-Feil syndrome.

    PubMed

    Karaca, Ender; Yuregir, Ozge O; Bozdogan, Sevcan T; Aslan, Huseyin; Pehlivan, Davut; Jhangiani, Shalini N; Akdemir, Zeynep C; Gambin, Tomasz; Bayram, Yavuz; Atik, Mehmed M; Erdin, Serkan; Muzny, Donna; Gibbs, Richard A; Lupski, James R

    2015-11-01

    Klippel-Feil syndrome is a rare disorder represented by a subgroup of segmentation defects of the vertebrae and characterized by fusion of the cervical vertebrae, low posterior hairline, and short neck with limited motion. Both autosomal dominant and recessive inheritance patterns were reported in families with Klippel-Feil. Mutated genes for both dominant (GDF6 and GDF3) and recessive (MEOX1) forms of Klippel-Feil syndrome have been shown to be involved in somite development via transcription regulation and signaling pathways. Heterotaxy arises from defects in proteins that function in the development of left-right asymmetry of the developing embryo. We describe a consanguineous family with a male proband who presents with classical Klippel-Feil syndrome together with heterotaxy (situs inversus totalis). The present patient also had Sprengel's deformity, deformity of the sternum, and a solitary kidney. Using exome sequencing, we identified a homozygous frameshift mutation (c.299delT; p.L100fs) in RIPPLY2, a gene shown to play a crucial role in somitogenesis and participate in the Notch signaling pathway via negatively regulating Tbx6. Our data confirm RIPPLY2 as a novel gene for autosomal recessive Klippel-Feil syndrome, and in addition-from a mechanistic standpoint-suggest the possibility that mutations in RIPPLY2 could also lead to heterotaxy. © 2015 Wiley Periodicals, Inc. PMID:26238661

  12. Total alkaloids of Rubus alceifolius Poir inhibit tumor angiogenesis through suppression of the Notch signaling pathway in a mouse model of hepatocellular carcinoma.

    PubMed

    Zhao, Jinyan; Lin, Wei; Cao, Zhiyun; Zhuang, Qunchuan; Zheng, Liangpu; Peng, Jun; Hong, Zhenfeng

    2015-01-01

    Angiogenesis, which has a critical role in human tumor growth and development, is tightly regulated by the Notch signaling pathway. Total alkaloids are active components of the plant Rubus alceifolius Poir, which is used for the treatment of various types of cancer. A previous study by our group showed that the total alkaloids of Rubus alceifolius Poir (TARAP) induced hepatocellular carcinoma (HCC) cell apoptosis through the activation of the mitochondria-dependent pathway in vitro and in vivo, as well as inhibited angiogenesis in a chick embryo chorioallantoic membrane model. In the present study, to further analyze the specific mechanisms underlying the antitumor activity of TARAP, a HCC xenograft mouse model was used to assess the effect of TARAP on angiogenesis in vivo. TARAP was found to suppress the expression of vascular endothelial growth factor (VEGF) A and VEGF receptor-2 in tumor tissues, which resulted in the inhibition of tumor angiogenesis. In addition, TARAP treatment was observed to inhibit the expression of Notch1, delta-like ligand 4 and jagged 1, which are key mediators of the Notch signaling pathway. The present study identified that the inhibition of tumor angiogenesis through the suppression of the Notch signaling pathway may be one of the mechanisms through which TARAP may be effective in the treatment of cancer.

  13. Increased expression of Hes5 protein in Notch signaling pathway in the hippocampus of mice offspring of dams fed a high-fat diet during pregnancy and suckling.

    PubMed

    Mendes-da-Silva, Cristiano; Lemes, Simone Ferreira; Baliani, Tanyara da Silva; Versutti, Milena Diorio; Torsoni, Marcio Alberto

    2015-02-01

    Maternal high-fat diet (HFD) impairs hippocampal development of offspring promoting decreased proliferation of neural progenitors, in neuronal differentiation, in dendritic spine density and synaptic plasticity reducing neurogenic capacity. Notch signaling pathway participates in molecular mechanisms of the neurogenesis. The activation of Notch signaling leads to the upregulation of Hes5, which inhibits the proliferation and differentiation of neural progenitors. This study aimed to investigate the Notch/Hes pathway activation in the hippocampus of the offspring of dams fed an HFD. Female Swiss mice were fed a control diet (CD) and an HFD from pre-mating until suckling. The bodyweight and mass of adipose tissue in the mothers and pups were also measured. The mRNA and protein expression of Notch1, Hes5, Mash1, and Delta1 in the hippocampus was assessed by RT-PCR and western blotting, respectively. Dams fed the HFD and their pups had an increased bodyweight and amount of adipose tissue. Furthermore, the offspring of mothers fed the HFD exhibited an increased Hes5 expression in the hippocampus compared with CD offspring. In addition, HFD offspring also expressed increased amounts of Notch1 and Hes5 mRNA, whereas Mash1 expression was decreased. However, the expression of Delta1 did not change significantly. We propose that the overexpression of Hes5, a Notch effector, downregulates the expression of the proneural gene Mash1 in the offspring of obese mothers, delaying cellular differentiation. These results provide further evidence that an offspring's hippocampus is molecularly susceptible to maternal HFD and suggest that Notch1 signaling in this brain region is important for neuronal differentiation.

  14. Notch signaling in prostate cancer: refining a therapeutic opportunity

    PubMed Central

    Su, Qingtai; Xin, Li

    2016-01-01

    Summary Notch is an evolutionarily conserved signaling pathway that plays a critical role in specifying cell fate and regulating tissue homeostasis and carcinogenesis. Studies using organ cultures and genetically engineered mouse models have demonstrated that Notch signaling regulates prostate development and homeostasis. However, the role of the Notch signaling pathway in prostate cancer remains inconclusive. Many published studies have documented consistent deregulation of major Notch signaling components in human prostate cancer cell lines, mouse models for prostate cancers, and human prostate cancer specimens at both the mRNA and the protein levels. However, functional studies in human cancer cells by modulation of Notch pathway elements suggest both tumor suppressive and oncogenic roles of Notch. These controversies may originate from our inadequate understanding of the regulation of Notch signaling under versatile genetic contexts, and reflect the multifaceted and pleiotropic roles of Notch in regulating different aspects of prostate cancer cell biology, such as proliferation, metastasis, and chemo-resistance. Future comprehensive studies using various mouse models for prostate cancer may help clarify the role of Notch signaling in prostate cancer and provide a solid basis for determining whether and how Notch should be employed as a therapeutic target for prostate cancer. PMID:26521657

  15. Notch signaling in cardiovascular disease and calcification.

    PubMed

    Rusanescu, Gabriel; Weissleder, Ralph; Aikawa, Elena

    2008-08-01

    Recent increase in human lifespan has shifted the spectrum of aging-related disorders to an unprecedented upsurge in cardiovascular diseases, especially calcific aortic valve stenosis, which has an 80% risk of progression to heart failure and death. A current therapeutic option for calcified valves is surgical replacement, which provides only temporary relief. Recent progress in cardiovascular research has suggested that arterial and valve calcification are the result of an active process of osteogenic differentiation, induced by a pro-atherogenic inflammatory response. At molecular level, the calcification process is regulated by a network of signaling pathways, including Notch, Wnt and TGFbeta/BMP pathways, which control the master regulator of osteogenesis Cbfa1/Runx2. Genetic and in vitro studies have implicated Notch signaling in the regulation of macrophage activation and cardiovascular calcification. Individuals with inactivating Notch1 mutations have a high rate of cardiovascular disorders, including valve stenosis and calcification. This article reviews recent progress in the mechanism of cardiovascular calcification and discusses potential molecular mechanisms involved, focusing on Notch receptors. We propose a calcification model where extreme increases in vascular wall cell density due to inflammation-induced cell proliferation can trigger an osteogenic differentiation program mediated by Notch receptors. PMID:19936191

  16. Notching on Cancer’s Door: Notch Signaling in Brain Tumors

    PubMed Central

    Teodorczyk, Marcin; Schmidt, Mirko H. H.

    2015-01-01

    Notch receptors play an essential role in the regulation of central cellular processes during embryonic and postnatal development. The mammalian genome encodes for four Notch paralogs (Notch 1–4), which are activated by three Delta-like (Dll1/3/4) and two Serrate-like (Jagged1/2) ligands. Further, non-canonical Notch ligands such as epidermal growth factor like protein 7 (EGFL7) have been identified and serve mostly as antagonists of Notch signaling. The Notch pathway prevents neuronal differentiation in the central nervous system by driving neural stem cell maintenance and commitment of neural progenitor cells into the glial lineage. Notch is therefore often implicated in the development of brain tumors, as tumor cells share various characteristics with neural stem and progenitor cells. Notch receptors are overexpressed in gliomas and their oncogenicity has been confirmed by gain- and loss-of-function studies in vitro and in vivo. To this end, special attention is paid to the impact of Notch signaling on stem-like brain tumor-propagating cells as these cells contribute to growth, survival, invasion, and recurrence of brain tumors. Based on the outcome of ongoing studies in vivo, Notch-directed therapies such as γ-secretase inhibitors and blocking antibodies have entered and completed various clinical trials. This review summarizes the current knowledge on Notch signaling in brain tumor formation and therapy. PMID:25601901

  17. Acquisition of epithelial-mesenchymal transition phenotype of gemcitabine-resistant pancreatic cancer cells is linked with activation of the notch signaling pathway.

    PubMed

    Wang, Zhiwei; Li, Yiwei; Kong, Dejuan; Banerjee, Sanjeev; Ahmad, Aamir; Azmi, Asfar Sohail; Ali, Shadan; Abbruzzese, James L; Gallick, Gary E; Sarkar, Fazlul H

    2009-03-15

    Despite rapid advances in many fronts, pancreatic cancer (PC) remains one of the most difficult human malignancies to treat due, in part, to de novo and acquired chemoresistance and radioresistance. Gemcitabine alone or in combination with other conventional therapeutics is the standard of care for the treatment of advanced PC without any significant improvement in the overall survival of patients diagnosed with this deadly disease. Previous studies have shown that PC cells that are gemcitabine-resistant (GR) acquired epithelial-mesenchymal transition (EMT) phenotype, which is reminiscent of "cancer stem-like cells"; however, the molecular mechanism that led to EMT phenotype has not been fully investigated. The present study shows that Notch-2 and its ligand, Jagged-1, are highly up-regulated in GR cells, which is consistent with the role of the Notch signaling pathway in the acquisition of EMT and cancer stem-like cell phenotype. We also found that the down-regulation of Notch signaling was associated with decreased invasive behavior of GR cells. Moreover, down-regulation of Notch signaling by siRNA approach led to partial reversal of the EMT phenotype, resulting in the mesenchymal-epithelial transition, which was associated with decreased expression of vimentin, ZEB1, Slug, Snail, and nuclear factor-kappaB. These results provide molecular evidence showing that the activation of Notch signaling is mechanistically linked with chemoresistance phenotype (EMT phenotype) of PC cells, suggesting that the inactivation of Notch signaling by novel strategies could be a potential targeted therapeutic approach for overcoming chemoresistance toward the prevention of tumor progression and/or treatment of metastatic PC.

  18. Tools and methods for studying Notch signaling in Drosophila melanogaster

    PubMed Central

    Zacharioudaki, Evanthia; Bray, Sarah J.

    2014-01-01

    Notch signaling involves a highly conserved pathway that mediates communication between neighboring cells. Activation of Notch by its ligands, results in the release of the Notch intracellular domain (NICD), which enters the nucleus and regulates transcription. This pathway has been implicated in many developmental decisions and diseases (including cancers) over the past decades. The simplicity of the Notch pathway in Drosophila melanogaster, in combination with the availability of powerful genetics, make this an attractive model for studying fundamental principles of Notch regulation and function. In this article we present some of the established and emerging tools that are available to monitor and manipulate the Notch pathway in Drosophila and discuss their strengths and weaknesses. PMID:24704358

  19. Α-arrestins - new players in Notch and GPCR signaling pathways in mammals.

    PubMed

    Puca, Loredana; Brou, Christel

    2014-04-01

    For many years, β-arrestins have been known to be involved in G-protein-coupled receptor (GPCR) desensitization. However, β-arrestins belong to a family of proteins that act as multifunctional scaffolding proteins, in particular during trafficking of transmembrane receptors. The arrestin family comprises visual arrestins, β-arrestins and α-arrestins. In mammals, the functions of the α-arrestins are beginning to be elucidated, and they are described as versatile adaptors that link GPCRs or the Notch receptor to E3 ubiquitin ligases and endocytic factors. These α-arrestins can act in sequence, complementarily or cooperatively with β-arrestins in trafficking and ubiquitylation events. This Commentary will summarize the recent advances in our understanding of the functions and properties of these α-arrestin proteins in comparison to β-arrestins, and will highlight a new hypothesis linking their functional complementarity to their physical interactions. α- and β-arrestins could form transient and versatile heterodimers that form a bridge between cargo and E3 ubiquitin ligases, thus allowing trafficking to proceed.

  20. Oncogenic role of the Notch pathway in primary liver cancer

    PubMed Central

    LU, JIE; XIA, YUJING; CHEN, KAN; ZHENG, YUANYUAN; WANG, JIANRONG; LU, WENXIA; YIN, QIN; WANG, FAN; ZHOU, YINGQUN; GUO, CHUANYONG

    2016-01-01

    Primary liver cancer, which includes hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and fibrolamellar HCC, is one of the most common malignancies and the third leading cause of cancer-associated mortality, worldwide. Despite the development of novel therapies, the prognosis of liver cancer patients remains extremely poor. Thus, investigation of the genetic background and molecular mechanisms underlying the development and progression of this disease has gained significant attention. The Notch signaling pathway is a crucial determinant of cell fate during development and disease in several organs. In the liver, Notch signaling is involved in biliary tree development and tubulogenesis, and is also significant in the development of HCC and ICC. These findings suggest that the modulation of Notch pathway activity may have therapeutic relevance. The present review summarizes Notch signaling during HCC and ICC development and discusses the findings of recent studies regarding Notch expression, which reveal novel insights into its function in liver cancer progression. PMID:27347091

  1. Notch signaling activation in pediatric low-grade astrocytoma.

    PubMed

    Brandt, William D; Schreck, Karisa C; Bar, Eli E; Taylor, Isabella; Marchionni, Luigi; Raabe, Eric; Eberhart, Charles G; Rodriguez, Fausto J

    2015-02-01

    Pilocytic astrocytoma (PA) is the most common primary brain tumor in children; various signaling pathways have been implicated in its biology. The Notch signaling pathway has been found to play a role in the development, stem cell biology, and pathogenesis of several cancers, but its role in PA has not been investigated. We studied alterations in Notch signaling components in tumor tissue from 18 patients with PA and 4 with other low-grade astrocytomas to identify much needed therapeutic targets. We found that Notch pathway members were overexpressed at the mRNA (NOTCH1, NOTCH2, HEY1, HEY2) and protein (HES1) levels in PAs at various anatomic sites compared with non-neoplastic brain samples. These changes were not associated with specific BRAF alterations. Inhibiting the Notch pathway in the pediatric low-grade astrocytoma cell lines Res186 and Res259 using either RNA interference or a γ-secretase inhibitor resulted in variable, but significant, reduction in cell growth and migration. This study suggests a potential role for Notch signaling in pediatric low-grade astrocytoma tumorigenesis and that Notch signaling may be a viable pathway therapeutic target. PMID:25575134

  2. Evidence for the Induction of Key Components of the NOTCH Signaling Pathway via Deltamethrin and Azamethiphos Treatment in the Sea Louse Caligus rogercresseyi

    PubMed Central

    Boltaña, Sebastian; Chávez-Mardones, Jaqueline; Valenzuela-Muñoz, Valentina; Gallardo-Escárate, Cristian

    2016-01-01

    The extensive use of organophosphates and pyrethroids in the aquaculture industry has negatively impacted parasite sensitivity to the delousing effects of these antiparasitics, especially among sea lice species. The NOTCH signaling pathway is a positive regulator of ABC transporter subfamily C expression and plays a key role in the generation and modulation of pesticide resistance. However, little is known about the molecular mechanisms behind pesticide resistance, partly due to the lack of genomic and molecular information on the processes involved in the resistance mechanism of sea lice. Next-generation sequencing technologies provide an opportunity for rapid and cost-effective generation of genome-scale data. The present study, through RNA-seq analysis, determined that the sea louse Caligus rogercresseyi (C. rogercresseyi) specifically responds to the delousing drugs azamethiphos and deltamethrin at the transcriptomic level by differentially activating mRNA of the NOTCH signaling pathway and of ABC genes. These results suggest that frequent antiparasitic application may increase the activity of inhibitory mRNA components, thereby promoting inhibitory NOTCH output and conditions for increased resistance to delousing drugs. Moreover, data analysis underscored that key functions of NOTCH/ABC components were regulated during distinct phases of the drug response, thus indicating resistance modifications in C. rogercresseyi resulting from the frequent use of organophosphates and pyrethroids. PMID:27187362

  3. Notch signaling: its roles and therapeutic potential in hematological malignancies

    PubMed Central

    Gu, Yisu

    2016-01-01

    Notch is a highly conserved signaling system that allows neighboring cells to communicate, thereby controlling their differentiation, proliferation and apoptosis, with the outcome of its activation being highly dependent on signal strength and cell type. As such, there is growing evidence that disturbances in physiological Notch signaling contribute to cancer development and growth through various mechanisms. Notch was first reported to contribute to tumorigenesis in the early 90s, through identification of the involvement of the Notch1 gene in the chromosomal translocation t(7;9)(q34;q34.3), found in a small subset of T-cell acute lymphoblastic leukemia. Since then, Notch mutations and aberrant Notch signaling have been reported in numerous other precursor and mature hematological malignancies, of both myeloid and lymphoid origin, as well as many epithelial tumor types. Of note, Notch has been reported to have both oncogenic and tumor suppressor roles, dependent on the cancer cell type. In this review, we will first give a general description of the Notch signaling pathway, and its physiologic role in hematopoiesis. Next, we will review the role of aberrant Notch signaling in several hematological malignancies. Finally, we will discuss current and potential future therapeutic approaches targeting this pathway. PMID:26934331

  4. 3,6-dihydroxyflavone suppresses the epithelial-mesenchymal transition in breast cancer cells by inhibiting the Notch signaling pathway

    PubMed Central

    Chen, Junli; Chang, Hui; Peng, Xiaoli; Gu, Yeyun; Yi, Long; Zhang, Qianyong; Zhu, Jundong; Mi, Mantian

    2016-01-01

    The epithelial to mesenchymal transition (EMT) is a critical developmental program in cancer stem cell (CSC) maintenance and in cancer metastasis. Here, our study found that 3,6-DHF could effectively inhibit EMT in BC cells in vitro and in vivo. 3,6-DHF effectively inhibits the formation and proliferation of BCSCs, and consequently reduces the tumor-initiating capacity of tumor cells in NOD/SCID mice. Optical in vivo imaging of cancer metastasis showed that 3,6-DHF administration suppresses the lung metastasis of BC cells in vivo. Further studies indicated that 3,6-DHF down-regulates Notch1, NICD, Hes-1 and c-Myc, consequently decreasing the formation of the functional transcriptional unit of NICD-CSL-MAML, causing Notch signaling inactivation in BC cells. Over-expression of Notch1 or inhibition of miR-34a significantly reduced the inhibitory effects of 3,6-DHF on EMT, CSCs, as well as cells migration and invasion in BC cells. These data indicated that 3,6-DHF effectively inhibits EMT and CSCs, as well as cells migration and invasion in BC cells, in which miR-34a-mediated Notch1 down-regulation plays a crucial role. PMID:27345219

  5. 14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    SciTech Connect

    Xin, Ying; Lu, Qingxian; Li, Qiutang

    2010-02-19

    14-3-3{sigma} (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3{sigma} mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3{sigma} activity in corneal epithelial cells by overexpressing dominative negative 14-3-3{sigma} led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3{sigma} mutant-expressing corneal epithelial cells. We conclude that 14-3-3{sigma} is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

  6. Effect of IL-17 monoclonal antibody Secukinumab combined with IL-35 blockade of Notch signaling pathway on the invasive capability of hepatoma cells.

    PubMed

    Li, H Ch; Zhang, Y X; Liu, Y; Wang, Q Sh

    2016-07-14

    We investigated the effect of the IL-17 monoclonal antibody Secukinumab combined with IL-35 in the blockade of the Notch signaling pathway on the invasive capability of hepatoma cells. We examined the effects of IL-17 antibody or IL-35 treatment alone or in combination on cell invasion and migration capabilities with Transwell chambers. The mRNA levels of Hes1, Hes5, and Hey1 were tested using quantitative polymerase chain reaction. The protein expression of N1ICD, Snail, and E-cadherin protein expressions were measured with western blot. The expression of Hes1, Hes5, Hey1 and N1ICD were all very high in hepatoma cell lines, and were positively correlated with the invasive migration capabilities of the cells. The combination of IL-17 monoclonal antibody Secukinumab with IL-35 could effectively inhibit the Notch signaling pathway, as well as the invasive migration of the cells. Snail and E-cadherin are involved in the migration of hepatoma cells, and it has been established that Snail can regulate the expression of E-cadherin. IL-17 monoclonal antibody Secukinumab combined with IL-35 can increase E-cadherin and decrease Snail expression, which are positively correlated with cell invasive migration capabilities. Overall, treatment with both IL-17 antibody and IL-35 is more effective than each treatment alone. Notch signaling is activated in hepatoma cell lines and increases with the enhancement of cell invasive migration capabilities. IL-17 monoclonal antibody Secukinumab combined with IL-35 can block the Notch signaling pathway, simultaneously reducing the invasive migration capability of hepatoma cells.

  7. Dishevelled limits Notch signalling through inhibition of CSL

    PubMed Central

    Collu, Giovanna M.; Hidalgo-Sastre, Ana; Acar, Ahmet; Bayston, Laura; Gildea, Clara; Leverentz, Michael K.; Mills, Christopher G.; Owens, Thomas W.; Meurette, Olivier; Dorey, Karel; Brennan, Keith

    2012-01-01

    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. PMID:23132247

  8. Notch Signaling in Meibomian Gland Epithelial Cell Differentiation

    PubMed Central

    Gidfar, Sanaz; Afsharkhamseh, Neda; Sanjari, Sara; Djalilian, Ali R.

    2016-01-01

    Purpose Notch1 was previously shown to play a critical role in murine meibomian gland function and maintenance. In this study, we have examined the expression and activation of Notch pathway in human meibomian gland epithelial cells in vitro. Methods An immortalized human meibomian gland epithelial cell (HMGEC) line was cultured under proliferative and differentiative conditions. Expression of Notch receptors and ligands were evaluated by quantitative PCR and Western blot. The effect of Notch inhibition and induction on oil production was also assessed. Results Human meibomian gland epithelial cell expressed Notch1, Notch2, Notch3, Jagged1, Jagged2, Delta-like 1, and Delta-like 3. The level of cleaved (activated) Notch1 strongly increased with differentiation. The expression of Notch3 was inversely correlated with proliferation. Induction and inhibition of Notch1 led to an increase and decrease in the amount of oil production, respectively. Conclusions Notch signaling appears to play an important role in human meibomian gland epithelial differentiation and oil production. This may provide a potential therapeutic pathway for treating meibomian gland dysfunction. PMID:26943148

  9. Uncovering Notch pathway in the parasitic flatworm Schistosoma mansoni.

    PubMed

    Magalhães, Lizandra G; Morais, Enyara R; Machado, Carla B; Gomes, Matheus S; Cabral, Fernanda J; Souza, Julia M; Soares, Cláudia S; Sá, Renata G; Castro-Borges, William; Rodrigues, Vanderlei

    2016-10-01

    Several signaling molecules that govern development in higher animals have been identified in the parasite Schistosoma mansoni, including the transforming growth factor β, protein tyrosine kinases, nuclear hormone receptors, among others. The Notch pathway is a highly conserved signaling mechanism which is involved in a wide variety of developmental processes including embryogenesis and oogenesis in worms and flies. Here we aimed to provide the molecular reconstitution of the Notch pathway in S. mansoni using the available transcriptome and genome databases. Our results also revealed the presence of the transcripts coded for SmNotch, SmSu(H), SmHes, and the gamma-secretase complex (SmNicastrin, SmAph-1, and SmPen-2), throughout all the life stages analyzed. Besides, it was observed that the viability and separation of adult worm pairs were not affected by treatment with N-[N(3,5)-difluorophenacetyl)-L-Alanyl]-S-phenylglycine t-butyl ester (DAPT), a Notch pathway inhibitor. Moreover, DAPT treatment decreased the production of phenotypically normal eggs and arrested their development in culture. Our results also showed a significant decrease in SmHes transcript levels in both adult worms and eggs treated with DAPT. These results provide, for the first time, functional validation of the Notch pathway in S. mansoni and suggest its involvement in parasite oogenesis and embryogenesis. Given the complexity of the Notch pathway, further experiments shall highlight the full repertoire of Notch-mediated cellular processes throughout the S. mansoni life cycle.

  10. Notch signaling regulates gastric antral LGR5 stem cell function

    PubMed Central

    Demitrack, Elise S; Gifford, Gail B; Keeley, Theresa M; Carulli, Alexis J; VanDussen, Kelli L; Thomas, Dafydd; Giordano, Thomas J; Liu, Zhenyi; Kopan, Raphael; Samuelson, Linda C

    2015-01-01

    The major signaling pathways regulating gastric stem cells are unknown. Here we report that Notch signaling is essential for homeostasis of LGR5+ antral stem cells. Pathway inhibition reduced proliferation of gastric stem and progenitor cells, while activation increased proliferation. Notch dysregulation also altered differentiation, with inhibition inducing mucous and endocrine cell differentiation while activation reduced differentiation. Analysis of gastric organoids demonstrated that Notch signaling was intrinsic to the epithelium and regulated growth. Furthermore, in vivo Notch manipulation affected the efficiency of organoid initiation from glands and single Lgr5-GFP stem cells, suggesting regulation of stem cell function. Strikingly, constitutive Notch activation in LGR5+ stem cells induced tissue expansion via antral gland fission. Lineage tracing using a multi-colored reporter demonstrated that Notch-activated stem cells rapidly generate monoclonal glands, suggesting a competitive advantage over unmanipulated stem cells. Notch activation was associated with increased mTOR signaling, and mTORC1 inhibition normalized NICD-induced increases in proliferation and gland fission. Chronic Notch activation induced undifferentiated, hyper-proliferative polyps, suggesting that aberrant activation of Notch in gastric stem cells may contribute to gastric tumorigenesis. PMID:26271103

  11. Regulation of Monocarboxylic Acid Transporter 1 Trafficking by the Canonical Wnt/β-Catenin Pathway in Rat Brain Endothelial Cells Requires Cross-talk with Notch Signaling.

    PubMed

    Liu, Zejian; Sneve, Mary; Haroldson, Thomas A; Smith, Jeffrey P; Drewes, Lester R

    2016-04-01

    The transport of monocarboxylate fuels such as lactate, pyruvate, and ketone bodies across brain endothelial cells is mediated by monocarboxylic acid transporter 1 (MCT1). Although the canonical Wnt/β-catenin pathway is required for rodent blood-brain barrier development and for the expression of associated nutrient transporters, the role of this pathway in the regulation of brain endothelial MCT1 is unknown. Here we report expression of nine members of the frizzled receptor family by the RBE4 rat brain endothelial cell line. Furthermore, activation of the canonical Wnt/β-catenin pathway in RBE4 cells via nuclear β-catenin signaling with LiCl does not alter brain endothelialMct1mRNA but increases the amount of MCT1 transporter protein. Plasma membrane biotinylation studies and confocal microscopic examination of mCherry-tagged MCT1 indicate that increased transporter results from reduced MCT1 trafficking from the plasma membrane via the endosomal/lysosomal pathway and is facilitated by decreased MCT1 ubiquitination following LiCl treatment. Inhibition of the Notch pathway by the γ-secretase inhibitorN-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester negated the up-regulation of MCT1 by LiCl, demonstrating a cross-talk between the canonical Wnt/β-catenin and Notch pathways. Our results are important because they show, for the first time, the regulation of MCT1 in cerebrovascular endothelial cells by the multifunctional canonical Wnt/β-catenin and Notch signaling pathways.

  12. Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia.

    PubMed

    Ye, Qi; Jiang, Jue; Zhan, Guanqun; Yan, Wanyao; Huang, Liang; Hu, Yufeng; Su, Hexiu; Tong, Qingyi; Yue, Ming; Li, Hua; Yao, Guangmin; Zhang, Yonghui; Liu, Hudan

    2016-01-01

    Aberrant activation of the NOTCH signaling pathway is crucial for the onset and progression of T cell leukemia. Yet recent studies also suggest a tumor suppressive role of NOTCH signaling in acute myeloid leukemia (AML) and reactivation of this pathway offers an attractive opportunity for anti-AML therapies. N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid that we previously isolated from Zephyranthes candida, exhibiting inhibitory activities in a variety of cancer cells, particularly those from AML. Here, we report NMHC not only selectively inhibits AML cell proliferation in vitro but also hampers tumor development in a human AML xenograft model. Genome-wide gene expression profiling reveals that NMHC activates the NOTCH signaling. Combination of NMHC and recombinant human NOTCH ligand DLL4 achieves a remarkable synergistic effect on NOTCH activation. Moreover, pre-inhibition of NOTCH by overexpression of dominant negative MAML alleviates NMHC-mediated cytotoxicity in AML. Further mechanistic analysis using structure-based molecular modeling as well as biochemical assays demonstrates that NMHC docks in the hydrophobic cavity within the NOTCH1 negative regulatory region (NRR), thus promoting NOTCH1 proteolytic cleavage. Our findings thus establish NMHC as a potential NOTCH agonist that holds great promises for future development as a novel agent beneficial to patients with AML. PMID:27211848

  13. Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia

    PubMed Central

    Ye, Qi; Jiang, Jue; Zhan, Guanqun; Yan, Wanyao; Huang, Liang; Hu, Yufeng; Su, Hexiu; Tong, Qingyi; Yue, Ming; Li, Hua; Yao, Guangmin; Zhang, Yonghui; Liu, Hudan

    2016-01-01

    Aberrant activation of the NOTCH signaling pathway is crucial for the onset and progression of T cell leukemia. Yet recent studies also suggest a tumor suppressive role of NOTCH signaling in acute myeloid leukemia (AML) and reactivation of this pathway offers an attractive opportunity for anti-AML therapies. N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid that we previously isolated from Zephyranthes candida, exhibiting inhibitory activities in a variety of cancer cells, particularly those from AML. Here, we report NMHC not only selectively inhibits AML cell proliferation in vitro but also hampers tumor development in a human AML xenograft model. Genome-wide gene expression profiling reveals that NMHC activates the NOTCH signaling. Combination of NMHC and recombinant human NOTCH ligand DLL4 achieves a remarkable synergistic effect on NOTCH activation. Moreover, pre-inhibition of NOTCH by overexpression of dominant negative MAML alleviates NMHC-mediated cytotoxicity in AML. Further mechanistic analysis using structure-based molecular modeling as well as biochemical assays demonstrates that NMHC docks in the hydrophobic cavity within the NOTCH1 negative regulatory region (NRR), thus promoting NOTCH1 proteolytic cleavage. Our findings thus establish NMHC as a potential NOTCH agonist that holds great promises for future development as a novel agent beneficial to patients with AML. PMID:27211848

  14. Notch signaling deregulation in multiple myeloma: A rational molecular target

    PubMed Central

    Garavelli, Silvia; Platonova, Natalia; Paoli, Alessandro; Basile, Andrea; Taiana, Elisa; Neri, Antonino; Chiaramonte, Raffaella

    2015-01-01

    Despite recent therapeutic advances, multiple myeloma (MM) is still an incurable neoplasia due to intrinsic or acquired resistance to therapy. Myeloma cell localization in the bone marrow milieu allows direct interactions between tumor cells and non-tumor bone marrow cells which promote neoplastic cell growth, survival, bone disease, acquisition of drug resistance and consequent relapse. Twenty percent of MM patients are at high-risk of treatment failure as defined by tumor markers or presentation as plasma cell leukemia. Cumulative evidences indicate a key role of Notch signaling in multiple myeloma onset and progression. Unlike other Notch-related malignancies, where the majority of patients carry gain-of-function mutations in Notch pathway members, in MM cell Notch signaling is aberrantly activated due to an increased expression of Notch receptors and ligands; notably, this also results in the activation of Notch signaling in surrounding stromal cells which contributes to myeloma cell proliferation, survival and migration, as well as to bone disease and intrinsic and acquired pharmacological resistance. Here we review the last findings on the mechanisms and the effects of Notch signaling dysregulation in MM and provide a rationale for a therapeutic strategy aiming at inhibiting Notch signaling, along with a complete overview on the currently available Notch-directed approaches. PMID:26308486

  15. A dual role for NOTCH signaling in joint cartilage maintenance and osteoarthritis.

    PubMed

    Liu, Zhaoyang; Chen, Jianquan; Mirando, Anthony J; Wang, Cuicui; Zuscik, Michael J; O'Keefe, Regis J; Hilton, Matthew J

    2015-07-21

    Loss of NOTCH signaling in postnatal murine joints results in osteoarthritis, indicating a requirement for NOTCH during maintenance of joint cartilage. However, NOTCH signaling components are substantially increased in abundance in posttraumatic osteoarthritis in humans and mice, suggesting either a reparative or a pathological role for NOTCH activation in osteoarthritis. We investigated a potential dual role for NOTCH in joint maintenance and osteoarthritis by generating two mouse models overexpressing the NOTCH1 intracellular domain (NICD) within postnatal joint cartilage. The first mouse model exhibited sustained NOTCH activation to resemble pathological NOTCH signaling, whereas the second model had transient NOTCH activation, which more closely reflected physiological NOTCH signaling. Sustained NOTCH signaling in joint cartilage led to an early and progressive osteoarthritic-like pathology, whereas transient NOTCH activation enhanced the synthesis of cartilage matrix and promoted joint maintenance under normal physiological conditions. Through RNA-sequencing, immunohistochemical, and biochemical approaches, we identified several targets that could be responsible for NOTCH-mediated cartilage degradation, fibrosis, and osteoarthritis progression. These targets included components of the interleukin-6 (IL-6)-signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase signaling pathways, which may also contribute to the posttraumatic development of osteoarthritis. Together, these data suggest a dual role for the NOTCH pathway in joint cartilage, and they identify downstream effectors of NOTCH signaling as potential targets for disease-modifying osteoarthritis drugs.

  16. Modulation of Notch signaling by antibodies specific for the extracellular negative regulatory region of NOTCH3.

    PubMed

    Li, Kang; Li, Yucheng; Wu, Wenjuan; Gordon, Wendy R; Chang, David W; Lu, Mason; Scoggin, Shane; Fu, Tihui; Vien, Long; Histen, Gavin; Zheng, Ji; Martin-Hollister, Rachel; Duensing, Thomas; Singh, Sanjaya; Blacklow, Stephen C; Yao, Zhengbin; Aster, Jon C; Zhou, Bin-Bing S

    2008-03-21

    The Notch pathway regulates the development of many tissues and cell types and is involved in a variety of human diseases, making it an attractive potential therapeutic target. This promise has been limited by the absence of potent inhibitors or agonists that are specific for individual human Notch receptors (NOTCH1-4). Using an unbiased functional screening, we identified monoclonal antibodies that specifically inhibit or induce activating proteolytic cleavages in NOTCH3. Remarkably, the most potent inhibitory and activating antibodies bind to overlapping epitopes within a juxtamembrane negative regulatory region that protects NOTCH3 from proteolysis and activation in its resting autoinhibited state. The inhibitory antibodies revert phenotypes conveyed on 293T cells by NOTCH3 signaling, such as increased cellular proliferation, survival, and motility, whereas the activating antibody mimics some of the effects of ligand-induced Notch activation. These findings provide insights into the mechanisms of Notch autoinhibition and activation and pave the way for the further development of specific antibody-based modulators of the Notch receptors, which are likely to be of utility in a wide range of experimental and therapeutic settings. PMID:18182388

  17. Genome-wide expression profiling in the Drosophila eye reveals unexpected repression of Notch signaling by the JAK/STAT pathway

    PubMed Central

    Flaherty, Maria Sol; Zavadil, Jiri; Ekas, Laura A.; Bach, Erika A.

    2010-01-01

    Although the JAK/STAT pathway regulates numerous processes in vertebrates and invertebrates through modulating transcription, its functionally-relevant transcriptional targets remain largely unknown. With one jak and one stat (stat92E), Drosophila provides a powerful system for finding new JAK/STAT target genes. Genome-wide expression profiling on eye discs in which Stat92E is hyperactivated, revealed 584 differentially-regulated genes, including known targets domeless, socs36E and wingless. Other differentially-regulated genes (chinmo, lama, Mo25, Imp-L2, Serrate, Delta) were validated and may represent new Stat92E targets. Genetic experiments revealed that Stat92E cell-autonomously represses Serrate, which encodes a Notch ligand. Loss of Stat92E led to de-repression of Serrate in the dorsal eye, resulting in ectopic Notch signaling and aberrant eye growth there. Thus, our micro-array documents a new Stat92E target gene and a previously-unidentified inhibitory action of Stat92E on Notch signaling. These data suggest that this study will be a useful resource for the identification of additional Stat92E targets. PMID:19504457

  18. The Wnt and Delta-Notch signalling pathways interact to direct pair-rule gene expression via caudal during segment addition in the spider Parasteatoda tepidariorum.

    PubMed

    Schönauer, Anna; Paese, Christian L B; Hilbrant, Maarten; Leite, Daniel J; Schwager, Evelyn E; Feitosa, Natália Martins; Eibner, Cornelius; Damen, Wim G M; McGregor, Alistair P

    2016-07-01

    In short-germ arthropods, posterior segments are added sequentially from a segment addition zone (SAZ) during embryogenesis. Studies in spiders such as Parasteatoda tepidariorum have provided insights into the gene regulatory network (GRN) underlying segment addition, and revealed that Wnt8 is required for dynamic Delta (Dl) expression associated with the formation of new segments. However, it remains unclear how these pathways interact during SAZ formation and segment addition. Here, we show that Delta-Notch signalling is required for Wnt8 expression in posterior SAZ cells, but represses the expression of this Wnt gene in anterior SAZ cells. We also found that these two signalling pathways are required for the expression of the spider orthologues of even-skipped (eve) and runt-1 (run-1), at least in part via caudal (cad). Moreover, it appears that dynamic expression of eve in this spider does not require a feedback loop with run-1, as is found in the pair-rule circuit of the beetle Tribolium Taken together, our results suggest that the development of posterior segments in Parasteatoda is directed by dynamic interactions between Wnt8 and Delta-Notch signalling that are read out by cad, which is necessary but probably not sufficient to regulate the expression of eve and run-1 Our study therefore provides new insights towards better understanding the evolution and developmental regulation of segmentation in other arthropods, including insects. PMID:27287802

  19. The Wnt and Delta-Notch signalling pathways interact to direct pair-rule gene expression via caudal during segment addition in the spider Parasteatoda tepidariorum.

    PubMed

    Schönauer, Anna; Paese, Christian L B; Hilbrant, Maarten; Leite, Daniel J; Schwager, Evelyn E; Feitosa, Natália Martins; Eibner, Cornelius; Damen, Wim G M; McGregor, Alistair P

    2016-07-01

    In short-germ arthropods, posterior segments are added sequentially from a segment addition zone (SAZ) during embryogenesis. Studies in spiders such as Parasteatoda tepidariorum have provided insights into the gene regulatory network (GRN) underlying segment addition, and revealed that Wnt8 is required for dynamic Delta (Dl) expression associated with the formation of new segments. However, it remains unclear how these pathways interact during SAZ formation and segment addition. Here, we show that Delta-Notch signalling is required for Wnt8 expression in posterior SAZ cells, but represses the expression of this Wnt gene in anterior SAZ cells. We also found that these two signalling pathways are required for the expression of the spider orthologues of even-skipped (eve) and runt-1 (run-1), at least in part via caudal (cad). Moreover, it appears that dynamic expression of eve in this spider does not require a feedback loop with run-1, as is found in the pair-rule circuit of the beetle Tribolium Taken together, our results suggest that the development of posterior segments in Parasteatoda is directed by dynamic interactions between Wnt8 and Delta-Notch signalling that are read out by cad, which is necessary but probably not sufficient to regulate the expression of eve and run-1 Our study therefore provides new insights towards better understanding the evolution and developmental regulation of segmentation in other arthropods, including insects.

  20. Notch1 Pathway Protects against Burn-Induced Myocardial Injury by Repressing Reactive Oxygen Species Production through JAK2/STAT3 Signaling

    PubMed Central

    Cai, Weixia; Yang, Xuekang; Han, Shichao; Guo, Haitao; Zheng, Zhao; Wang, Hongtao; Guan, Hao; Jia, Yanhui; Gao, Jianxin; Yang, Tao; Zhu, Xiongxiang; Hu, Dahai

    2016-01-01

    Oxidative stress plays an important role in burn-induced myocardial injury, but the cellular mechanisms that control reactive oxygen species (ROS) production and scavenging are not fully understood. This study demonstrated that blockade of Notch signaling via knockout of the transcription factor RBP-J or a pharmacological inhibitor aggravated postburn myocardial injury, which manifested as deteriorated serum CK, CK-MB, and LDH levels and increased apoptosis in vitro and in vivo. Interruption of Notch signaling increased intracellular ROS production, and a ROS scavenger reversed the exacerbated myocardial injury after Notch signaling blockade. These results suggest that Notch signaling deficiency aggravated postburn myocardial injury through increased ROS levels. Notch signaling blockade also decreased MnSOD expression in vitro and in vivo. Notably, Notch signaling blockade downregulated p-JAK2 and p-STAT3 expression. Inhibition of JAK2/STAT3 signaling with AG490 markedly decreased MnSOD expression, increased ROS production, and aggravated myocardial injury. AG490 plus GSI exerted no additional effects. These results demonstrate that Notch signaling protects against burn-induced myocardial injury through JAK2/STAT3 signaling, which activates the expression of MnSOD and leads to decreased ROS levels. PMID:27057278

  1. Notch3 signaling promotes the development of pulmonary arterial hypertension.

    PubMed

    Li, Xiaodong; Zhang, Xiaoxue; Leathers, Robin; Makino, Ayako; Huang, Chengqun; Parsa, Pouria; Macias, Jesus; Yuan, Jason X-J; Jamieson, Stuart W; Thistlethwaite, Patricia A

    2009-11-01

    Notch receptor signaling is implicated in controlling smooth muscle cell proliferation and in maintaining smooth muscle cells in an undifferentiated state. Pulmonary arterial hypertension is characterized by excessive vascular resistance, smooth muscle cell proliferation in small pulmonary arteries, leading to elevation of pulmonary vascular resistance, right ventricular failure and death. Here we show that human pulmonary hypertension is characterized by overexpression of NOTCH3 in small pulmonary artery smooth muscle cells and that the severity of disease in humans and rodents correlates with the amount of NOTCH3 protein in the lung. We further show that mice with homozygous deletion of Notch3 do not develop pulmonary hypertension in response to hypoxic stimulation and that pulmonary hypertension can be successfully treated in mice by administration of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase inhibitor that blocks activation of Notch3 in smooth muscle cells. We show a mechanistic link from NOTCH3 receptor signaling through the Hairy and enhancer of Split-5 (HES-5) protein to smooth muscle cell proliferation and a shift to an undifferentiated smooth muscle cell phenotype. These results suggest that the NOTCH3-HES-5 signaling pathway is crucial for the development of pulmonary arterial hypertension and provide a target pathway for therapeutic intervention. PMID:19855400

  2. NUMB is a break of WNT-Notch signaling cycle.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2006-09-01

    Notch, FGF and WNT signaling pathways cross-talk during embryogenesis, tissue regeneration and carcinogenesis. Notch-ligand binding to Notch receptors leads to the cleavage of Notch receptors and the following nuclear translocation of Notch intracellular domain (NICD) to induce transcriptional activation of Notch target genes. Notch signaling inhibitors, NUMB and NUMB-like (NUMBL), are docking proteins with PTB domain. We searched for the TCF/LEF-binding site within the promoter region of NUMB and NUMBL genes. Because two TCF/LEF-binding sites were identified within human NUMB promoter based on bioinformatics and human intelligence (Humint), comparative integromics analyses on NUMB orthologs were further performed. Chimpanzee NUBM gene, consisting of 13 exons, was identified within NW_115880.1 genome sequence. XM_510045.1 was not the correct coding sequence for chimpanzee NUMB. Chimpanzee NUMB gene was found to encode a 651-amino-acid protein showing 99.5, 93.9 and 82.6% total-amino-acid identity with human NUMB, mouse Numb and chicken numb, respectively. Human NUMB mRNA was expressed in placenta, ES cells, neural tissues, trachea, testis, uterus, thymus, coronary artery as well as in a variety of tumors, such as cervical cancer, tong tumor, brain tumor, colorectal and breast cancer. Although distal TCF/LEF-binding site within human NUMB promoter was conserved only among primate NUMB orthologs, proximal TCF/LEF-binding site was conserved among primate and rodent NUMB orthologs. NUMB, JAG1, FGF18, FGF20 and SPRY4 are potent targets of the canonical WNT signaling pathway in progenitor cells. NUMB inhibits Notch signaling in progenitor cells to induce differentiation, while JAG1 activates Notch signaling in stem cells to maintain self-renewal potential. Because Notch signaling inhibitor NUMB was identified as the safe apparatus for the WNT - Notch signaling cycle, epigenetic silencing, deletion and loss-of-function mutation of NUMB gene could lead to carcinogenesis

  3. A role of the LIN-12/Notch signaling pathway in diversifying the non-striated egg-laying muscles in C. elegans.

    PubMed

    Hale, Jared J; Amin, Nirav M; George, Carolyn; Via, Zachary; Shi, Herong; Liu, Jun

    2014-05-15

    The proper formation and function of an organ is dependent on the specification and integration of multiple cell types and tissues. An example of this is the Caenorhabditis elegans hermaphrodite egg-laying system, which requires coordination between the vulva, uterus, neurons, and musculature. While the genetic constituents of the first three components have been well studied, little is known about the molecular mechanisms underlying the specification of the egg-laying musculature. The egg-laying muscles are non-striated in nature and consist of sixteen cells, four each of type I and type II vulval muscles and uterine muscles. These 16 non-striated muscles exhibit distinct morphology, location, synaptic connectivity and function. Using an RNAi screen targeting the putative transcription factors in the C. elegans genome, we identified a number of novel factors important for the diversification of these different types of egg-laying muscles. In particular, we found that RNAi knockdown of lag-1, which encodes the sole C. elegans ortholog of the transcription factor CSL (CBF1, Suppressor of Hairless, LAG-1), an effector of the LIN-12/Notch pathway, led to the production of extra type I vulval muscles. Similar phenotypes were also observed in animals with down-regulation of the Notch receptor LIN-12 and its DSL (Delta, Serrate, LAG-2) ligand LAG-2. The extra type I vulval muscles in animals with reduced LIN-12/Notch signaling resulted from a cell fate transformation of type II vulval muscles to type I vulval muscles. We showed that LIN-12/Notch was activated in the undifferentiated type II vulval muscle cells by LAG-2/DSL that is likely produced by the anchor cell (AC). Our findings provide additional evidence highlighting the roles of LIN-12/Notch signaling in coordinating the formation of various components of the functional C. elegans egg-laying system. We also identify multiple new factors that play critical roles in the proper specification of the different types

  4. Notch signalling mediates reproductive constraint in the adult worker honeybee

    PubMed Central

    Duncan, Elizabeth J.; Hyink, Otto; Dearden, Peter K.

    2016-01-01

    The hallmark of eusociality is the reproductive division of labour, in which one female caste reproduces, while reproduction is constrained in the subordinate caste. In adult worker honeybees (Apis mellifera) reproductive constraint is conditional: in the absence of the queen and brood, adult worker honeybees activate their ovaries and lay haploid male eggs. Here, we demonstrate that chemical inhibition of Notch signalling can overcome the repressive effect of queen pheromone and promote ovary activity in adult worker honeybees. We show that Notch signalling acts on the earliest stages of oogenesis and that the removal of the queen corresponds with a loss of Notch protein in the germarium. We conclude that the ancient and pleiotropic Notch signalling pathway has been co-opted into constraining reproduction in worker honeybees and we provide the first molecular mechanism directly linking ovary activity in adult worker bees with the presence of the queen. PMID:27485026

  5. Notch signalling mediates reproductive constraint in the adult worker honeybee.

    PubMed

    Duncan, Elizabeth J; Hyink, Otto; Dearden, Peter K

    2016-01-01

    The hallmark of eusociality is the reproductive division of labour, in which one female caste reproduces, while reproduction is constrained in the subordinate caste. In adult worker honeybees (Apis mellifera) reproductive constraint is conditional: in the absence of the queen and brood, adult worker honeybees activate their ovaries and lay haploid male eggs. Here, we demonstrate that chemical inhibition of Notch signalling can overcome the repressive effect of queen pheromone and promote ovary activity in adult worker honeybees. We show that Notch signalling acts on the earliest stages of oogenesis and that the removal of the queen corresponds with a loss of Notch protein in the germarium. We conclude that the ancient and pleiotropic Notch signalling pathway has been co-opted into constraining reproduction in worker honeybees and we provide the first molecular mechanism directly linking ovary activity in adult worker bees with the presence of the queen. PMID:27485026

  6. Notch pathway activation targets AML-initiating cell homeostasis and differentiation

    PubMed Central

    Ntziachristos, Panagiotis; Ndiaye-Lobry, Delphine; Oh, Philmo; Cimmino, Luisa; Zhu, Nan; Araldi, Elisa; Hu, Wenhuo; Freund, Jacquelyn; Abdel-Wahab, Omar; Ibrahim, Sherif; Skokos, Dimitris; Armstrong, Scott A.; Levine, Ross L.; Park, Christopher Y.

    2013-01-01

    Notch signaling pathway activation is known to contribute to the pathogenesis of a spectrum of human malignancies, including T cell leukemia. However, recent studies have implicated the Notch pathway as a tumor suppressor in myeloproliferative neoplasms and several solid tumors. Here we report a novel tumor suppressor role for Notch signaling in acute myeloid leukemia (AML) and demonstrate that Notch pathway activation could represent a therapeutic strategy in this disease. We show that Notch signaling is silenced in human AML samples, as well as in AML-initiating cells in an animal model of the disease. In vivo activation of Notch signaling using genetic Notch gain of function models or in vitro using synthetic Notch ligand induces rapid cell cycle arrest, differentiation, and apoptosis of AML-initiating cells. Moreover, we demonstrate that Notch inactivation cooperates in vivo with loss of the myeloid tumor suppressor Tet2 to induce AML-like disease. These data demonstrate a novel tumor suppressor role for Notch signaling in AML and elucidate the potential therapeutic use of Notch receptor agonists in the treatment of this devastating leukemia. PMID:23359070

  7. PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner.

    PubMed

    Sjöqvist, Marika; Antfolk, Daniel; Ferraris, Saima; Rraklli, Vilma; Haga, Cecilia; Antila, Christian; Mutvei, Anders; Imanishi, Susumu Y; Holmberg, Johan; Jin, Shaobo; Eriksson, John E; Lendahl, Urban; Sahlgren, Cecilia

    2014-04-01

    Activation of Notch signaling requires intracellular routing of the receptor, but the mechanisms controlling the distinct steps in the routing process is poorly understood. We identify PKCζ as a key regulator of Notch receptor intracellular routing. When PKCζ was inhibited in the developing chick central nervous system and in cultured myoblasts, Notch-stimulated cells were allowed to undergo differentiation. PKCζ phosphorylates membrane-tethered forms of Notch and regulates two distinct routing steps, depending on the Notch activation state. When Notch is activated, PKCζ promotes re-localization of Notch from late endosomes to the nucleus and enhances production of the Notch intracellular domain, which leads to increased Notch activity. In the non-activated state, PKCζ instead facilitates Notch receptor internalization, accompanied with increased ubiquitylation and interaction with the endosomal sorting protein Hrs. Collectively, these data identify PKCζ as a key regulator of Notch trafficking and demonstrate that distinct steps in intracellular routing are differentially modulated depending on Notch signaling status.

  8. From Fly Wings to Targeted Cancer Therapies: A Centennial for Notch Signaling

    PubMed Central

    Ntziachristos, Panagiotis; Lim, Jing Shan; Sage, Julien; Aifantis, Iannis

    2014-01-01

    Since Notch phenotypes in Drosophila melanogaster were identified 100 years, Notch signaling has been extensively characterized as a regulator of cell fate decisions in a variety of organisms and tissues. However, in the past 20 years, accumulating evidence has linked alterations in the Notch pathway to tumorigenesis. In this Perspective, we discuss the pro-tumorigenic and tumor suppressive functions of Notch signaling and dissect the molecular mechanisms that underlie these functions in hematopoietic cancers and solid tumors. Finally, we link these mechanisms and observations to possible therapeutic strategies targeting the Notch pathway in human cancers. PMID:24651013

  9. Notch signaling: switching an oncogene to a tumor suppressor

    PubMed Central

    Lobry, Camille; Oh, Philmo; Mansour, Marc R.; Look, A. Thomas

    2014-01-01

    The Notch signaling pathway is a regulator of self-renewal and differentiation in several tissues and cell types. Notch is a binary cell-fate determinant, and its hyperactivation has been implicated as oncogenic in several cancers including breast cancer and T-cell acute lymphoblastic leukemia (T-ALL). Recently, several studies also unraveled tumor-suppressor roles for Notch signaling in different tissues, including tissues where it was before recognized as an oncogene in specific lineages. Whereas involvement of Notch as an oncogene in several lymphoid malignancies (T-ALL, B-chronic lymphocytic leukemia, splenic marginal zone lymphoma) is well characterized, there is growing evidence involving Notch signaling as a tumor suppressor in myeloid malignancies. It therefore appears that Notch signaling pathway’s oncogenic or tumor-suppressor abilities are highly context dependent. In this review, we summarize and discuss latest advances in the understanding of this dual role in hematopoiesis and the possible consequences for the treatment of hematologic malignancies. PMID:24608975

  10. Cellular Notch responsiveness is defined by phosphoinositide 3-kinase-dependent signals

    PubMed Central

    Mckenzie, Grahame; Ward, George; Stallwood, Yvette; Briend, Emmanuel; Papadia, Sofia; Lennard, Andrew; Turner, Martin; Champion, Brian; Hardingham, Giles E

    2006-01-01

    Background Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways. Results We show that cellular responsiveness to Notch signals depends on the activity of the PI3K-Akt pathway in cells as diverse as CHO cells, primary T-cells and hippocampal neurons. Induction of the endogenous PI3K-Akt pathway in CHO cells (by the insulin pathway), in T-cells (via TCR activation) or in neurons (via TrKB activation) potentiates Notch-dependent responses. We propose that the PI3K-Akt pathway exerts its influence on Notch primarily via inhibition of GSK3-beta, a kinase known to phosphorylate and regulate Notch signals. Conclusion The PI3K-Akt pathway acts as a "gain control" for Notch signal responses. Since physiological levels of intracellular Notch are often low, coincidence with PI3K-activation may be crucial for induction of Notch-dependent responses. PMID:16507111

  11. Notch1 endocytosis is induced by ligand and is required for signal transduction.

    PubMed

    Chapman, G; Major, J A; Iyer, K; James, A C; Pursglove, S E; Moreau, J L M; Dunwoodie, S L

    2016-01-01

    The Notch signalling pathway is widely utilised during embryogenesis in situations where cell-cell interactions are important for cell fate specification and differentiation. DSL ligand endocytosis into the ligand-expressing cell is an important aspect of Notch signalling because it is thought to supply the force needed to separate the Notch heterodimer to initiate signal transduction. A functional role for receptor endocytosis during Notch signal transduction is more controversial. Here we have used live-cell imaging to examine trafficking of the Notch1 receptor in response to ligand binding. Contact with cells expressing ligands induced internalisation and intracellular trafficking of Notch1. Notch1 endocytosis was accompanied by transendocytosis of ligand into the Notch1-expressing signal-receiving cell. Ligand caused Notch1 endocytosis into SARA-positive endosomes in a manner dependent on clathrin and dynamin function. Moreover, inhibition of endocytosis in the receptor-expressing cell impaired ligand-induced Notch1 signalling. Our findings resolve conflicting observations from mammalian and Drosophila studies by demonstrating that ligand-dependent activation of Notch1 signalling requires receptor endocytosis. Endocytosis of Notch1 may provide a force on the ligand:receptor complex that is important for potent signal transduction.

  12. Characterization of Notch Signaling During Osteogenic Differentiation in Human Osteosarcoma Cell Line MG63.

    PubMed

    Ongaro, Alessia; Pellati, Agnese; Bagheri, Leila; Rizzo, Paola; Caliceti, Cristiana; Massari, Leo; De Mattei, Monica

    2016-12-01

    Osteogenic differentiation is a multi-step process controlled by a complex molecular framework. Notch is an evolutionarily conserved intercellular signaling pathway playing a prominent role in cell fate and differentiation, although the mechanisms by which this pathway regulates osteogenesis remain controversial. This study aimed to investigate, in vitro, the involvement of Notch pathway during all the developmental stages of osteogenic differentiation in human osteosarcoma cell line MG63. Cells were cultured in basal condition (control) and in osteoinductive medium (OM). Notch inhibitors were also added in OM to block Notch pathway. During osteogenic differentiation, early (alkaline phosphatase activity and collagen type I) and late osteogenic markers (osteocalcin levels and matrix mineralization), as well as the gene expression of the main osteogenic transcription factors (Runx2, Osterix, and Dlx5) increased. Time dependent changes in the expression of specific Notch receptors were identified in OM versus control with a significant reduction in the expression of Notch1 and Notch3 receptors in the early phase of differentiation, and an increase of Notch2 and Notch4 receptors in the late phase. Among Notch nuclear target genes, Hey1 expression was significantly higher in OM than control, while Hes5 expression decreased. Osteogenic markers were reduced and Hey1 was significantly inhibited by Notch inhibitors, suggesting a role for Notch through the canonical pathway. In conclusion, Notch pathway might be involved with a dual role in osteogenesis of MG63, through the activation of Notch2, Notch4, and Hey1, inducing osteoblast differentiation and the depression of Notch1, Notch3, and Hes5, maintaining an undifferentiated status. J. Cell. Physiol. 231: 2652-2663, 2016. © 2016 Wiley Periodicals, Inc. PMID:26946465

  13. Notch1 signaling stimulates proliferation of immature cardiomyocytes.

    PubMed

    Collesi, Chiara; Zentilin, Lorena; Sinagra, Gianfranco; Giacca, Mauro

    2008-10-01

    The identification of the molecular mechanisms controlling cardiomyocyte proliferation during the embryonic, fetal, and early neonatal life appears of paramount interest in regard to exploiting this information to promote cardiac regeneration. Here, we show that the proliferative potential of neonatal rat cardiomyocytes is powerfully stimulated by the sustained activation of the Notch pathway. We found that Notch1 is expressed in proliferating ventricular immature cardiac myocytes (ICMs) both in vitro and in vivo, and that the number of Notch1-positive cells in the heart declines with age. Notch1 expression in ICMs paralleled the expression of its Jagged1 ligand on non-myocyte supporting cells. The inhibition of Notch signaling in ICMs blocked their proliferation and induced apoptosis; in contrast, its activation by Jagged1 or by the constitutive expression of its activated form using an adeno-associated virus markedly stimulated proliferative signaling and promoted ICM expansion. Maintenance or reactivation of Notch signaling in cardiac myocytes might represent an interesting target for innovative regenerative therapy. PMID:18824567

  14. Activation of Notch1 signaling is required for β-catenin–mediated human primary melanoma progression

    PubMed Central

    Balint, Klara; Xiao, Min; Pinnix, Chelsea C.; Soma, Akinobu; Veres, Imre; Juhasz, Istvan; Brown, Eric J.; Capobianco, Anthony J.; Herlyn, Meenhard; Liu, Zhao-Jun

    2005-01-01

    Notch is a highly conserved transmembrane receptor that determines cell fate. Notch signaling denotes cleavage of the Notch intracellular domain, its translocation to the nucleus, and subsequent activation of target gene transcription. Involvement of Notch signaling in several cancers is well known, but its role in melanoma remains poorly characterized. Here we show that the Notch1 pathway is activated in human melanoma. Blocking Notch signaling suppressed whereas constitutive activation of the Notch1 pathway enhanced primary melanoma cell growth both in vitro and in vivo yet had little effect on metastatic melanoma cells. Activation of Notch1 signaling enabled primary melanoma cells to gain metastatic capability. Furthermore, the oncogenic effect of Notch1 on primary melanoma cells was mediated by β-catenin, which was upregulated following Notch1 activation. Inhibiting β-catenin expression reversed Notch1-enhanced tumor growth and metastasis. Our data therefore suggest a β-catenin–dependent, stage-specific role for Notch1 signaling in promoting the progression of primary melanoma. PMID:16239965

  15. Notch signaling in mammalian hair cell regeneration

    PubMed Central

    Slowik, Amber D.; Bermingham-McDonogh, Olivia

    2014-01-01

    In the inner ear, Notch signaling has been shown to have two key developmental roles. The first occurs early in otic development and defines the prosensory domains that will develop into the six sensory organs of the inner ear. The second role occurs later in development and establishes the mosaic-like pattern of the mechanosensory hair cells and their surrounding support cells through the more well-characterized process of lateral inhibition. These dual developmental roles have inspired several different strategies to regenerate hair cells in the mature inner ear organs. These strategies include (1) modulation of Notch signaling in inner ear stem cells in order to increase hair cell yield, (2) activation of Notch signaling in order to promote the formation of ectopic sensory regions in normally non-sensory regions within the inner ear, and (3) inhibition of Notch signaling to disrupt lateral inhibition and allow support cells to transdifferentiate into hair cells. In this review, we summarize some of the promising studies that have used these various strategies for hair cell regeneration through modulation of Notch signaling and some of the challenges that remain in developing therapies based on hair cell regeneration. PMID:25328289

  16. NOTCH signaling in skeletal progenitors is critical for fracture repair

    PubMed Central

    Wang, Cuicui; Inzana, Jason A.; Mirando, Anthony J.; Liu, Zhaoyang; Shen, Jie; O’Keefe, Regis J.; Awad, Hani A.; Hilton, Matthew J.

    2016-01-01

    Fracture nonunions develop in 10%–20% of patients with fractures, resulting in prolonged disability. Current data suggest that bone union during fracture repair is achieved via proliferation and differentiation of skeletal progenitors within periosteal and soft tissues surrounding bone, while bone marrow stromal/stem cells (BMSCs) and other skeletal progenitors may also contribute. The NOTCH signaling pathway is a critical maintenance factor for BMSCs during skeletal development, although the precise role for NOTCH and the requisite nature of BMSCs following fracture is unknown. Here, we evaluated whether NOTCH and/or BMSCs are required for fracture repair by performing nonstabilized and stabilized fractures on NOTCH-deficient mice with targeted deletion of RBPjk in skeletal progenitors, maturing osteoblasts, and committed chondrocytes. We determined that removal of NOTCH signaling in BMSCs and subsequent depletion of this population result in fracture nonunion, as the fracture repair process was normal in animals harboring either osteoblast- or chondrocyte-specific deletion of RBPjk. Together, this work provides a genetic model of a fracture nonunion and demonstrates the requirement for NOTCH and BMSCs in fracture repair, irrespective of fracture stability and vascularity. PMID:26950423

  17. Role of Wnt and Notch signaling in regulating hair cell regeneration in the cochlea.

    PubMed

    Waqas, Muhammad; Zhang, Shasha; He, Zuhong; Tang, Mingliang; Chai, Renjie

    2016-09-01

    Sensory hair cells in the inner ear are responsible for sound recognition. Damage to hair cells in adult mammals causes permanent hearing impairment because these cells cannot regenerate. By contrast, newborn mammals possess limited regenerative capacity because of the active participation of various signaling pathways, including Wnt and Notch signaling. The Wnt and Notch pathways are highly sophisticated and conserved signaling pathways that control multiple cellular events necessary for the formation of sensory hair cells. Both signaling pathways allow resident supporting cells to regenerate hair cells in the neonatal cochlea. In this regard, Wnt and Notch signaling has gained increased research attention in hair cell regeneration. This review presents the current understanding of the Wnt and Notch signaling pathways in the auditory portion of the inner ear and discusses the possibilities of controlling these pathways with the hair cell fate determiner Atoh1 to regulate hair cell regeneration in the mammalian cochlea.

  18. Role of Wnt and Notch signaling in regulating hair cell regeneration in the cochlea.

    PubMed

    Waqas, Muhammad; Zhang, Shasha; He, Zuhong; Tang, Mingliang; Chai, Renjie

    2016-09-01

    Sensory hair cells in the inner ear are responsible for sound recognition. Damage to hair cells in adult mammals causes permanent hearing impairment because these cells cannot regenerate. By contrast, newborn mammals possess limited regenerative capacity because of the active participation of various signaling pathways, including Wnt and Notch signaling. The Wnt and Notch pathways are highly sophisticated and conserved signaling pathways that control multiple cellular events necessary for the formation of sensory hair cells. Both signaling pathways allow resident supporting cells to regenerate hair cells in the neonatal cochlea. In this regard, Wnt and Notch signaling has gained increased research attention in hair cell regeneration. This review presents the current understanding of the Wnt and Notch signaling pathways in the auditory portion of the inner ear and discusses the possibilities of controlling these pathways with the hair cell fate determiner Atoh1 to regulate hair cell regeneration in the mammalian cochlea. PMID:27527363

  19. Roles of Pofut1 and O-fucose in mammalian Notch signaling.

    PubMed

    Stahl, Mark; Uemura, Kazuhide; Ge, Changhui; Shi, Shaolin; Tashima, Yuko; Stanley, Pamela

    2008-05-16

    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.

  20. Nas transgenic mouse line allows visualization of Notch pathway activity in vivo

    PubMed Central

    Souilhol, Céline; Cormier, Sarah; Monet, Marie; Vandormael-Pournin, Sandrine; Joutel, Anne; Babinet, Charles; Cohen-Tannoudji, Michel

    2006-01-01

    The Notch signalling pathway plays multiple and important roles in mammals. However, several aspects of its action, in particular the precise mapping of its sites of activity, remain unclear. To address this issue, we have generated a transgenic line carrying a construct consisting of a nls-lacZ reporter gene under the control of a minimal promoter and multiple RBP-Jκ binding sites. Here we show that this transgenic line, we named NAS for Notch Activity Sensor, displays an expression profile that is consistent with current knowledge on Notch activity sites in mice, even though it may not report on all these sites. Moreover, we observe that NAS transgene expression is abolished in a RBP-Jκ deficient background indicating that it indeed requires Notch/RBP-Jκ signalling pathway activity. Thus, the NAS transgenic line constitutes a valuable and versatile tool to gain further insights into the complex and various functions of the Notch signalling pathway. PMID:16708386

  1. Notch-1 Signalling Is Activated in Brain Arteriovenous Malformations in Humans

    ERIC Educational Resources Information Center

    ZhuGe, Qichuan; Zhong, Ming; Zheng, WeiMing; Yang, Guo-Yuan; Mao, XiaoOu; Xie, Lin; Chen, Gourong; Chen, Yongmei; Lawton, Michael T.; Young, William L.; Greenberg, David A.; Jin, Kunlin

    2009-01-01

    A role for the Notch signalling pathway in the formation of arteriovenous malformations during development has been suggested. However, whether Notch signalling is involved in brain arteriovenous malformations in humans remains unclear. Here, we performed immunohistochemistry on surgically resected brain arteriovenous malformations and found that,…

  2. Notch signaling represses GATA4-induced expression of genes involved in steroid biosynthesis

    PubMed Central

    George, Rajani M.; Hahn, Katherine L.; Rawls, Alan; Viger, Robert S.; Wilson-Rawls, Jeanne

    2015-01-01

    Notch2 and Notch3 and genes of the Notch signaling network are dynamically expressed in developing follicles, where they are essential for granulosa cell proliferation and meiotic maturation. Notch receptors, ligands, and downstream effector genes are also expressed in testicular Leydig cells, predicting a potential role in regulating steroidogenesis. In this study, we sought to determine if Notch signaling in small follicles regulates the proliferation response of granulosa cells to follicle stimulating hormone and represses the up-regulation steroidogenic gene expression that occurs in response to FSH as the follicle grows. Inhibition of Notch signaling in small preantral follicles led to the up-regulation of the expression of genes in the steroid biosynthetic pathway. Similarly, progesterone secretion by MA-10 Leydig cells was significantly inhibited by constitutively active Notch. Together, these data indicated that Notch signaling inhibits steroidogenesis. GATA4 has been shown to be a positive regulator of steroidogenic genes, including steroidogenic acute regulatory protein, P450 aromatase, and 3B-hydroxysteroid dehydrogenase. We observed that Notch downstream effectors HEY1, HEY2, and HEYL are able to differentially regulate these GATA4-dependent promoters. These data are supported by the presence of HEY/HES binding sites in these promoters. These studies indicate that Notch signaling has a role in the complex regulation of the steroidogenic pathway. PMID:26183893

  3. Inhibition of Notch signaling pathway attenuates sympathetic hyperinnervation together with the augmentation of M2 macrophages in rats post-myocardial infarction.

    PubMed

    Yin, Jie; Hu, Hesheng; Li, Xiaolu; Xue, Mei; Cheng, Wenjuan; Wang, Ye; Xuan, Yongli; Li, Xinran; Yang, Na; Shi, Yugen; Yan, Suhua

    2016-01-01

    Inflammation-dominated sympathetic sprouting adjacent to the necrotic region following myocardial infarction (MI) has been implicated in the etiology of arrhythmias resulting in sudden cardiac death; however, the mechanisms responsible remain to be elucidated. Although being a key immune mediator, the role of Notch has yet to be explored. We investigated whether Notch regulates macrophage responses to inflammation and affects cardiac sympathetic reinnervation in rats undergoing MI. MI was induced by coronary artery ligation. A high level of Notch intracellular domain was observed in the macrophages that infiltrated the infarct area at 3 days post-MI. The administration of the Notch inhibitor N-N-(3,5-difluorophenacetyl-L-alanyl)-S-phenylglycine-t-butyl ester (DAPT) (intravenously 30 min before MI and then daily until death) decreased the number of macrophages and significantly increased the M2 macrophage activation profile in the early stages and attenuated the expression of nerve growth factor (NGF). Eventually, NGF-induced sympathetic hyperinnervation was blunted, as assessed by the immunofluorescence of tyrosine hydroxylase. At 7 days post-MI, the arrhythmia score of programmed electric stimulation in the vehicle-treated infarcted rats was higher than that in rats treated with DAPT. Further deterioration in cardiac function and decreases in the plasma levels of TNF-α and IL-1β were also detected. In vitro studies revealed that LPS/IFN-γ upregulated the surface expression of NGF in M1 macrophages in a Notch-dependent manner. We concluded that Notch inhibition during the acute inflammatory response phase is associated with the downregulation of NGF, probably through a macrophage-dependent pathway, thus preventing the process of sympathetic hyperinnervation.

  4. Inhibition of Notch signaling pathway attenuates sympathetic hyperinnervation together with the augmentation of M2 macrophages in rats post-myocardial infarction.

    PubMed

    Yin, Jie; Hu, Hesheng; Li, Xiaolu; Xue, Mei; Cheng, Wenjuan; Wang, Ye; Xuan, Yongli; Li, Xinran; Yang, Na; Shi, Yugen; Yan, Suhua

    2016-01-01

    Inflammation-dominated sympathetic sprouting adjacent to the necrotic region following myocardial infarction (MI) has been implicated in the etiology of arrhythmias resulting in sudden cardiac death; however, the mechanisms responsible remain to be elucidated. Although being a key immune mediator, the role of Notch has yet to be explored. We investigated whether Notch regulates macrophage responses to inflammation and affects cardiac sympathetic reinnervation in rats undergoing MI. MI was induced by coronary artery ligation. A high level of Notch intracellular domain was observed in the macrophages that infiltrated the infarct area at 3 days post-MI. The administration of the Notch inhibitor N-N-(3,5-difluorophenacetyl-L-alanyl)-S-phenylglycine-t-butyl ester (DAPT) (intravenously 30 min before MI and then daily until death) decreased the number of macrophages and significantly increased the M2 macrophage activation profile in the early stages and attenuated the expression of nerve growth factor (NGF). Eventually, NGF-induced sympathetic hyperinnervation was blunted, as assessed by the immunofluorescence of tyrosine hydroxylase. At 7 days post-MI, the arrhythmia score of programmed electric stimulation in the vehicle-treated infarcted rats was higher than that in rats treated with DAPT. Further deterioration in cardiac function and decreases in the plasma levels of TNF-α and IL-1β were also detected. In vitro studies revealed that LPS/IFN-γ upregulated the surface expression of NGF in M1 macrophages in a Notch-dependent manner. We concluded that Notch inhibition during the acute inflammatory response phase is associated with the downregulation of NGF, probably through a macrophage-dependent pathway, thus preventing the process of sympathetic hyperinnervation. PMID:26491050

  5. Gliolectin positively regulates Notch signalling during wing-vein specification in Drosophila.

    PubMed

    Prasad, Naveen; Shashidhara, Lingadahalli S

    2015-01-01

    Notch signalling is essential for animal development. It integrates multiple pathways controlling cell fate and specification. Here we report the genetic characterization of Gliolectin, presumably a lectin, a cytoplasmic protein, significantly enriched in Golgi bodies. Its expression overlaps with regions where Notch is activated. Loss of gliolectin function results in ectopic veins, while gain of its function causes loss of wing veins. It positively regulates Enhancer of split mβ, a target of Notch signalling. These observations suggest that it is a positive regulator of Notch signalling during wing development in Drosophila. PMID:26505251

  6. Functional redundancy of the Notch pathway in ovarian cancer cell lines

    PubMed Central

    Silva, Fernanda; Félix, Ana; Serpa, Jacinta

    2016-01-01

    Epithelial ovarian cancer is the most lethal gynecologic malignancy, despite advances in treatment. The most common histological type, high-grade ovarian serous carcinoma (OSC) is usually diagnosed at an advanced stage, and although these types of tumors frequently respond to surgery and platinum-based chemotherapy, they usually recur. Ovarian clear cell carcinoma (OCCC) is an unusual histological type, which is known to be intrinsically chemoresistant and is associated with poor prognosis in advanced stages. In recent years, genetic alterations and epigenetic modulation of signaling pathways have been reported in OSC and OCCC, including the overexpression of Notch pathway elements and histone deacetylases. Histone deacetylase inhibitors (HDACis), including vorinostat (suberoylanilide hydroxamic acid), alter the transcription of genes involved in cell growth, survival and apoptosis, and have become an attractive therapeutic approach. However, no previous work has addressed the effect of HDACis, and in particular vorinostat, on Notch signaling in ovarian cancer. Therefore, the present study aimed to investigate the modulation of the Notch pathway by vorinostat in ovarian cancer. Using immunofluorescence and quantitative polymerase chain reaction, the present results revealed that vorinostat activated the Notch pathway in OCCC and OSC cell lines, through different Notch ligands. In OCCC, the activation of the Notch pathway appeared to occur through Delta-like (Dll) ligands 1, 2 and 3, whereas in OSC Dll1 and Jagged 1 and 2 ligands were involved. The activation of the Notch pathway by vorinostat, in OCCC and OSC cell lines, culminated in the increased expression of the same downstream transcription factors, hairy enhancer of split (Hes) 1 and 5, and Hes-related proteins 1 and 2. In conclusion, vorinostat modulates the expression of several downstream targets of the Notch pathway and independent Notch receptors and ligands that are expressed in OSC and OCCC. This

  7. Functional redundancy of the Notch pathway in ovarian cancer cell lines

    PubMed Central

    Silva, Fernanda; Félix, Ana; Serpa, Jacinta

    2016-01-01

    Epithelial ovarian cancer is the most lethal gynecologic malignancy, despite advances in treatment. The most common histological type, high-grade ovarian serous carcinoma (OSC) is usually diagnosed at an advanced stage, and although these types of tumors frequently respond to surgery and platinum-based chemotherapy, they usually recur. Ovarian clear cell carcinoma (OCCC) is an unusual histological type, which is known to be intrinsically chemoresistant and is associated with poor prognosis in advanced stages. In recent years, genetic alterations and epigenetic modulation of signaling pathways have been reported in OSC and OCCC, including the overexpression of Notch pathway elements and histone deacetylases. Histone deacetylase inhibitors (HDACis), including vorinostat (suberoylanilide hydroxamic acid), alter the transcription of genes involved in cell growth, survival and apoptosis, and have become an attractive therapeutic approach. However, no previous work has addressed the effect of HDACis, and in particular vorinostat, on Notch signaling in ovarian cancer. Therefore, the present study aimed to investigate the modulation of the Notch pathway by vorinostat in ovarian cancer. Using immunofluorescence and quantitative polymerase chain reaction, the present results revealed that vorinostat activated the Notch pathway in OCCC and OSC cell lines, through different Notch ligands. In OCCC, the activation of the Notch pathway appeared to occur through Delta-like (Dll) ligands 1, 2 and 3, whereas in OSC Dll1 and Jagged 1 and 2 ligands were involved. The activation of the Notch pathway by vorinostat, in OCCC and OSC cell lines, culminated in the increased expression of the same downstream transcription factors, hairy enhancer of split (Hes) 1 and 5, and Hes-related proteins 1 and 2. In conclusion, vorinostat modulates the expression of several downstream targets of the Notch pathway and independent Notch receptors and ligands that are expressed in OSC and OCCC. This

  8. Notch Signaling Mediates Skeletal Muscle Atrophy in Cancer Cachexia Caused by Osteosarcoma.

    PubMed

    Mu, Xiaodong; Agarwal, Rashmi; March, Daniel; Rothenberg, Adam; Voigt, Clifford; Tebbets, Jessica; Huard, Johnny; Weiss, Kurt

    2016-01-01

    Skeletal muscle atrophy in cancer cachexia is mediated by the interaction between muscle stem cells and various tumor factors. Although Notch signaling has been known as a key regulator of both cancer development and muscle stem cell activity, the potential involvement of Notch signaling in cancer cachexia and concomitant muscle atrophy has yet to be elucidated. The murine K7M2 osteosarcoma cell line was used to generate an orthotopic model of sarcoma-associated cachexia, and the role of Notch signaling was evaluated. Skeletal muscle atrophy was observed in the sarcoma-bearing mice, and Notch signaling was highly active in both tumor tissues and the atrophic skeletal muscles. Systemic inhibition of Notch signaling reduced muscle atrophy. In vitro coculture of osteosarcoma cells with muscle-derived stem cells (MDSCs) isolated from normal mice resulted in decreased myogenic potential of MDSCs, while the application of Notch inhibitor was able to rescue this repressed myogenic potential. We further observed that Notch-activating factors reside in the exosomes of osteosarcoma cells, which activate Notch signaling in MDSCs and subsequently repress myogenesis. Our results revealed that signaling between tumor and muscle via the Notch pathway may play an important role in mediating the skeletal muscle atrophy seen in cancer cachexia. PMID:27378829

  9. Notch Signaling Mediates Skeletal Muscle Atrophy in Cancer Cachexia Caused by Osteosarcoma

    PubMed Central

    Agarwal, Rashmi; March, Daniel; Voigt, Clifford

    2016-01-01

    Skeletal muscle atrophy in cancer cachexia is mediated by the interaction between muscle stem cells and various tumor factors. Although Notch signaling has been known as a key regulator of both cancer development and muscle stem cell activity, the potential involvement of Notch signaling in cancer cachexia and concomitant muscle atrophy has yet to be elucidated. The murine K7M2 osteosarcoma cell line was used to generate an orthotopic model of sarcoma-associated cachexia, and the role of Notch signaling was evaluated. Skeletal muscle atrophy was observed in the sarcoma-bearing mice, and Notch signaling was highly active in both tumor tissues and the atrophic skeletal muscles. Systemic inhibition of Notch signaling reduced muscle atrophy. In vitro coculture of osteosarcoma cells with muscle-derived stem cells (MDSCs) isolated from normal mice resulted in decreased myogenic potential of MDSCs, while the application of Notch inhibitor was able to rescue this repressed myogenic potential. We further observed that Notch-activating factors reside in the exosomes of osteosarcoma cells, which activate Notch signaling in MDSCs and subsequently repress myogenesis. Our results revealed that signaling between tumor and muscle via the Notch pathway may play an important role in mediating the skeletal muscle atrophy seen in cancer cachexia. PMID:27378829

  10. PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner

    PubMed Central

    Sjöqvist, Marika; Antfolk, Daniel; Ferraris, Saima; Rraklli, Vilma; Haga, Cecilia; Antila, Christian; Mutvei, Anders; Imanishi, Susumu Y; Holmberg, Johan; Jin, Shaobo; Eriksson, John E; Lendahl, Urban; Sahlgren, Cecilia

    2014-01-01

    Activation of Notch signaling requires intracellular routing of the receptor, but the mechanisms controlling the distinct steps in the routing process is poorly understood. We identify PKCζ as a key regulator of Notch receptor intracellular routing. When PKCζ was inhibited in the developing chick central nervous system and in cultured myoblasts, Notch-stimulated cells were allowed to undergo differentiation. PKCζ phosphorylates membrane-tethered forms of Notch and regulates two distinct routing steps, depending on the Notch activation state. When Notch is activated, PKCζ promotes re-localization of Notch from late endosomes to the nucleus and enhances production of the Notch intracellular domain, which leads to increased Notch activity. In the non-activated state, PKCζ instead facilitates Notch receptor internalization, accompanied with increased ubiquitylation and interaction with the endosomal sorting protein Hrs. Collectively, these data identify PKCζ as a key regulator of Notch trafficking and demonstrate that distinct steps in intracellular routing are differentially modulated depending on Notch signaling status. PMID:24662486

  11. Regulation of Notch signaling and endocytosis by the Lgl neoplastic tumor suppressor

    PubMed Central

    Portela, Marta; Parsons, Linda M; Grzeschik, Nicola A; Richardson, Helena E

    2015-01-01

    The evolutionarily conserved neoplastic tumor suppressor protein, Lethal (2) giant larvae (Lgl), plays roles in cell polarity and tissue growth via regulation of the Hippo pathway. In our recent study, we showed that in the developing Drosophila eye epithelium, depletion of Lgl leads to increased ligand-dependent Notch signaling. lgl mutant tissue also exhibits an accumulation of early endosomes, recycling endosomes, early-multivesicular body markers and acidic vesicles. We showed that elevated Notch signaling in lgl− tissue can be rescued by feeding larvae the vesicle de-acidifying drug chloroquine, revealing that Lgl attenuates Notch signaling by limiting vesicle acidification. Strikingly, chloroquine also rescued the lgl− overgrowth phenotype, suggesting that the Hippo pathway defects were also rescued. In this extraview, we provide additional data on the regulation of Notch signaling and endocytosis by Lgl, and discuss possible mechanisms by which Lgl depletion contributes to signaling pathway defects and tumorigenesis. PMID:25789785

  12. Special AT-rich sequence-binding protein-1 participates in the maintenance of breast cancer stem cells through regulation of the Notch signaling pathway and expression of Snail1 and Twist1

    PubMed Central

    SUN, ZHENGKUI; ZHANG, CHAO; ZOU, XUESEN; JIANG, GUIXIANG; XU, ZONGQUAN; LI, WENTING; XIE, HUI

    2015-01-01

    The stem cell populations in cancerous tissues and cell lines vary widely and are often associated with aggressive cases of breast cancer. Despite research on the topic, the mechanism underlying the regulation of the breast cancer stem cell (BCSC) population within tumors remains to be fully elucidated. To investigate the function of special AT-rich sequence-binding protein-1 (SATB1) in the maintenance of the BCSC population, SATB1 was overexpressed with lentivirus in MCF-7 cells or knocked down with shRNA-lentivirus in BT-549 cells. The effects of SATB1 overexpression or knockdown on mammosphere formation, the size of the of BCSC population, cell invasion and tumorigenesis were investigated. Activation of the Notch signaling pathway and expression of Snail1 and Twist1 were also examined in the cells. Overexpression of SATB1 in MCF-7 cells was observed to increase mammosphere formation, the size of the BCSC population, cell invasion and tumorigenesis, accompanied by an increase in the activation of Notch signaling and expression levels of Snail1 and Twist1. Conversely, knockdown of SATB1 in BT-549 cells produced the opposite effects. The results indicated that expression of SATB1 may increase the size of the BCSC population via the activation of the Notch signaling pathway and by increasing expression levels of Snail1 and Twist1. PMID:25586771

  13. Non-Canonical Notch Signaling Drives Activation and Differentiation of Peripheral CD4+ T Cells

    PubMed Central

    Dongre, Anushka; Surampudi, Lalitha; Lawlor, Rebecca G.; Fauq, Abdul H.; Miele, Lucio; Golde, Todd E.; Minter, Lisa M.; Osborne, Barbara A.

    2014-01-01

    Cleavage of the Notch receptor via a γ-secretase, results in the release of the active intra-cellular domain of Notch that migrates to the nucleus and interacts with RBP-Jκ, resulting in the activation of downstream target genes. This canonical Notch signaling pathway has been documented to influence T cell development and function. However, the mechanistic details underlying this process remain obscure. In addition to RBP-Jκ, the intra-cellular domain of Notch also interacts with other proteins in the cytoplasm and nucleus, giving rise to the possibility of an alternate, RBP-Jκ independent Notch pathway. However, the contribution of such RBP-Jκ independent, “non-canonical” Notch signaling in regulating peripheral T cell responses is unknown. In this report, we specifically demonstrate the requirement of Notch1 for regulating signal strength and signaling events distal to the T cell receptor in peripheral CD4+ T cells. By using mice with a conditional deletion in Notch1 or RBP-Jκ, we show that Notch1 regulates activation and proliferation of CD4+ T cells independently of RBP-Jκ. Furthermore, differentiation to TH1 and iTreg lineages although Notch dependent, is RBP-Jκ independent. Our striking observations demonstrate that many of the cell-intrinsic functions of Notch occur independently of RBP-Jκ. Such non-canonical regulation of these processes likely occurs through NF-κ B. This reveals a previously unknown, novel role of non-canonical Notch signaling in regulating peripheral T cell responses. PMID:24611064

  14. Notch3/Jagged1 circuitry reinforces notch signaling and sustains T-ALL.

    PubMed

    Pelullo, Maria; Quaranta, Roberta; Talora, Claudio; Checquolo, Saula; Cialfi, Samantha; Felli, Maria Pia; te Kronnie, Geertruy; Borga, Chiara; Besharat, Zein Mersini; Palermo, Rocco; Di Marcotullio, Lucia; Capobianco, Anthony J; Gulino, Alberto; Screpanti, Isabella; Bellavia, Diana

    2014-12-01

    Deregulated Notch signaling has been extensively linked to T-cell acute lymphoblastic leukemia (T-ALL). Here, we show a direct relationship between Notch3 receptor and Jagged1 ligand in human cell lines and in a mouse model of T-ALL. We provide evidence that Notch-specific ligand Jagged1 is a new Notch3 signaling target gene. This essential event justifies an aberrant Notch3/Jagged1 cis-expression inside the same cell. Moreover, we demonstrate in Notch3-IC-overexpressing T lymphoma cells that Jagged1 undergoes a raft-associated constitutive processing. The proteolytic cleavage allows the Jagged1 intracellular domain to empower Notch signaling activity and to increase the transcriptional activation of Jagged1 itself (autocrine effect). On the other hand, the release of the soluble Jagged1 extracellular domain has a positive impact on activating Notch signaling in adjacent cells (paracrine effect), finally giving rise to a Notch3/Jagged1 auto-sustaining loop that supports the survival, proliferation, and invasion of lymphoma cells and contributes to the development and progression of Notch-dependent T-ALL. These observations are also supported by a study conducted on a cohort of patients in which Jagged1 expression is associated to adverse prognosis. PMID:25499214

  15. Inhibition of fibroblast growth by Notch1 signaling is mediated by induction of Wnt11-dependent WISP-1.

    PubMed

    Liu, Zhao-Jun; Li, Yan; Tan, Yurong; Xiao, Min; Zhang, Jialin; Radtke, Freddy; Velazquez, Omaida C

    2012-01-01

    Fibroblasts are an integral component of stroma and important source of growth factors and extracellular matrix (ECM). They play a prominent role in maintaining tissue homeostasis and in wound healing and tumor growth. Notch signaling regulates biological function in a variety of cells. To elucidate the physiological function of Notch signaling in fibroblasts, we ablated Notch1 in mouse (Notch1(Flox/Flox)) embryonic fibroblasts (MEFs). Notch1-deficient (Notch1(-/-)) MEFs displayed faster growth and motility rate compared to Notch1(Flox/Flox) MEFs. Such phenotypic changes, however, were reversible by reconstitution of Notch1 activation via overexpression of the intracellular domain of Notch1 (NICD1) in Notch1-deficient MEFs. In contrast, constitutive activation of Notch1 signaling by introducing NICD1 into primary human dermal fibroblasts (FF2441), which caused pan-Notch activation, inhibited cell growth and motility, whereas cellular inhibition was relievable when the Notch activation was countered with dominant-negative mutant of Master-mind like 1 (DN-MAML-1). Functionally, "Notch-activated" stromal fibroblasts could inhibit tumor cell growth/invasion. Moreover, Notch activation induced expression of Wnt-induced secreted proteins-1 (WISP-1/CCN4) in FF2441 cells while deletion of Notch1 in MEFs resulted in an opposite effect. Notably, WISP-1 suppressed fibroblast proliferation, and was responsible for mediating Notch1's inhibitory effect since siRNA-mediated blockade of WISP-1 expression could relieve cell growth inhibition. Notch1-induced WISP-1 expression appeared to be Wnt11-dependent, but Wnt1-independent. Blockade of Wnt11 expression resulted in decreased WISP-1 expression and liberated Notch-induced cell growth inhibition. These findings indicated that inhibition of fibroblast proliferation by Notch pathway activation is mediated, at least in part, through regulating Wnt1-independent, but Wnt11-dependent WISP-1 expression.

  16. Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival

    PubMed Central

    Lina, Taslima T.; Dunphy, Paige S.; Luo, Tian

    2016-01-01

    ABSTRACT Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival. PMID:27381289

  17. Regulation of Breast Cancer Stem Cell Activity by Signalling Through the Notch4 Receptor

    PubMed Central

    Harrison, Hannah; Farnie, Gillian; Howell, Sacha J; Rock, Rebecca E; Stylianou, Spyros; Brennan, Keith R; Bundred, Nigel J; Clarke, Robert B

    2012-01-01

    The Notch receptor signalling pathway plays an important role in breast development, regulation of stem cells and differentiation of luminal progenitor cells. The pathway also plays a significant role in breast cancer development and progression. However, which of the Notch receptors that regulate breast cancer stem cells is unknown. We assessed stem cell activity in breast cancer cell lines and nine primary human tumour samples. In vitro and in vivo breast cancer stem cell activity was enriched using selection of anoikis resistant cells or cells expressing the membrane phenotype ESA+/CD44+/CD24low. We compared the activation of Notch receptors in the breast cancer stem cell-enriched population to luminally differentiated cells and studied the effects of pathway inhibition, both in vitro and in vivo. We find that Notch4 signalling activity is 8-fold higher in the breast cancer stem cell-enriched cells compared to the differentiated cells while Notch1 activation is 4-fold lower in breast cancer stem cells. Furthermore, pharmacological or genetic Notch inhibition markedly reduced breast cancer stem cell activity in vitro, and significantly reduced tumour formation in vivo. Importantly, cells with Notch4 knock-down using specific shRNA formed fewer mammosphere colonies than Notch1 knock-down cells. In vivo Notch1 knock-down, like pharmacological inhibition, reduced the number and size of tumours but Notch4 knock-down suppressed tumour initiation completely. Our findings indicate that Notch4-targeted therapies will be more effective than targeting Notch1 in suppressing breast cancer recurrence initiated by breast cancer stem cells. PMID:20068161

  18. Notch signaling from the endosome requires a conserved dileucine motif

    PubMed Central

    Zheng, Li; Saunders, Cosmo A.; Sorensen, Erika B.; Waxmonsky, Nicole C.; Conner, Sean D.

    2013-01-01

    Notch signaling is reliant on γ-secretase–mediated processing, although the subcellular location where γ-secretase cleaves Notch to initiate signaling remains unresolved. Accumulating evidence demonstrates that Notch signaling is modulated by endocytosis and endosomal transport. In this study, we investigated the relationship between Notch transport itinerary and signaling capacity. In doing so, we discovered a highly conserved dileucine sorting signal encoded within the cytoplasmic tail that directs Notch to the limiting membrane of the lysosome for signaling. Mutating the dileucine motif led to receptor accumulation in cation-dependent mannose-phosphate receptor–positive tubular early endosomes and a reduction in Notch signaling capacity. Moreover, truncated receptor forms that mimic activated Notch were readily cleaved by γ-secretase within the endosome; however, the cleavage product was proteasome-sensitive and failed to contribute to robust signaling. Collectively these results indicate that Notch signaling from the lysosome limiting membrane is conserved and that receptor targeting to this compartment is an active process. Moreover, the data support a model in which Notch signaling in mammalian systems is initiated from either the plasma membrane or lysosome, but not the early endosome. PMID:23171551

  19. Hippo signaling is required for Notch-dependent smooth muscle differentiation of neural crest

    PubMed Central

    Manderfield, Lauren J.; Aghajanian, Haig; Engleka, Kurt A.; Lim, Lillian Y.; Liu, Feiyan; Jain, Rajan; Li, Li; Olson, Eric N.; Epstein, Jonathan A.

    2015-01-01

    Notch signaling has well-defined roles in the assembly of arterial walls and in the development of the endothelium and smooth muscle of the vasculature. Hippo signaling regulates cellular growth in many tissues, and contributes to regulation of organ size, in addition to other functions. Here, we show that the Notch and Hippo pathways converge to regulate smooth muscle differentiation of the neural crest, which is crucial for normal development of the aortic arch arteries and cranial vasculature during embryonic development. Neural crest-specific deletion of the Hippo effectors Yap and Taz produces neural crest precursors that migrate normally, but fail to produce vascular smooth muscle, and Notch target genes such as Jagged1 fail to activate normally. We show that Yap is normally recruited to a tissue-specific Jagged1 enhancer by directly interacting with the Notch intracellular domain (NICD). The Yap-NICD complex is recruited to chromatin by the DNA-binding protein Rbp-J in a Tead-independent fashion. Thus, Hippo signaling can modulate Notch signaling outputs, and components of the Hippo and Notch pathways physically interact. Convergence of Hippo and Notch pathways by the mechanisms described here might be relevant for the function of these signaling cascades in many tissues and in diseases such as cancer. PMID:26253400

  20. Expression patterns of Notch1, Notch2, and Notch3 suggest multiple functional roles for the Notch-DSL signaling system during brain development.

    PubMed

    Irvin, D K; Zurcher, S D; Nguyen, T; Weinmaster, G; Kornblum, H I

    2001-07-23

    The Notch-DSL signaling system consists of multiple receptors and ligands, and plays many roles in development. The function of Notch receptors and ligands in mammalian brain, however, is poorly understood. In the current study, we examined the expression patterns for three receptors of this system, Notch1, 2, and 3, in late embryonic and postnatal rat brain by in situ hybridization. The three receptors have overlapping but different patterns of expression. Messenger RNA for all three proteins is found in postnatal central nervous system (CNS) germinal zones and, in early postnatal life, within numerous cells throughout the CNS. Within zones of cellular proliferation of the postnatal brain, Notch1 mRNA is found in both the subventricular and the ventricular germinal zones, whereas Notch2 and Notch3 mRNAs are more highly localized to the ventricular zones. Both Notch1 and Notch3 mRNAs are expressed along the inner aspect of the dentate gyrus, a site of adult neurogenesis. Notch2 mRNA is expressed in the external granule cell layer of the developing cerebellum. In several brain areas, Notch1 and Notch2 mRNAs are relatively concentrated in white matter, whereas Notch3 mRNA is not. Neurosphere cultures (which contain CNS stem cells), purified astrocyte cultures, and striatal neuron-enriched cultures express Notch1 mRNA. However, in these latter cultures, Notch1 mRNA is produced by nestin-containing cells, rather than by postmitotic neurons. Taken together, these results support multiple roles for Notch1, 2, and 3 receptor activation during CNS development, particularly during gliogenesis.

  1. Pleiotropic roles of Notch signaling in normal, malignant, and developmental hematopoiesis in the human

    PubMed Central

    Kushwah, Rahul; Guezguez, Borhane; Lee, Jung Bok; Hopkins, Claudia I; Bhatia, Mickie

    2014-01-01

    The Notch signaling pathway is evolutionarily conserved across species and plays an important role in regulating cell differentiation, proliferation, and survival. It has been implicated in several different hematopoietic processes including early hematopoietic development as well as adult hematological malignancies in humans. This review focuses on recent developments in understanding the role of Notch signaling in the human hematopoietic system with an emphasis on hematopoietic initiation from human pluripotent stem cells and regulation within the bone marrow. Based on recent insights, we summarize potential strategies for treatment of human hematological malignancies toward the concept of targeting Notch signaling for fate regulation. PMID:25252682

  2. miRNA-34c regulates Notch signaling during bone development

    PubMed Central

    Bae, Yangjin; Yang, Tao; Zeng, Huan-Chang; Campeau, Philippe M.; Chen, Yuqing; Bertin, Terry; Dawson, Brian C.; Munivez, Elda; Tao, Jianning; Lee, Brendan H.

    2012-01-01

    During bone homeostasis, osteoblast and osteoclast differentiation is coupled and regulated by multiple signaling pathways and their downstream transcription factors. Here, we show that microRNA 34 (miR-34) is significantly induced by BMP2 during osteoblast differentiation. In vivo, osteoblast-specific gain of miR-34c in mice leads to an age-dependent osteoporosis due to the defective mineralization and proliferation of osteoblasts and increased osteoclastogenesis. In osteoblasts, miR-34c targets multiple components of the Notch signaling pathway, including Notch1, Notch2 and Jag1 in a direct manner, and influences osteoclast differentiation in a non-cell-autonomous fashion. Taken together, our results demonstrate that miR-34c is critical during osteoblastogenesis in part by regulating Notch signaling in bone homeostasis. Furthermore, miR-34c-mediated post-transcriptional regulation of Notch signaling in osteoblasts is one possible mechanism to modulate the proliferative effect of Notch in the committed osteoblast progenitors which may be important in the pathogenesis of osteosarcomas. Therefore, understanding the functional interaction of miR-34 and Notch signaling in normal bone development and in bone cancer could potentially lead to therapies modulating miR-34 signaling. PMID:22498974

  3. Notch pathway regulates female germ cell meiosis progression and early oogenesis events in fetal mouse.

    PubMed

    Feng, Yan-Min; Liang, Gui-Jin; Pan, Bo; Qin, Xun-Si; Zhang, Xi-Feng; Chen, Chun-Lei; Li, Lan; Cheng, Shun-Feng; De Felici, Massimo; Shen, Wei

    2014-01-01

    A critical process of early oogenesis is the entry of mitotic oogonia into meiosis, a cell cycle switch regulated by a complex gene regulatory network. Although Notch pathway is involved in numerous important aspects of oogenesis in invertebrate species, whether it plays roles in early oogenesis events in mammals is unknown. Therefore, the rationale of the present study was to investigate the roles of Notch signaling in crucial processes of early oogenesis, such as meiosis entry and early oocyte growth. Notch receptors and ligands were localized in mouse embryonic female gonads and 2 Notch inhibitors, namely DAPT and L-685,458, were used to attenuate its signaling in an in vitro culture system of ovarian tissues from 12.5 days post coitum (dpc) fetus. The results demonstrated that the expression of Stra8, a master gene for germ cell meiosis, and its stimulation by retinoic acid (RA) were reduced after suppression of Notch signaling, and the other meiotic genes, Dazl, Dmc1, and Rec8, were abolished or markedly decreased. Furthermore, RNAi of Notch1 also markedly inhibited the expression of Stra8 and SCP3 in cultured female germ cells. The increased methylation status of CpG islands within the Stra8 promoter of the oocytes was observed in the presence of DAPT, indicating that Notch signaling is probably necessary for maintaining the epigenetic state of this gene in a way suitable for RA stimulation. Furthermore, in the presence of Notch inhibitors, progression of oocytes through meiosis I was markedly delayed. At later culture periods, the rate of oocyte growth was decreased, which impaired subsequent primordial follicle assembly in cultured ovarian tissues. Taken together, these results suggested new roles of the Notch signaling pathway in female germ cell meiosis progression and early oogenesis events in mammals.

  4. Inhibition of gamma-secretase affects proliferation of leukemia and hepatoma cell lines through Notch signaling.

    PubMed

    Suwanjunee, Saipin; Wongchana, Wipawee; Palaga, Tanapat

    2008-06-01

    Notch signaling is a well-conserved pathway playing crucial roles in regulating cell fate decision, proliferation, and apoptosis during the development of multiple cell lineages. Aberration in Notch signaling is associated with tumorigenesis of tissues from various origins. To investigate the role Notch signaling plays in the proliferation of cancer cell lines, the expression profiles of Notch1 in six human cancer cell lines (Jurkat, HepG2, SW620, KATOIII, A375, BT474) were examined. All cell lines differentially expressed Notch1, and only Jurkat and SW620 expressed cleaved Notch1 (Val1744). Among the six cell lines tested, only Jurkat and HepG2 showed a decrease in cell proliferation during 4 days of treatment with a gamma-secretase inhibitor (GSI). This is the first report on the anti-proliferative effects of GSI on a human hepatoma cell line. These two cell lines expressed Notch1-3, Jagged1, Jagged2, Dlk1 and Hes1. GSI treatment led to a decrease in Hes1 expression in both cell lines. Surprisingly, GSI treatment resulted in the accumulation of Notch1 protein upon treatment. During this period, GSI treatment did not induce apoptosis, but caused cell cycle arrest in both cell lines. This was also correlated with decreased c-myc expression. Forced expression of activated intracellular Notch1 completely abrogated GSI sensitivity in both cell lines. These results clearly demonstrate that Notch signaling positively regulates cell proliferation in Jurkat and HepG2 cell lines and that GSI treatment inhibits tumor cell proliferation through the suppression of Notch signaling. PMID:18418214

  5. Inhibition of CK2α down-regulates Notch1 signalling in lung cancer cells

    PubMed Central

    Zhang, Shulin; Long, Hao; Yang, Yi-Lin; Wang, Yucheng; Hsieh, David; Li, Weiming; Au, Alfred; Stoppler, Hubert J; Xu, Zhidong; Jablons, David M; You, Liang

    2013-01-01

    Protein kinase CK2 is frequently elevated in a variety of human cancers. The Notch1 signalling pathway has been implicated in stem cell maintenance and its aberrant activation has been shown in several types of cancer including lung cancer. Here, we show, for the first time, that CK2α is a positive regulator of Notch1 signalling in lung cancer cell lines A549 and H1299. We found that Notch1 protein level was reduced after CK2α silencing. Down-regulation of Notch1 transcriptional activity was demonstrated after the silencing of CK2α in lung cancer cells. Furthermore, small-molecule CK2α inhibitor CX-4945 led to a dose-dependent inhibition of Notch1 transcriptional activity. Conversely, forced overexpression of CK2α resulted in an increase in Notch1 transcriptional activity. Finally, the inhibition of CK2α led to a reduced proportion of stem-like CD44 + /CD24− cell population. Thus, we report that the inhibition of CK2α down-regulates Notch1 signalling and subsequently reduces a cancer stem-like cell population in human lung cancer cells. Our data suggest that CK2α inhibitors may be beneficial to the lung cancer patients with activated Notch1 signalling. PMID:23651443

  6. Notch signaling indirectly promotes chondrocyte hypertrophy via regulation of BMP signaling and cell cycle arrest

    PubMed Central

    Shang, Xifu; Wang, Jinwu; Luo, Zhengliang; Wang, Yongjun; Morandi, Massimo M.; Marymont, John V.; Hilton, Matthew J.; Dong, Yufeng

    2016-01-01

    Cell cycle regulation is critical for chondrocyte differentiation and hypertrophy. Recently we identified the Notch signaling pathway as an important regulator of chondrocyte proliferation and differentiation during mouse cartilage development. To investigate the underlying mechanisms, we assessed the role for Notch signaling regulation of the cell cycle during chondrocyte differentiation. Real-time RT-PCR data showed that over-expression of the Notch Intracellular Domain (NICD) significantly induced the expression of p57, a cell cycle inhibitor, in chondrocytes. Flow cytometric analyses further confirmed that over-expression of NICD in chondrocytes enhances the G0/G1 cell cycle transition and cell cycle arrest. In contrast, treatment of chondrocytes with the Notch inhibitor, DAPT, decreased both endogenous and BMP2-induced SMAD 1/5/8 phosphorylation and knockdown of SMAD 1/5/8 impaired NICD-induced chondrocyte differentiation and p57 expression. Co-immunoprecipitation using p-SMAD 1/5/8 and NICD antibodies further showed a strong interaction of these proteins during chondrocyte maturation. Finally, RT-PCR and Western blot results revealed a significant reduction in the expression of the SMAD-related phosphatase, PPM1A, following NICD over-expression. Taken together, our results demonstrate that Notch signaling induces cell cycle arrest and thereby initiates chondrocyte hypertrophy via BMP/SMAD-mediated up-regulation of p57. PMID:27146698

  7. Biological therapy induces expression changes in Notch pathway in psoriasis.

    PubMed

    Skarmoutsou, Evangelia; Trovato, Chiara; Granata, Mariagrazia; Rossi, Giulio A; Mosca, Ambra; Longo, Valentina; Gangemi, Pietro; Pettinato, Maurizio; D'Amico, Fabio; Mazzarino, Maria Clorinda

    2015-12-01

    Psoriasis is a chronic inflammatory skin disease, characterized by hyperproliferation of keratinocytes and by skin infiltration of activated T cells. To date, the pathophysiology of psoriasis has not yet been fully elucidated. The Notch pathway plays a determinant role in cell fate determination, proliferation, differentiation, immune cell development and function. Many biological agents, used in the treatment of psoriasis, include TFN-α inhibitors, such as etanercept, adalimumab, and anti IL-12/IL-23 p40 antibody, such as ustekinumab. This study aimed to determine mRNA expression levels by real-time RT-PCR, and protein expression levels, analysed by Western blot and immunohistochemistry, of some components of the Notch pathway, such as NOTCH1, NOTCH2, JAGGED1, and HES1 after biological treatments in psoriatic patients. mRNA and protein levels of NOTCH1, NOTCH2, JAGGED1 and HES1 were upregulated in skin samples from untreated psoriatic patients compared with normal controls. Biological therapy showed to downregulate differently the protein expression levels of the molecules under study. Our study suggests that Notch pathway components might be a potential therapeutic target against psoriasis.

  8. Notch1 Pathway Activity Determines the Regulatory Role of Cancer-Associated Fibroblasts in Melanoma Growth and Invasion

    PubMed Central

    Shao, Hongwei; Kong, Ranran; Ferrari, Massimiliano L.; Radtke, Freddy; Capobianco, Anthony J.; Liu, Zhao-Jun

    2015-01-01

    Cancer-associated fibroblasts (CAF) play a crucial role in regulating cancer progression, yet the molecular determinant that governs the tumor regulatory role of CAF remains unknown. Using a mouse melanoma model in which exogenous melanoma cells were grafted on the skin of two lines of mice where the genetic activation or inactivation of Notch1 signaling specifically occurs in natural host stromal fibroblasts, we demonstrated that Notch1 pathway activity could determine the tumor-promoting or tumor-suppressing phenotype in CAF. CAF carrying elevated Notch1 activity significantly inhibited melanoma growth and invasion, while those with a null Notch1 promoted melanoma invasion. These findings identify the Notch1 pathway as a molecular determinant that controls the regulatory role of CAF in melanoma skin growth and invasion, unveiling Notch1 signaling as a potential therapeutic target for melanoma and potentially other solid tumors. PMID:26562315

  9. Notch signaling change in pulmonary vascular remodeling in rats with pulmonary hypertension and its implication for therapeutic intervention.

    PubMed

    Qiao, Lina; Xie, Liang; Shi, Kun; Zhou, Tongfu; Hua, Yimin; Liu, Hanmin

    2012-01-01

    Pulmonary hypertension (PH) is a fatal disease that lacks an effective therapy. Notch signaling pathway plays a crucial role in the angiogenesis and vascular remodeling. However, its roles in vascular remodeling in PH have not been well studied. In the current study, using hypoxia-induced PH model in rat, we examined the expression of Notch and its downstream factors. Then, we used vessel strip culture system and γ-secretase inhibitor DAPT, a Notch signaling inhibitor to determine the effect of Notch signaling in vascular remodeling and its potential therapeutic value. Our results indicated that Notch 1-4 were detected in the lung tissue with variable levels in different cell types such as smooth muscle cells and endothelial cells of pulmonary artery, bronchia, and alveoli. In addition, following the PH induction, all of Notch1, Notch3, Notch4 receptor, and downstream factor, HERP1 in pulmonary arteries, mRNA expressions were increased with a peak at 1-2 weeks. Furthermore, the vessel wall thickness from rats with hypoxia treatment increased after cultured for 8 days, which could be decreased approximately 30% by DAPT, accompanied with significant increase of expression level of apoptotic factors (caspase-3 and Bax) and transformation of vascular smooth muscle cell (VSMC) phenotype from synthetic towards contractile. In conclusion, the current study suggested Notch pathway plays an important role in pulmonary vascular remodeling in PH and targeting Notch signaling pathway could be a valuable approach to design new therapy for PH. PMID:23251561

  10. Notch1 Pathway Activation Results from the Epigenetic Abrogation of Notch-Related MicroRNAs in Mycosis Fungoides.

    PubMed

    Gallardo, Fernando; Sandoval, Juan; Díaz-Lagares, Angel; Garcia, Ricard; D'Altri, Teresa; González, Jessica; Alegre, Victor; Servitje, Octavio; Crujeiras, Ana-Belén; Stefánsson, Ólafur-Andri; Espinet, Blanca; Hernández, Maria-Inmaculada; Bellosillo, Beatriz; Esteller, Manel; Pujol, Ramon-Maria; Bigas, Anna; Espinosa, Lluis

    2015-12-01

    Notch is a family of transmembrane receptors that participate in the regulation of cell differentiation, proliferation, and stemness. Notch pathway activation has also been found associated with different human cancers including primary cutaneous T-cell lymphomas (CTCL). The elucidation of the mechanisms driving Notch activation in these particular diseases has remained elusive. Here we studied the possibility that DNA methylation at Notch pathway gene promoters and/or deregulation of Notch-associated microRNAs contribute to activate Notch in mycosis fungoides (MF). By genome-wide DNA methylation analysis, we failed to detect any consistent methylation at the Notch1, the Notch-ligand Jagged1, or the Notch-target Hes1 gene promoters, but found a significant methylation of the Notch-related microRNAs, in particular miR-200c and miR-124. Downregulation of miR-200c is associated with overexpression of Jagged1, concomitant to Notch1 activation. CTCL cell lines were infected with lentiviral vector encoding for miR-200c and ectopic expression of miR-200c in CTCL lines resulted in Jagged1 protein downregulation associated with a reduction in the levels of active Notch1. Our study deciphers an epigenetic mechanism regulating the Notch pathway in (MF) that might contribute to the future design of more specific therapeutic strategies. PMID:26302069

  11. Aberrant Notch signaling in glioblastoma stem cells contributes to tumor recurrence and invasion.

    PubMed

    Yu, Jian-Bo; Jiang, Hao; Zhan, Ren-Ya

    2016-08-01

    Upregulation of the Notch signaling pathway in cancer stem cells and side population (SP) cells has a major role in maintenance, self-renewal and chemoresistance. The present study isolated a cancer stem cell-like SP accounting for 4.1% of a glioblastoma cell population using a Hoechst 33342 dye exclusion assay. In this glioblastoma SP, the expression of of Notch1 signaling proteins Notch1 intracellular domain and Hes‑1 was markedly upregulated. Furthermore, knockdown of Notch1 by RNA interference significantly diminished the neurosphere formation ability, self‑renewal and chemoresistance of the SP cells. In addition, the expression of the stem‑cell surface genes Oct‑4, Sox2 and Nanog in SP cells was significantly reduced and the sensitivity to the SP cells to chemotherapeutics was enhanced following Notch1 knockdown. In conclusion, the results of the present study suggested that upregulation of Notch1 is involved in the chemotherapy resistance and tumor recurrence of glioblastoma. Hence, the development of novel anti‑cancer drugs targeting the Notch1 signaling pathway may be a promising strategy for curing glioblastoma. PMID:27315154

  12. 6-Shogaol Inhibits Breast Cancer Cells and Stem Cell-Like Spheroids by Modulation of Notch Signaling Pathway and Induction of Autophagic Cell Death

    PubMed Central

    Ray, Anasuya; Vasudevan, Smreti; Sengupta, Suparna

    2015-01-01

    Cancer stem cells (CSCs) pose a serious obstacle to cancer therapy as they can be responsible for poor prognosis and tumour relapse. In this study, we have investigated inhibitory activity of the ginger-derived compound 6-shogaol against breast cancer cells both in monolayer and in cancer-stem cell-like spheroid culture. The spheroids were generated from adherent breast cancer cells. 6-shogaol was effective in killing both breast cancer monolayer cells and spheroids at doses that were not toxic to noncancerous cells. The percentages of CD44+CD24-/low cells and the secondary sphere content were reduced drastically upon treatment with 6-shogaol confirming its action on CSCs. Treatment with 6-shogaol caused cytoplasmic vacuole formation and cleavage of microtubule associated protein Light Chain3 (LC3) in both monolayer and spheroid culture indicating that it induced autophagy. Kinetic analysis of the LC3 expression and a combination treatment with chloroquine revealed that the autophagic flux instigated cell death in 6-shogaol treated breast cancer cells in contrast to the autophagy inhibitor chloroquine. Furthermore, 6-shogaol-induced cell death got suppressed in the presence of chloroquine and a very low level of apoptosis was exhibited even after prolonged treatment of the compound, suggesting that autophagy is the major mode of cell death induced by 6-shogaol in breast cancer cells. 6-shogaol reduced the expression levels of Cleaved Notch1 and its target proteins Hes1 and Cyclin D1 in spheroids, and the reduction was further pronounced in the presence of a γ-secretase inhibitor. Secondary sphere formation in the presence of the inhibitor was also further reduced by 6-shogaol. Together, these results indicate that the inhibitory action of 6-shogaol on spheroid growth and sustainability is conferred through γ-secretase mediated down-regulation of Notch signaling. The efficacy of 6-shogaol in monolayer and cancer stem cell-like spheroids raise hope for its

  13. Direct regulation of interleukin-6 expression by Notch signaling in macrophages.

    PubMed

    Wongchana, Wipawee; Palaga, Tanapat

    2012-03-01

    Interleukin-6 (IL-6) is a pleiotropic, pro-inflammatory cytokine produced by various types of cells, including macrophages. Within the IL-6 gene promoter region, the signature binding motif of CBF1/Su(H)/Lag-1 (CSL), a key DNA-binding protein in the Notch signaling pathway, was identified and found to overlap with a consensus nuclear factor (NF)-κB-binding site. Notch signaling is highly conserved and is involved in the regulation of biological functions in immune cells. In this study, we investigated the role of Notch signaling in the regulation of the IL-6 transcript in murine macrophages. The upregulation of Notch1 protein levels and the appearance of cleaved Notch1 (Val1744) correlated well with the increased IL-6 mRNA expression levels in murine primary bone marrow-derived macrophages (BMMφ) after activation by lipopolysaccharide (LPS) together with interferon-gamma (IFN-γ). Treatment of BMMφ with the γ-secretase inhibitor IL-CHO to suppress the transduction of Notch signaling resulted in a partial decrease in the level of IL-6 mRNA and the amount of IL-6 protein produced. In contrast, the overexpression of a constitutively activated intracellular Notch1 protein (N(IC)) in the RAW264.7 macrophage-like cell line resulted in significantly higher IL-6 transcript expression levels than in cells transfected with the empty vector control. The NF-κB inhibitor completely abrogated IL-6 mRNA expression induced by the overexpression of N(IC). Chromatin immunoprecipitation (ChIP) using an anti-Notch1 antibody demonstrated that Notch1 is associated with the IL-6 promoter in RAW264.7 cells activated by LPS/IFN-γ but not in unstimulated cells. Taken together, these results strongly suggest that Notch1 positively regulates IL-6 expression via NF-κB in activated macrophages.

  14. Direct regulation of interleukin-6 expression by Notch signaling in macrophages

    PubMed Central

    Wongchana, Wipawee; Palaga, Tanapat

    2012-01-01

    Interleukin-6 (IL-6) is a pleiotropic, pro-inflammatory cytokine produced by various types of cells, including macrophages. Within the IL-6 gene promoter region, the signature binding motif of CBF1/Su(H)/Lag-1 (CSL), a key DNA-binding protein in the Notch signaling pathway, was identified and found to overlap with a consensus nuclear factor (NF)-κB-binding site. Notch signaling is highly conserved and is involved in the regulation of biological functions in immune cells. In this study, we investigated the role of Notch signaling in the regulation of the IL-6 transcript in murine macrophages. The upregulation of Notch1 protein levels and the appearance of cleaved Notch1 (Val1744) correlated well with the increased IL-6 mRNA expression levels in murine primary bone marrow-derived macrophages (BMMφ) after activation by lipopolysaccharide (LPS) together with interferon-gamma (IFN-γ). Treatment of BMMφ with the γ-secretase inhibitor IL-CHO to suppress the transduction of Notch signaling resulted in a partial decrease in the level of IL-6 mRNA and the amount of IL-6 protein produced. In contrast, the overexpression of a constitutively activated intracellular Notch1 protein (NIC) in the RAW264.7 macrophage-like cell line resulted in significantly higher IL-6 transcript expression levels than in cells transfected with the empty vector control. The NF-κB inhibitor completely abrogated IL-6 mRNA expression induced by the overexpression of NIC. Chromatin immunoprecipitation (ChIP) using an anti-Notch1 antibody demonstrated that Notch1 is associated with the IL-6 promoter in RAW264.7 cells activated by LPS/IFN-γ but not in unstimulated cells. Taken together, these results strongly suggest that Notch1 positively regulates IL-6 expression via NF-κB in activated macrophages. PMID:21983868

  15. Notch Signaling and Hes Labeling in the Normal and Drug-Damaged Organ of Corti

    PubMed Central

    Batts, Shelley A.; Shoemaker, Christopher R.; Raphael, Yehoash

    2009-01-01

    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. PMID:19185606

  16. Novel genes upregulated when NOTCH signalling is disrupted during hypothalamic development

    PubMed Central

    2013-01-01

    Background The generation of diverse neuronal types and subtypes from multipotent progenitors during development is crucial for assembling functional neural circuits in the adult central nervous system. It is well known that the Notch signalling pathway through the inhibition of proneural genes is a key regulator of neurogenesis in the vertebrate central nervous system. However, the role of Notch during hypothalamus formation along with its downstream effectors remains poorly defined. Results Here, we have transiently blocked Notch activity in chick embryos and used global gene expression analysis to provide evidence that Notch signalling modulates the generation of neurons in the early developing hypothalamus by lateral inhibition. Most importantly, we have taken advantage of this model to identify novel targets of Notch signalling, such as Tagln3 and Chga, which were expressed in hypothalamic neuronal nuclei. Conclusions These data give essential advances into the early generation of neurons in the hypothalamus. We demonstrate that inhibition of Notch signalling during early development of the hypothalamus enhances expression of several new markers. These genes must be considered as important new targets of the Notch/proneural network. PMID:24360028

  17. Notch signaling promotes osteoclast maturation and resorptive activity.

    PubMed

    Ashley, Jason W; Ahn, Jaimo; Hankenson, Kurt D

    2015-11-01

    The role of Notch signaling in osteoclast differentiation is controversial with conflicting experimental evidence indicating both stimulatory and inhibitory roles. Differences in experimental protocols and in vivo versus in vitro models may explain the discrepancies between studies. In this study, we investigated cell autonomous roles of Notch signaling in osteoclast differentiation and function by altering Notch signaling during osteoclast differentiation using stimulation with immobilized ligands Jagged1 or Delta-like1 or by suppression with γ-secretase inhibitor DAPT or transcriptional inhibitor SAHM1. Stimulation of Notch signaling in committed osteoclast precursors resulted in larger osteoclasts with a greater number of nuclei and resorptive activity whereas suppression resulted in smaller osteoclasts with fewer nuclei and suppressed resorptive activity. Conversely, stimulation of Notch signaling in osteoclast precursors prior to induction of osteoclastogenesis resulted in fewer osteoclasts. Our data support a mechanism of context-specific Notch signaling effects wherein Notch stimulation inhibits commitment to osteoclast differentiation, but enhances the maturation and function of committed precursors.

  18. Notch Signalling Is Required for the Formation of Structurally Stable Muscle Fibres in Zebrafish

    PubMed Central

    Pascoal, Susana; Esteves de Lima, Joana; Leslie, Jonathan D.; Hughes, Simon M.; Saúde, Leonor

    2013-01-01

    Background Accurate regulation of Notch signalling is central for developmental processes in a variety of tissues, but its function in pectoral fin development in zebrafish is still unknown. Methodology/Principal Findings Here we show that core elements necessary for a functional Notch pathway are expressed in developing pectoral fins in or near prospective muscle territories. Blocking Notch signalling at different levels of the pathway consistently leads to the formation of thin, wavy, fragmented and mechanically weak muscles fibres and loss of stress fibres in endoskeletal disc cells in pectoral fins. Although the structural muscle genes encoding Desmin and Vinculin are normally transcribed in Notch-disrupted pectoral fins, their proteins levels are severely reduced, suggesting that weak mechanical forces produced by the muscle fibres are unable to stabilize/localize these proteins. Moreover, in Notch signalling disrupted pectoral fins there is a decrease in the number of Pax7-positive cells indicative of a defect in myogenesis. Conclusions/Significance We propose that by controlling the differentiation of myogenic progenitor cells, Notch signalling might secure the formation of structurally stable muscle fibres in the zebrafish pectoral fin. PMID:23840804

  19. The canonical Notch pathway effector RBP-J regulates neuronal plasticity and expression of GABA transporters in hippocampal networks.

    PubMed

    Liu, Shuxi; Wang, Yue; Worley, Paul F; Mattson, Mark P; Gaiano, Nicholas

    2015-05-01

    Activation of the Notch pathway in neurons is essential for learning and memory in various species from invertebrates to mammals. However, it remains unclear how Notch signaling regulates neuronal plasticity, and whether the transcriptional regulator and canonical pathway effector RBP-J plays a role. Here, we report that conditional disruption of RBP-J in the postnatal hippocampus leads to defects in long-term potentiation, long-term depression, and in learning and memory. Using gene expression profiling and chromatin immunoprecipitation, we identified two GABA transporters, GAT2 and BGT1, as putative Notch/RBP-J pathway targets, which may function downstream of RBP-J to limit the accumulation of GABA in the Schaffer collateral pathway. Our results reveal an essential role for canonical Notch/RBP-J signaling in hippocampal synaptic plasticity and suggest that role, at least in part, is mediated by the regulation of GABAergic signaling. PMID:25515406

  20. The canonical Notch pathway effector RBP-J regulates neuronal plasticity and expression of GABA transporters in hippocampal networks

    PubMed Central

    Liu, Shuxi; Wang, Yue; Worley, Paul F.; Mattson, Mark P.; Gaiano, Nicholas

    2014-01-01

    Activation of the Notch pathway in neurons is essential for learning and memory in various species from invertebrates to mammals. However, it remains unclear how Notch signaling regulates neuronal plasticity, and whether the transcriptional regulator and canonical pathway effector RBP-J plays a role. Here we report that conditional disruption of RBP-J in the postnatal hippocampus leads to defects in long-term potentiation (LTP), long-term depression (LTD), and in learning and memory. Using gene expression profiling and chromatin immunoprecipitation, we identified two GABA transporters, GAT2 and BGT1, as putative Notch/RBP-J pathway targets, which may function downstream of RBP-J to limit the accumulation of GABA in the Schaffer collateral pathway. Our results reveal an essential role for canonical Notch/RBP-J signaling in hippocampal synaptic plasticity and suggest that role, at least in part, is mediated by the regulation of GABAergic signaling. PMID:25515406

  1. The canonical Notch pathway effector RBP-J regulates neuronal plasticity and expression of GABA transporters in hippocampal networks.

    PubMed

    Liu, Shuxi; Wang, Yue; Worley, Paul F; Mattson, Mark P; Gaiano, Nicholas

    2015-05-01

    Activation of the Notch pathway in neurons is essential for learning and memory in various species from invertebrates to mammals. However, it remains unclear how Notch signaling regulates neuronal plasticity, and whether the transcriptional regulator and canonical pathway effector RBP-J plays a role. Here, we report that conditional disruption of RBP-J in the postnatal hippocampus leads to defects in long-term potentiation, long-term depression, and in learning and memory. Using gene expression profiling and chromatin immunoprecipitation, we identified two GABA transporters, GAT2 and BGT1, as putative Notch/RBP-J pathway targets, which may function downstream of RBP-J to limit the accumulation of GABA in the Schaffer collateral pathway. Our results reveal an essential role for canonical Notch/RBP-J signaling in hippocampal synaptic plasticity and suggest that role, at least in part, is mediated by the regulation of GABAergic signaling.

  2. Notch signalling drives bone marrow stromal cell-mediated chemoresistance in acute myeloid leukemia

    PubMed Central

    Kamga, Paul Takam; Bassi, Giulio; Cassaro, Adriana; Midolo, Martina; Di Trapani, Mariano; Gatti, Alessandro; Carusone, Roberta; Resci, Federica; Perbellini, Omar; Gottardi, Michele; Bonifacio, Massimiliano; Kamdje, Armel Hervé Nwabo; Ambrosetti, Achille; Krampera, Mauro

    2016-01-01

    Both preclinical and clinical investigations suggest that Notch signalling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms. However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-κB. These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML. PMID:26967055

  3. Competition in Notch Signaling with Cis Enriches Cell Fate Decisions

    PubMed Central

    Formosa-Jordan, Pau; Ibañes, Marta

    2014-01-01

    Notch signaling is involved in cell fate choices during the embryonic development of Metazoa. Commonly, Notch signaling arises from the binding of the Notch receptor to its ligands in adjacent cells driving cell-to-cell communication. Yet, cell-autonomous control of Notch signaling through both ligand-dependent and ligand-independent mechanisms is known to occur as well. Examples include Notch signaling arising in the absence of ligand binding, and cis-inhibition of Notch signaling by titration of the Notch receptor upon binding to its ligands within a single cell. Increasing experimental evidences support that the binding of the Notch receptor with its ligands within a cell (cis-interactions) can also trigger a cell-autonomous Notch signal (cis-signaling), whose potential effects on cell fate decisions and patterning remain poorly understood. To address this question, herein we mathematically and computationally investigate the cell states arising from the combination of cis-signaling with additional Notch signaling sources, which are either cell-autonomous or involve cell-to-cell communication. Our study shows that cis-signaling can switch from driving cis-activation to effectively perform cis-inhibition and identifies under which conditions this switch occurs. This switch relies on the competition between Notch signaling sources, which share the same receptor but differ in their signaling efficiency. We propose that the role of cis-interactions and their signaling on fine-grained patterning and cell fate decisions is dependent on whether they drive cis-inhibition or cis-activation, which could be controlled during development. Specifically, cis-inhibition and not cis-activation facilitates patterning and enriches it by modulating the ratio of cells in the high-ligand expression state, by enabling additional periodic patterns like stripes and by allowing localized patterning highly sensitive to the precursor state and cell-autonomous bistability. Our study

  4. Notch1, Notch2, and Epstein-Barr virus-encoded nuclear antigen 2 signaling differentially affects proliferation and survival of Epstein-Barr virus-infected B cells.

    PubMed

    Kohlhof, Hella; Hampel, Franziska; Hoffmann, Reinhard; Burtscher, Helmut; Weidle, Ulrich H; Hölzel, Michael; Eick, Dirk; Zimber-Strobl, Ursula; Strobl, Lothar J

    2009-05-28

    The canonical mode of transcriptional activation by both the Epstein-Barr viral protein, Epstein-Barr virus-encoded nuclear antigen 2 (EBNA2), and an activated Notch receptor (Notch-IC) requires their recruitment to RBPJ, suggesting that EBNA2 uses the Notch pathway to achieve B-cell immortalization. To gain further insight into the biologic equivalence between Notch-IC and EBNA2, we performed a genome-wide expression analysis, revealing that Notch-IC and EBNA2 exhibit profound differences in the regulation of target genes. Whereas Notch-IC is more potent in regulating genes associated with differentiation and development, EBNA2 is more potent in inducing viral and cellular genes involved in proliferation, survival, and chemotaxis. Because both EBNA2 and Notch-IC induced the expression of cell cycle-associated genes, we analyzed whether Notch1-IC or Notch2-IC can replace EBNA2 in B-cell immortalization. Although Notch-IC could drive quiescent B cells into the cell cycle, B-cell immortalization was not maintained, partially due to an increased apoptosis rate in Notch-IC-expressing cells. Expression analysis revealed that both EBNA2 and Notch-IC induced the expression of proapoptotic genes, but only in EBNA2-expressing cells were antiapoptotic genes strongly up-regulated. These findings suggest that Notch signaling in B cells and B-cell lymphomas is only compatible with proliferation if pathways leading to antiapototic signals are active. PMID:19339697

  5. Reduced Notch signalling leads to postnatal skeletal muscle hypertrophy in Pofut1cax/cax mice.

    PubMed

    Al Jaam, Bilal; Heu, Katy; Pennarubia, Florian; Segelle, Alexandre; Magnol, Laetitia; Germot, Agnès; Legardinier, Sébastien; Blanquet, Véronique; Maftah, Abderrahman

    2016-09-01

    Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O-fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1(cax/cax) (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1(cax/cax) mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1(cax/cax) SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7(+)/MYOD(-) progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O-fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice. PMID:27628322

  6. Reduced Notch signalling leads to postnatal skeletal muscle hypertrophy in Pofut1cax/cax mice

    PubMed Central

    Al Jaam, Bilal; Heu, Katy; Pennarubia, Florian; Segelle, Alexandre; Magnol, Laetitia; Germot, Agnès; Blanquet, Véronique; Maftah, Abderrahman

    2016-01-01

    Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O-fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1cax/cax (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1cax/cax mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1cax/cax SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7+/MYOD− progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O-fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice. PMID:27628322

  7. Expression of Notch pathway genes in the embryonic mouse metanephros suggests a role in proximal tubule development.

    PubMed

    Leimeister, Cornelia; Schumacher, Nina; Gessler, Manfred

    2003-10-01

    The interaction of neighboring cells via Notch signalling leads to cell fate determination, differentiation and patterning of highly organized tissues. Mice with targeted disruption of genes from the Notch signal transduction pathway display defects in the developing somites, neurogenic structures, blood vessels, heart and other organs. Recent studies have added requirements for Notch signalling during kidney, pancreas and thymus morphogenesis. Here, we describe the expression of all four receptors (Notch1-4), the five transmembrane ligands (Dll1, 3, 4, Jag1 and Jag2), intracellular effectors (the Hey genes) and extracellular modulators (Lfng, Mfng, Rfng) in the developing mouse metanephros. Our results point to a Lfng-dependent role for Notch signalling in the development of nephron segments, especially the proximal tubules.

  8. The Mammalian Orthologs of Drosophila Lgd, CC2D1A and CC2D1B, Function in the Endocytic Pathway, but Their Individual Loss of Function Does Not Affect Notch Signalling

    PubMed Central

    Drusenheimer, Nadja; Migdal, Bernhard; Jäckel, Sandra; Tveriakhina, Lena; Scheider, Kristina; Schulz, Katharina; Gröper, Jieny; Köhrer, Karl; Klein, Thomas

    2015-01-01

    CC2D1A and CC2D1B belong to the evolutionary conserved Lgd protein family with members in all multi-cellular animals. Several functions such as centrosomal cleavage, involvement in signalling pathways, immune response and synapse maturation have been described for CC2D1A. Moreover, the Drosophila melanogaster ortholog Lgd was shown to be involved in the endosomal trafficking of the Notch receptor and other transmembrane receptors and physically interacts with the ESCRT-III component Shrub/CHMP4. To determine if this function is conserved in mammals we generated and characterized Cc2d1a and Cc2d1b conditional knockout mice. While Cc2d1b deficient mice displayed no obvious phenotype, we found that Cc2d1a deficient mice as well as conditional mutants that lack CC2D1A only in the nervous system die shortly after birth due to respiratory distress. This finding confirms the suspicion that the breathing defect is caused by the central nervous system. However, an involvement in centrosomal function could not be confirmed in Cc2d1a deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific Cc2d1a mutant mice. These mice did not display any alterations in goblet cell number, proliferating cell number or expression of the Notch reporter Hes1-emGFP, suggesting that CC2D1A is not required for Notch signalling. However, our EM analysis revealed that the average size of endosomes of Cc2d1a mutant cells, but not Cc2d1b mutant cells, is increased, indicating a defect in endosomal morphogenesis. We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced. This indicates that CC2D1A cycles between the cytosol and the endosomal membrane. Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd. Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT

  9. Metabolic Syndrome Impairs Notch Signaling and Promotes Apoptosis in Chronically Ischemic Myocardium

    PubMed Central

    Elmadhun, Nassrene Y.; Sabe, Ashraf A.; Lassaletta, Antonio D.; Chu, Louis M.; Kondra, Katelyn; Sturek, Michael; Sellke, Frank W.

    2014-01-01

    Objective Impaired angiogenesis is a known consequence of metabolic syndrome (MetS), however, the mechanism is not fully understood. Recent studies have shown that the Notch signaling pathway is an integral component of cardiac angiogenesis. We tested in a clinically relevant swine model the effects of MetS on Notch and apoptosis signaling in chronically ischemic myocardium. Methods Ossabaw swine were fed either a regular diet (CTL, n=8) or a high-cholesterol diet (MetS, n=8) to induce MetS. An ameroid constrictor was placed to induce chronic myocardial ischemia. Eleven weeks later, animals underwent cardiac harvest of the ischemic myocardium. Results There was down-regulation of pro-angiogenesis proteins Notch2, Notch4, Jagged2, Ang1 and ENOS in the MetS group compared to CTL. There was also up-regulation of pro-apoptosis protein Caspase8, and down-regulation of anti-angiogenesis protein pFOX03, and pro-survival proteins pP38 and HSP90 in the MetS group. Cell death was increased in the MetS group compared to CTL. Both CTL and MetS groups had similar arteriolar count and capillary density, and Notch3 and Jagged1 were both similarly concentrated in the smooth muscle wall in both groups. Conclusions MetS in chronic myocardial ischemia significantly impairs Notch signaling by down regulating Notch receptors, ligands and pro-angiogenesis proteins. MetS also increases apoptosis signaling, decreases survival signaling and increases cell death in chronically ischemic myocardium. Although short-term angiogenesis appears unaffected in this model of early MetS, the molecular signals for angiogenesis are impaired, thus suggesting that inhibition of Notch signaling may underlie decreased angiogenesis in later stages of MetS. PMID:25037620

  10. Prolactin signaling enhances colon cancer stemness by modulating Notch signaling in a Jak2-STAT3/ERK manner.

    PubMed

    Neradugomma, Naveen K; Subramaniam, Dharmalingam; Tawfik, Ossama W; Goffin, Vincent; Kumar, T Rajendra; Jensen, Roy A; Anant, Shrikant

    2014-04-01

    Prolactin (PRL) is a secretory cytokine produced by various tissues. Binding to the cognate PRL receptor (PRLR), it activates intracellular signaling via janus kinase (JAK), extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription (STAT) proteins. PRL regulates diverse activities under normal and abnormal conditions, including malignancies. Previous clinical data suggest serum PRL levels are elevated in colorectal cancer (CRC) patients. In this study, we first determined the expression of PRL and PRLR in colon cancer tissue and cell lines. Higher levels of PRLR expression were observed in the cancer cells and cell lines compared with normal colonic epithelial cells. Incubation of colon cancer cells with PRL-induced JAK2, STAT3 and ERK1/2 phosphorylation and increased expression of Jagged 1, which is a Notch-1 receptor ligand. Notch signaling regulates CRC stem cell population. We observed increased accumulation of the cleaved/active form of Notch-1 receptor (Notch intracellular domain) and increased expression of Notch responsive genes HEY1, HES1 and stem cell marker genes DCLK1, LGR5, ALDH1 and CD44. Finally, inhibiting PRL induced JAK2-STAT3 and JAK2-ERK1/2 using AG490 and PD98059, respectively, leads to complete abrogation of Notch signaling, suggesting a role for this pathway in regulating CRC stem cells. Together, our results demonstrate that cytokine signaling induced by PRL is active in colorectal cancers and may provide a novel target for therapeutic intervention.

  11. Lhx2 Is an Essential Factor for Retinal Gliogenesis and Notch Signaling

    PubMed Central

    de Melo, Jimmy; Zibetti, Cristina; Clark, Brian S.; Hwang, Woochang; Miranda-Angulo, Ana L.; Qian, Jiang

    2016-01-01

    Müller glia (MG) are the only glial cell type produced by the neuroepithelial progenitor cells that generate the vertebrate retina. MG are required to maintain retinal homeostasis and support the survival of retinal neurons. Furthermore, in certain vertebrate classes, MG function as adult stem cells, mediating retinal regeneration in response to injury. However, the mechanisms that regulate MG development are poorly understood because there is considerable overlap in gene expression between retinal progenitor cells and differentiated MG. We show that the LIM homeodomain transcription factor Lhx2 is required for the development of MG in the mouse retina. Temporally controlled knock-out studies reveal a requirement for Lhx2 during all stages of MG development, ranging from the proliferation of gliocompetent retinal progenitors, activation of Müller-specific gene expression, and terminal differentiation of MG morphological features. We show that Lhx2 regulates gliogenesis in part by regulating directly the expression of Notch pathway genes including Notch1, Dll1, and Dll3 and gliogenic transcription factors such as Hes1, Hes5, Sox8, and Rax. Conditional knock-out of Lhx2 resulted in a rapid downregulation of Notch pathway genes and loss of Notch signaling. We further demonstrate that Müller gliogenesis induced by misexpression of the potently gliogenic Notch pathway transcriptional effector Hes5 requires Lhx2 expression. These results indicate that Lhx2 not only directly regulates expression of Notch signaling pathway components, but also acts together with the gliogenic Notch pathway to drive MG specification and differentiation. SIGNIFICANCE STATEMENT Müller glia (MG) are radial glial cells located in the vertebrate retina that are essential for the function and survival of retinal neurons. We found the LIM homeodomain transcription factor Lhx2 to be expressed in both retinal progenitor cells and MG. Using conditional knock-outs, we show that Lhx2 is required

  12. Iterative Role of Notch Signaling in Spinal Motor Neuron Diversification.

    PubMed

    Tan, G Christopher; Mazzoni, Esteban O; Wichterle, Hynek

    2016-07-26

    The motor neuron progenitor domain in the ventral spinal cord gives rise to multiple subtypes of motor neurons and glial cells. Here, we examine whether progenitors found in this domain are multipotent and which signals contribute to their cell-type-specific differentiation. Using an in vitro neural differentiation model, we demonstrate that motor neuron progenitor differentiation is iteratively controlled by Notch signaling. First, Notch controls the timing of motor neuron genesis by repressing Neurogenin 2 (Ngn2) and maintaining Olig2-positive progenitors in a proliferative state. Second, in an Ngn2-independent manner, Notch contributes to the specification of median versus hypaxial motor column identity and lateral versus medial divisional identity of limb-innervating motor neurons. Thus, motor neuron progenitors are multipotent, and their diversification is controlled by Notch signaling that iteratively increases cellular diversity arising from a single neural progenitor domain. PMID:27425621

  13. Proinflammatory cytokine tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) suppresses satellite cell self-renewal through inversely modulating Notch and NF-κB signaling pathways.

    PubMed

    Ogura, Yuji; Mishra, Vivek; Hindi, Sajedah M; Kuang, Shihuan; Kumar, Ashok

    2013-12-01

    Satellite cell self-renewal is an essential process to maintaining the robustness of skeletal muscle regenerative capacity. However, extrinsic factors that regulate self-renewal of satellite cells are not well understood. Here, we demonstrate that TWEAK cytokine reduces the proportion of Pax7(+)/MyoD(-) cells (an index of self-renewal) on myofiber explants and represses multiple components of Notch signaling in satellite cell cultures. The number of Pax7(+) cells is significantly increased in skeletal muscle of TWEAK knock-out (KO) mice compared with wild-type in response to injury. Furthermore, Notch signaling is significantly elevated in cultured satellite cells and in regenerating myofibers of TWEAK-KO mice. Forced activation of Notch signaling through overexpression of the Notch1 intracellular domain (N1ICD) rescued the TWEAK-mediated inhibition of satellite cell self-renewal. TWEAK also activates the NF-κB transcription factor in satellite cells and inhibition of NF-κB significantly improved the number of Pax7(+) cells in TWEAK-treated cultures. Furthermore, our results demonstrate that a reciprocal interaction between NF-κB and Notch signaling governs the inhibitory effect of TWEAK on satellite cell self-renewal. Collectively, our study demonstrates that TWEAK suppresses satellite cell self-renewal through activating NF-κB and repressing Notch signaling.

  14. Physiological Notch signaling promotes gliogenesis in the developing peripheral and central nervous systems

    PubMed Central

    Taylor, Merritt K.; Kelly, Yeager; Morrison, Sean J.

    2009-01-01

    Constitutive activation of the Notch pathway can promote gliogenesis by peripheral (PNS) and central (CNS) nervous system progenitors. This raises the question of whether physiological Notch signaling regulates gliogenesis in vivo. To test this, we conditionally deleted Rbpsuh (Rbpj) from mouse PNS or CNS progenitors using Wnt1-Cre or Nestin-Cre. Rbpsuh encodes a DNA-binding protein (RBP/J) that is required for canonical signaling by all Notch receptors. In most regions of the developing PNS and spinal cord, Rbpsuh deletion caused only mild defects in neurogenesis, but severe defects in gliogenesis. These resulted from defects in glial specification or differentiation, not premature depletion of neural progenitors, because we were able to culture undifferentiated progenitors from the PNS and spinal cord despite their failure to form glia in vivo. In spinal cord progenitors, Rbpsuh was required to maintain Sox9 expression during gliogenesis, demonstrating that Notch signaling promotes the expression of a glial-specification gene. These results demonstrate that physiological Notch signaling is required for gliogenesis in vivo, independent of the role of Notch in the maintenance of undifferentiated neural progenitors. PMID:17537790

  15. Activation of Notch1 signaling in stromal fibroblasts inhibits melanoma growth by upregulating WISP-1.

    PubMed

    Shao, H; Cai, L; Grichnik, J M; Livingstone, A S; Velazquez, O C; Liu, Z-J

    2011-10-20

    The tumor microenvironment is emerging as an important target for cancer therapy. Fibroblasts (Fbs) within the tumor stroma are critically involved in promoting tumor growth and angiogenesis through secretion of soluble factors, synthesis of extracellular matrix and direct cell-cell interaction. In this work, we aim to alter the biological activity of stromal Fbs by modulating the Notch1 signaling pathway. We show that Fbs engineered to constitutively activate the Notch1 pathway significantly inhibit melanoma growth and tumor angiogenesis. We determine that the inhibitory effect of 'Notch-engineered' Fbs is mediated by increased secretion of Wnt-induced secreted protein-1 (WISP-1) as the effects of Notch1 activation in Fbs are reversed by shRNA-mediated blockade of WISP-1. When 'Notch-engineered' Fbs are co-grafted with melanoma cells in SCID mice, shRNA-mediated blockade of WISP-1 reverses the tumor-suppressive phenotype of the 'Notch-engineered' Fbs, significantly increases melanoma growth and tumor angiogenesis. Consistent with these findings, supplement of recombinant WISP-1 protein inhibits melanoma cell growth in vitro. In addition, WISP-1 is modestly expressed in melanoma-activated Fbs but highly expressed in inactivated Fbs. Evaluation of human melanoma skin biopsies indicates that expression of WISP-1 is significantly lower in melanoma nests and surrounding areas filled with infiltrated immune cells than in the adjacent dermis unaffected by the melanoma. Overall, our study shows that constitutive activation of the Notch1 pathway confers Fbs with a suppressive phenotype to melanoma growth, partially through WISP-1. Thus, targeting tumor stromal Fbs by activating Notch signaling and/or increasing WISP-1 may represent a novel therapeutic approach to combat melanoma.

  16. Mind bomb1 is a ubiquitin ligase essential for mouse embryonic development and Notch signaling.

    PubMed

    Barsi, Julius C; Rajendra, Rashmi; Wu, Jiang I; Artzt, Karen

    2005-10-01

    The Notch-Delta signaling pathway controls many conserved cell determination events. While the Notch end is fairly well characterized, the Delta end remains poorly understood. Mind bomb1 (MIB1) is one of two E3 ligases known to ubiquitinate Delta. We report here that a targeted mutation of Mib1 in mice results in embryonic lethality by E10.5. Mutants exhibit multiple defects due to their inability to modulate Notch signaling. As histopathology revealed a strong neurogenic phenotype, this study concentrates on characterizing the Mib1 mutant by analyzing Notch pathway components in embryonic neuroepithelium prior to developmental arrest. Premature neurons were observed to undergo apoptosis soon after differentiation. Aberrant neurogenesis is a direct consequence of lowered Hes1 and Hes5 expression resulting from the inability to generate Notch1 intracellular domain (NICD1). We conclude that MIB1 activity is required for S3 cleavage of the Notch1 receptor. These results have direct implications for manipulating the differentiation of neuronal stem cells and provide a putative target for the modulation of specific tumors.

  17. Alterations in Notch signalling in skeletal muscles from mdx and dko dystrophic mice and patients with Duchenne muscular dystrophy.

    PubMed

    Church, Jarrod E; Trieu, Jennifer; Chee, Annabel; Naim, Timur; Gehrig, Stefan M; Lamon, Séverine; Angelini, Corrado; Russell, Aaron P; Lynch, Gordon S

    2014-04-01

    New Findings What is the central question of this study? The Notch signalling pathway plays an important role in muscle regeneration, and activation of the pathway has been shown to enhance muscle regeneration in aged mice. It is unknown whether Notch activation will have a similarly beneficial effect on muscle regeneration in the context of Duchenne muscular dystrophy (DMD). What is the main finding and its importance? Although expression of Notch signalling components is altered in both mouse models of DMD and in human DMD patients, activation of the Notch signalling pathway does not confer any functional benefit on muscles from dystrophic mice, suggesting that other signalling pathways may be more fruitful targets for manipulation in treating DMD. Abstract In Duchenne muscular dystrophy (DMD), muscle damage and impaired regeneration lead to progressive muscle wasting, weakness and premature death. The Notch signalling pathway represents a central regulator of gene expression and is critical for cellular proliferation, differentiation and apoptotic signalling during all stages of embryonic muscle development. Notch activation improves muscle regeneration in aged mice, but its potential to restore regeneration and function in muscular dystrophy is unknown. We performed a comprehensive examination of several genes involved in Notch signalling in muscles from dystrophin-deficient mdx and dko (utrophin- and dystrophin-null) mice and DMD patients. A reduction of Notch1 and Hes1 mRNA in tibialis anterior muscles of dko mice and quadriceps muscles of DMD patients and a reduction of Hes1 mRNA in the diaphragm of the mdx mice were observed, with other targets being inconsistent across species. Activation and inhibition of Notch signalling, followed by measures of muscle regeneration and function, were performed in the mouse models of DMD. Notch activation had no effect on functional regeneration in C57BL/10, mdx or dko mice. Notch inhibition significantly depressed the

  18. Systemic Inhibition of Canonical Notch Signaling Results in Sustained Callus Inflammation and Alters Multiple Phases of Fracture Healing

    PubMed Central

    Dishowitz, Michael I.; Mutyaba, Patricia L.; Takacs, Joel D.; Barr, Andrew M.; Engiles, Julie B.; Ahn, Jaimo; Hankenson, Kurt D.

    2013-01-01

    The Notch signaling pathway is an important regulator of embryological bone development, and many aspects of development are recapitulated during bone repair. We have previously reported that Notch signaling components are upregulated during bone fracture healing. However, the significance of the Notch pathway in bone regeneration has not been described. Therefore, the objective of this study was to determine the importance of Notch signaling in regulating bone fracture healing by using a temporally controlled inducible transgenic mouse model (Mx1-Cre;dnMAMLf/-) to impair RBPjκ-mediated canonical Notch signaling. The Mx1 promoter was synthetically activated resulting in temporally regulated systemic dnMAML expression just prior to creation of bilateral tibial fractures. This allowed for mice to undergo unaltered embryological and post-natal skeletal development. Results showed that systemic Notch inhibition prolonged expression of inflammatory cytokines and neutrophil cell inflammation, and reduced the proportion of cartilage formation within the callus at 10 days-post-fracture (dpf) Notch inhibition did not affect early bone formation at 10dpf, but significantly altered bone maturation and remodeling at 20dpf. Increased bone volume fraction in dnMAML fractures, which was due to a moderate decrease in callus size with no change in bone mass, coincided with increased trabecular thickness but decreased connectivity density, indicating that patterning of bone was altered. Notch inhibition decreased total osteogenic cell density, which was comprised of more osteocytes rather than osteoblasts. dnMAML also decreased osteoclast density, suggesting that osteoclast activity may also be important for altered fracture healing. It is likely that systemic Notch inhibition had both direct effects within cell types as well as indirect effects initiated by temporally upstream events in the fracture healing cascade. Surprisingly, Notch inhibition did not alter cell proliferation

  19. Activation of Notch1 synergizes with multiple pathways in promoting castration-resistant prostate cancer

    PubMed Central

    Stoyanova, Tanya; Riedinger, Mireille; Lin, Shu; Faltermeier, Claire M.; Smith, Bryan A.; Zhang, Kelvin X.; Going, Catherine C.; Goldstein, Andrew S.; Lee, John K.; Drake, Justin M.; Rice, Meghan A.; Hsu, En-Chi; Nowroozizadeh, Behdokht; Castor, Brandon; Orellana, Sandra Y.; Blum, Steven M.; Cheng, Donghui; Pienta, Kenneth J.; Reiter, Robert E.; Pitteri, Sharon J.; Huang, Jiaoti; Witte, Owen N.

    2016-01-01

    Metastatic castration-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-specific mortality. Defining new mechanisms that can predict recurrence and drive lethal CRPC is critical. Here, we demonstrate that localized high-risk prostate cancer and metastatic CRPC, but not benign prostate tissues or low/intermediate-risk prostate cancer, express high levels of nuclear Notch homolog 1, translocation-associated (Notch1) receptor intracellular domain. Chronic activation of Notch1 synergizes with multiple oncogenic pathways altered in early disease to promote the development of prostate adenocarcinoma. These tumors display features of epithelial-to-mesenchymal transition, a cellular state associated with increased tumor aggressiveness. Consistent with its activation in clinical CRPC, tumors driven by Notch1 intracellular domain in combination with multiple pathways altered in prostate cancer are metastatic and resistant to androgen deprivation. Our study provides functional evidence that the Notch1 signaling axis synergizes with alternative pathways in promoting metastatic CRPC and may represent a new therapeutic target for advanced prostate cancer. PMID:27694579

  20. Mechanism of Notch Pathway Activation and Its Role in the Regulation of Olfactory Plasticity in Drosophila melanogaster

    PubMed Central

    2016-01-01

    The neural plasticity of sensory systems is being increasingly recognized as playing a role in learning and memory. We have previously shown that Notch, part of an evolutionarily conserved intercellular signaling pathway, is required in adult Drosophila melanogaster olfactory receptor neurons (ORNs) for the structural and functional plasticity of olfactory glomeruli that is induced by chronic odor exposure. In this paper we address how long-term exposure to odor activates Notch and how Notch in conjunction with chronic odor mediates olfactory plasticity. We show that upon chronic odor exposure a non-canonical Notch pathway mediates an increase in the volume of glomeruli by a mechanism that is autonomous to ORNs. In addition to activating a pathway that is autonomous to ORNs, chronic odor exposure also activates the Notch ligand Delta in second order projection neurons (PNs), but this does not appear to require acetylcholine receptor activation in PNs. Delta on PNs then feeds back to activate canonical Notch signaling in ORNs, which restricts the extent of the odor induced increase in glomerular volume. Surprisingly, even though the pathway that mediates the increase in glomerular volume is autonomous to ORNs, nonproductive transsynaptic Delta/Notch interactions that do not activate the canonical pathway can block the increase in volume. In conjunction with chronic odor, the canonical Notch pathway also enhances cholinergic activation of PNs. We present evidence suggesting that this is due to increased acetylcholine release from ORNs. In regulating physiological plasticity, Notch functions solely by the canonical pathway, suggesting that there is no direct connection between morphological and physiological plasticity. PMID:26986723

  1. Mechanism of Notch Pathway Activation and Its Role in the Regulation of Olfactory Plasticity in Drosophila melanogaster.

    PubMed

    Kidd, Simon; Lieber, Toby

    2016-01-01

    The neural plasticity of sensory systems is being increasingly recognized as playing a role in learning and memory. We have previously shown that Notch, part of an evolutionarily conserved intercellular signaling pathway, is required in adult Drosophila melanogaster olfactory receptor neurons (ORNs) for the structural and functional plasticity of olfactory glomeruli that is induced by chronic odor exposure. In this paper we address how long-term exposure to odor activates Notch and how Notch in conjunction with chronic odor mediates olfactory plasticity. We show that upon chronic odor exposure a non-canonical Notch pathway mediates an increase in the volume of glomeruli by a mechanism that is autonomous to ORNs. In addition to activating a pathway that is autonomous to ORNs, chronic odor exposure also activates the Notch ligand Delta in second order projection neurons (PNs), but this does not appear to require acetylcholine receptor activation in PNs. Delta on PNs then feeds back to activate canonical Notch signaling in ORNs, which restricts the extent of the odor induced increase in glomerular volume. Surprisingly, even though the pathway that mediates the increase in glomerular volume is autonomous to ORNs, nonproductive transsynaptic Delta/Notch interactions that do not activate the canonical pathway can block the increase in volume. In conjunction with chronic odor, the canonical Notch pathway also enhances cholinergic activation of PNs. We present evidence suggesting that this is due to increased acetylcholine release from ORNs. In regulating physiological plasticity, Notch functions solely by the canonical pathway, suggesting that there is no direct connection between morphological and physiological plasticity. PMID:26986723

  2. Radiation-Induced Notch Signaling in Breast Cancer Stem Cells

    SciTech Connect

    Lagadec, Chann; Vlashi, Erina; Alhiyari, Yazeed; Phillips, Tiffany M.; Bochkur Dratver, Milana; Pajonk, Frank

    2013-11-01

    Purpose: To explore patterns of Notch receptor and ligand expression in response to radiation that could be crucial in defining optimal dosing schemes for γ-secretase inhibitors if combined with radiation. Methods and Materials: Using MCF-7 and T47D breast cancer cell lines, we used real-time reverse transcription–polymerase chain reaction to study the Notch pathway in response to radiation. Results: We show that Notch receptor and ligand expression during the first 48 hours after irradiation followed a complex radiation dose–dependent pattern and was most pronounced in mammospheres, enriched for breast cancer stem cells. Additionally, radiation activated the Notch pathway. Treatment with a γ-secretase inhibitor prevented radiation-induced Notch family gene expression and led to a significant reduction in the size of the breast cancer stem cell pool. Conclusions: Our results indicate that, if combined with radiation, γ-secretase inhibitors may prevent up-regulation of Notch receptor and ligand family members and thus reduce the number of surviving breast cancer stem cells.

  3. Mutations in TSPEAR, Encoding a Regulator of Notch Signaling, Affect Tooth and Hair Follicle Morphogenesis

    PubMed Central

    Samuelov, Liat; Bertolini, Marta; Weissglas-Volkov, Daphna; Eskin-Schwartz, Marina; Malchin, Natalia; Bochner, Ron; Fainberg, Gilad; Goldberg, Ilan; Sugawara, Koji; Tsuruta, Daisuke; Morasso, Maria; Shalev, Stavit; Gallo, Richard L.; Shomron, Noam; Paus, Ralf; Sprecher, Eli

    2016-01-01

    Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway. PMID:27736875

  4. Notch signaling regulates M2 type macrophage polarization during the development of proliferative vitreoretinopathy.

    PubMed

    Zhang, Jingjing; Zhou, Qingjun; Yuan, Gongqiang; Dong, Muchen; Shi, Weiyun

    2015-01-01

    Macrophages play an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). M2 macrophages can promote tissue remodeling and repair. In this study, CD206 positive M2 type macrophages were found in preretinal fibrous membranes of the mouse model of PVR induced by the intravitreal injection of retinal pigment epithelial (RPE) cells. Notch signaling determines M2 macrophage polarization. The specific inhibition of Notch signaling pathway by the intravitreal injection of γ-secretase inhibitor DAPT attenuated RPE cells-induced PVR formation as demonstrated by the decreased expression of α-SMA, and inhibited M2 type macrophage infiltation as demonstrated by the decreased expression of Arg-1. Notch signaling may modulate PVR formation by regulating M2 type macrophage polarization. PMID:26410397

  5. [Notch signaling in bone formation and related skeletal diseases].

    PubMed

    Ma, Hongwei; Wu, Yaqiong; Zhang, Haifeng

    2015-04-01

    Notch signaling is highly conserved in evolution and regarded as a key factor in cell fate determination. It mediates cell-to-cell interactions that are critical for embryonic development and tissue renewal, and is involved in the occurrence and metastasis of neoplasm. Recent researches have found that such signaling plays an important role in modulating the differentiation of chondrocytes, osteoblasts and osteoclasts. Dysfunction of Notch signaling can result in many skeletal diseases such as bone tumor, disorders of bone development or bone metabolism.

  6. Immunohistochemical localization of notch signaling molecules in ameloblastomas

    PubMed Central

    2011-01-01

    We examined Notch signaling molecules, Notch1 and Jagged1, in serial large cases of typical solid/multicystic ameloblastoma. In general, Notch positive staining products were frequently detected in the cytoplasms of the cells. In the same cells, Jagged positive staining were also frequently observed, while only occasionally positive in peripheral cells, especially in cuboidal cells. The results showed that these morphogenesis regulation factors are closely related to cytological differentiation in neoplastic cells of ameloblastoma. The Notch and Jagged positive-cell ratios were frequently positive, and the ratios were nearly the same between the varied histopathological, cytological patterns. However, the less-differentiated cells were fewer in number than that of well-differentiated cells. PMID:21810559

  7. Integrative genetic, epigenetic and pathological analysis of paraganglioma reveals complex dysregulation of NOTCH signaling.

    PubMed

    Cama, Alessandro; Verginelli, Fabio; Lotti, Lavinia Vittoria; Napolitano, Francesco; Morgano, Annalisa; D'Orazio, Andria; Vacca, Michele; Perconti, Silvia; Pepe, Felice; Romani, Federico; Vitullo, Francesca; di Lella, Filippo; Visone, Rosa; Mannelli, Massimo; Neumann, Hartmut P H; Raiconi, Giancarlo; Paties, Carlo; Moschetta, Antonio; Tagliaferri, Roberto; Veronese, Angelo; Sanna, Mario; Mariani-Costantini, Renato

    2013-10-01

    Head and neck paragangliomas, rare neoplasms of the paraganglia composed of nests of neurosecretory and glial cells embedded in vascular stroma, provide a remarkable example of organoid tumor architecture. To identify genes and pathways commonly deregulated in head and neck paraganglioma, we integrated high-density genome-wide copy number variation (CNV) analysis with microRNA and immunomorphological studies. Gene-centric CNV analysis of 24 cases identified a list of 104 genes most significantly targeted by tumor-associated alterations. The "NOTCH signaling pathway" was the most significantly enriched term in the list (P = 0.002 after Bonferroni or Benjamini correction). Expression of the relevant NOTCH pathway proteins in sustentacular (glial), chief (neuroendocrine) and endothelial cells was confirmed by immunohistochemistry in 47 head and neck paraganglioma cases. There were no relationships between level and pattern of NOTCH1/JAG2 protein expression and germline mutation status in the SDH genes, implicated in paraganglioma predisposition, or the presence/absence of immunostaining for SDHB, a surrogate marker of SDH mutations. Interestingly, NOTCH upregulation was observed also in cases with no evidence of CNVs at NOTCH signaling genes, suggesting altered epigenetic modulation of this pathway. To address this issue we performed microarray-based microRNA expression analyses. Notably 5 microRNAs (miR-200a,b,c and miR-34b,c), including those most downregulated in the tumors, correlated to NOTCH signaling and directly targeted NOTCH1 in in vitro experiments using SH-SY5Y neuroblastoma cells. Furthermore, lentiviral transduction of miR-200s and miR-34s in patient-derived primary tympano-jugular paraganglioma cell cultures was associated with NOTCH1 downregulation and increased levels of markers of cell toxicity and cell death. Taken together, our results provide an integrated view of common molecular alterations associated with head and neck paraganglioma and

  8. Family Based Whole Exome Sequencing Reveals the Multifaceted Role of Notch Signaling in Congenital Heart Disease

    PubMed Central

    Chetaille, Philippe; Prince, Andrea; Godard, Beatrice; Leclerc, Severine; Sobreira, Nara; Ling, Hua; Awadalla, Philip; Thibeault, Maryse; Khairy, Paul; Samuels, Mark E.; Andelfinger, Gregor

    2016-01-01

    Left-ventricular outflow tract obstructions (LVOTO) encompass a wide spectrum of phenotypically heterogeneous heart malformations which frequently cluster in families. We performed family based whole-exome and targeted re-sequencing on 182 individuals from 51 families with multiple affected members. Central to our approach is the family unit which serves as a reference to identify causal genotype-phenotype correlations. Screening a multitude of 10 overlapping phenotypes revealed disease associated and co-segregating variants in 12 families. These rare or novel protein altering mutations cluster predominantly in genes (NOTCH1, ARHGAP31, MAML1, SMARCA4, JARID2, JAG1) along the Notch signaling cascade. This is in line with a significant enrichment (Wilcoxon, p< 0.05) of variants with a higher pathogenicity in the Notch signaling pathway in patients compared to controls. The significant enrichment of novel protein truncating and missense mutations in NOTCH1 highlights the allelic and phenotypic heterogeneity in our pediatric cohort. We identified novel co-segregating pathogenic mutations in NOTCH1 associated with left and right-sided cardiac malformations in three independent families with a total of 15 affected individuals. In summary, our results suggest that a small but highly pathogenic fraction of family specific mutations along the Notch cascade are a common cause of LVOTO. PMID:27760138

  9. Involvement of the Notch pathway in terminal astrocytic differentiation: role of PKA.

    PubMed

    Angulo-Rojo, Carla; Manning-Cela, Rebeca; Aguirre, Adán; Ortega, Arturo; López-Bayghen, Esther

    2013-12-23

    The Notch pathway is a highly conserved signaling system essential for modulating neurogenesis and promoting astrogenesis. Similarly, the cAMP signaling cascade can promote astrocytic commitment in several cell culture models, such as the C6 glioma cell line. These cells have the capacity to differentiate into oligodendrocytes or astrocytes, characteristics that allow their use as a glial progenitor model. In this context, we explore here the plausible involvement of cAMP in Notch-dependent signal transactions. The exposure of C6 cells to a non-hydrolysable cAMP analogue resulted in a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD) and transcriptional activity. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage and is sensitive to inhibitors of the cAMP-dependent protein kinase, PKA. As expected, Notch cleavage and nuclear translocation resulted in the up-regulation of the mRNA levels of one of its target genes, the transcription factor Hair and enhancer of split 5. Moreover, the glutamate uptake activity, as well as the expression of astrocytic markers such as glial fibrillary acidic protein, S100β protein and GLAST was also enhanced in cAMP-exposed cells. Our results clearly suggest that during the process of C6 astrocytic differentiation, cAMP activates the PKA/γ-secretase/NICD/RBPJ(κ) pathway and Notch1 expression, leading to transcriptional activation of the genes responsible for glial progenitor cell fate decision.

  10. Involvement of the Notch pathway in terminal astrocytic differentiation: role of PKA

    PubMed Central

    Angulo-Rojo, Carla; Manning-Cela, Rebeca; Aguirre, Adán; Ortega, Arturo; López-Bayghen, Esther

    2013-01-01

    The Notch pathway is a highly conserved signaling system essential for modulating neurogenesis and promoting astrogenesis. Similarly, the cAMP signaling cascade can promote astrocytic commitment in several cell culture models, such as the C6 glioma cell line. These cells have the capacity to differentiate into oligodendrocytes or astrocytes, characteristics that allow their use as a glial progenitor model. In this context, we explore here the plausible involvement of cAMP in Notch-dependent signal transactions. The exposure of C6 cells to a non-hydrolysable cAMP analogue resulted in a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD) and transcriptional activity. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage and is sensitive to inhibitors of the cAMP-dependent protein kinase, PKA. As expected, Notch cleavage and nuclear translocation resulted in the up-regulation of the mRNA levels of one of its target genes, the transcription factor Hair and enhancer of split 5. Moreover, the glutamate uptake activity, as well as the expression of astrocytic markers such as glial fibrillary acidic protein, S100β protein and GLAST was also enhanced in cAMP-exposed cells. Our results clearly suggest that during the process of C6 astrocytic differentiation, cAMP activates the PKA/γ-secretase/NICD/RBPJκ pathway and Notch1 expression, leading to transcriptional activation of the genes responsible for glial progenitor cell fate decision. PMID:24286475

  11. The Notch ligand Delta-like 1 integrates inputs from TGFbeta/Activin and Wnt pathways

    SciTech Connect

    Bordonaro, Michael Tewari, Shruti Atamna, Wafa Lazarova, Darina L.

    2011-06-10

    Unlike the well-characterized nuclear function of the Notch intracellular domain, it has been difficult to identify a nuclear role for the ligands of Notch. Here we provide evidence for the nuclear function of the Notch ligand Delta-like 1 in colon cancer (CC) cells exposed to butyrate. We demonstrate that the intracellular domain of Delta-like 1 (Dll1icd) augments the activity of Wnt signaling-dependent reporters and that of the promoter of the connective tissue growth factor (CTGF) gene. Data suggest that Dll1icd upregulates CTGF promoter activity through both direct and indirect mechanisms. The direct mechanism is supported by co-immunoprecipitation of endogenous Smad2/3 proteins and Dll1 and by chromatin immunoprecipitation analyses that revealed the occupancy of Dll1icd on CTGF promoter sequences containing a Smad binding element. The indirect upregulation of CTGF expression by Dll1 is likely due to the ability of Dll1icd to increase Wnt signaling, a pathway that targets CTGF. CTGF expression is induced in butyrate-treated CC cells and results from clonal growth assays support a role for CTGF in the cell growth-suppressive role of butyrate. In conclusion, integration of the Notch, Wnt, and TGFbeta/Activin signaling pathways is in part mediated by the interactions of Dll1 with Smad2/3 and Tcf4.

  12. Dephosphorylated parafibromin is a transcriptional coactivator of the Wnt/Hedgehog/Notch pathways

    PubMed Central

    Kikuchi, Ippei; Takahashi-Kanemitsu, Atsushi; Sakiyama, Natsuki; Tang, Chao; Tang, Pei-Jung; Noda, Saori; Nakao, Kazuki; Kassai, Hidetoshi; Sato, Toshiro; Aiba, Atsu; Hatakeyama, Masanori

    2016-01-01

    Evolutionally conserved Wnt, Hedgehog (Hh) and Notch morphogen pathways play essential roles in the development, homeostasis and pathogenesis of multicellular organisms. Nevertheless, mechanisms that intracellularly coordinate these signal inputs remain poorly understood. Here we found that parafibromin, a component of the PAF complex, competitively interacts with β-catenin and Gli1, thereby potentiating transactivation of Wnt- and Hh-target genes in a mutually exclusive manner. Parafibromin also binds to the Notch intracellular domain (NICD), enabling concerted activation of Wnt- and Notch-target genes. The transcriptional platform function of parafibromin is potentiated by tyrosine dephosphorylation, mediated by SHP2 phosphatase, while it is attenuated by tyrosine phosphorylation, mediated by PTK6 kinase. Consequently, acute loss of parafibromin in mice disorganizes the normal epithelial architecture of the intestine, which requires coordinated activation/inactivation of Wnt, Hh and/or Notch signalling. Parafibromin integrates and converts signals conveyed by these morphogen pathways into appropriate transcriptional outputs in a tyrosine phosphorylation/dephosphorylation-regulated manner. PMID:27650679

  13. Dephosphorylated parafibromin is a transcriptional coactivator of the Wnt/Hedgehog/Notch pathways.

    PubMed

    Kikuchi, Ippei; Takahashi-Kanemitsu, Atsushi; Sakiyama, Natsuki; Tang, Chao; Tang, Pei-Jung; Noda, Saori; Nakao, Kazuki; Kassai, Hidetoshi; Sato, Toshiro; Aiba, Atsu; Hatakeyama, Masanori

    2016-09-21

    Evolutionally conserved Wnt, Hedgehog (Hh) and Notch morphogen pathways play essential roles in the development, homeostasis and pathogenesis of multicellular organisms. Nevertheless, mechanisms that intracellularly coordinate these signal inputs remain poorly understood. Here we found that parafibromin, a component of the PAF complex, competitively interacts with β-catenin and Gli1, thereby potentiating transactivation of Wnt- and Hh-target genes in a mutually exclusive manner. Parafibromin also binds to the Notch intracellular domain (NICD), enabling concerted activation of Wnt- and Notch-target genes. The transcriptional platform function of parafibromin is potentiated by tyrosine dephosphorylation, mediated by SHP2 phosphatase, while it is attenuated by tyrosine phosphorylation, mediated by PTK6 kinase. Consequently, acute loss of parafibromin in mice disorganizes the normal epithelial architecture of the intestine, which requires coordinated activation/inactivation of Wnt, Hh and/or Notch signalling. Parafibromin integrates and converts signals conveyed by these morphogen pathways into appropriate transcriptional outputs in a tyrosine phosphorylation/dephosphorylation-regulated manner.

  14. RAM-induced Allostery Facilitates Assembly of a Notch Pathway Active Transcription Complex

    SciTech Connect

    Friedmann, David R.; Wilson, Jeffrey J.; Kovall, Rhett A.

    2008-07-09

    The Notch pathway is a conserved cell-to-cell signaling mechanism, in which extracellular signals are transduced into transcriptional outputs through the nuclear effector CSL. CSL is converted from a repressor to an activator through the formation of the CSL-NotchIC-Mastermind ternary complex. The RAM (RBP-J associated molecule) domain of NotchIC avidly interacts with CSL; however, its role in assembly of the CSL-NotchIC-Mastermind ternary complex is not understood. Here we provide a comprehensive thermodynamic, structural, and biochemical analysis of the RAM-CSL interaction for components from both mouse and worm. Our binding data show that RAM and CSL form a high affinity complex in the presence or absence of DNA. Our structural studies reveal a striking distal conformational change in CSL upon RAM binding, which creates a docking site for Mastermind to bind to the complex. Finally, we show that the addition of a RAM peptide in trans facilitates formation of the CSL-NotchIC-Mastermind ternary complex in vitro.

  15. Aspartate mutations in presenilin and gamma-secretase inhibitors both impair notch1 proteolysis and nuclear translocation with relative preservation of notch1 signaling.

    PubMed

    Berezovska, O; Jack, C; McLean, P; Aster, J C; Hicks, C; Xia, W; Wolfe, M S; Kimberly, W T; Weinmaster, G; Selkoe, D J; Hyman, B T

    2000-08-01

    It has been hypothesized that a presenilin 1 (PS1)-related enzymatic activity is responsible for proteolytic cleavage of the C-terminal intracellular protein of Notch1, in addition to its role in beta-amyloid protein (Abeta) formation from the amyloid precursor protein (APP). We developed an assay to monitor ligand-induced Notch1 proteolysis and nuclear translocation in individual cells : Treatment of full-length Notch1-enhanced green fluorescent protein-transfected Chinese hamster ovary (CHO) cells with a soluble preclustered form of the physiologic ligand Delta leads to rapid accumulation of the C terminus of Notch1 in the nucleus and to transcriptional activation of a C-promoter binding factor 1 (CBF1) reporter construct. Nuclear translocation was blocked by cotransfection with Notch's physiologic inhibitor Numb. Using this assay, we now confirm and extend the observation that PS1 is involved in Notch1 nuclear translocation and signaling in mammalian cells. We demonstrate that the D257A and the D385A PS1 mutations, which had been shown previously to block APP gamma-secretase activity, also prevent Notch1 cleavage and translocation to the nucleus but do not alter Notch1 trafficking to the cell surface. We also show that two APP gamma-secretase inhibitors block Notch1 nuclear translocation with an IC(50) similar to that reported for APP gamma-secretase. Notch1 signaling, assessed by measuring the activity of CBF1, a downstream transcription factor, was impaired but not abolished by the PS1 aspartate mutations or gamma-secretase inhibitors. Our results support the hypotheses that (a) PS1-dependent APP gamma-secretase-like enzymatic activity is critical for both APP and Notch processing and (b) the Notch1 signaling pathway remains partially activated even when Notch1 proteolytic processing and nuclear translocation are markedly inhibited. The latter is an important finding from the perspective of therapeutic treatment of Alzheimer's disease by targeting gamma

  16. Increasing Notch signaling antagonizes PRC2-mediated silencing to promote reprograming of germ cells into neurons

    PubMed Central

    Seelk, Stefanie; Adrian-Kalchhauser, Irene; Hargitai, Balázs; Hajduskova, Martina; Gutnik, Silvia; Tursun, Baris; Ciosk, Rafal

    2016-01-01

    Cell-fate reprograming is at the heart of development, yet very little is known about the molecular mechanisms promoting or inhibiting reprograming in intact organisms. In the C. elegans germline, reprograming germ cells into somatic cells requires chromatin perturbation. Here, we describe that such reprograming is facilitated by GLP-1/Notch signaling pathway. This is surprising, since this pathway is best known for maintaining undifferentiated germline stem cells/progenitors. Through a combination of genetics, tissue-specific transcriptome analysis, and functional studies of candidate genes, we uncovered a possible explanation for this unexpected role of GLP-1/Notch. We propose that GLP-1/Notch promotes reprograming by activating specific genes, silenced by the Polycomb repressive complex 2 (PRC2), and identify the conserved histone demethylase UTX-1 as a crucial GLP-1/Notch target facilitating reprograming. These findings have wide implications, ranging from development to diseases associated with abnormal Notch signaling. DOI: http://dx.doi.org/10.7554/eLife.15477.001 PMID:27602485

  17. Opposing actions of endocannabinoids on cholangiocarcinoma growth is via the differential activation of Notch signaling

    SciTech Connect

    Frampton, Gabriel; Coufal, Monique; Li, Huang; Ramirez, Jonathan; DeMorrow, Sharon

    2010-05-15

    The endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2-AG) have opposing effects on cholangiocarcinoma growth. Implicated in cancer, Notch signaling requires the {gamma}-secretase complex for activation. The aims of this study were to determine if the opposing effects of endocannabinoids depend on the differential activation of the Notch receptors and to demonstrate that the differential activation of these receptors are due to presenilin 1 containing- and presenilin 2 containing-{gamma}-secretase complexes. Mz-ChA-1 cells were treated with AEA or 2-AG. Notch receptor expression, activation, and nuclear translocation were determined. Specific roles for Notch 1 and 2 on cannabinoid-induced effects were determined by transient transfection of Notch 1 or 2 shRNA vectors before stimulation with AEA or 2-AG. Expression of presenilin 1 and 2 was determined after AEA or 2-AG treatment, and the involvement of presenilin 1 and 2 in the cannabinoid-induced effects was demonstrated in cell lines with low presenilin 1 or 2 expression. Antiproliferative effects of AEA required increased Notch 1 mRNA, activation, and nuclear translocation, whereas the growth-promoting effects induced by 2-AG required increased Notch 2 mRNA expression, activation, and nuclear translocation. AEA increased presenilin 1 expression and recruitment into the {gamma}-secretase complex, whereas 2-AG increased expression and recruitment of presenilin 2. The development of novel therapeutic strategies aimed at modulating the endocannabinoid system or mimicking the mode of action of AEA on Notch signaling pathways would prove beneficial for cholangiocarcinoma management.

  18. NRF2-mediated Notch pathway activation enhances hematopoietic reconstitution following myelosuppressive radiation

    PubMed Central

    Kim, Jung-Hyun; Thimmulappa, Rajesh K.; Kumar, Vineet; Cui, Wanchang; Kumar, Sarvesh; Kombairaju, Ponvijay; Zhang, Hao; Margolick, Joseph; Matsui, William; Macvittie, Thomas; Malhotra, Sanjay V.; Biswal, Shyam

    2014-01-01

    A nuclear disaster may result in exposure to potentially lethal doses of ionizing radiation (IR). Hematopoietic acute radiation syndrome (H-ARS) is characterized by severe myelosuppression, which increases the risk of infection, bleeding, and mortality. Here, we determined that activation of nuclear factor erythroid-2–related factor 2 (NRF2) signaling enhances hematopoietic stem progenitor cell (HSPC) function and mitigates IR-induced myelosuppression and mortality. Augmenting NRF2 signaling in mice, either by genetic deletion of the NRF2 inhibitor Keap1 or by pharmacological NRF2 activation with 2-trifluoromethyl-2′-methoxychalone (TMC), enhanced hematopoietic reconstitution following bone marrow transplantation (BMT). Strikingly, even 24 hours after lethal IR exposure, oral administration of TMC mitigated myelosuppression and mortality in mice. Furthermore, TMC administration to irradiated transgenic Notch reporter mice revealed activation of Notch signaling in HSPCs and enhanced HSPC expansion by increasing Jagged1 expression in BM stromal cells. Administration of a Notch inhibitor ablated the effects of TMC on hematopoietic reconstitution. Taken together, we identified a mechanism by which NRF2-mediated Notch signaling improves HSPC function and myelosuppression following IR exposure. Our data indicate that targeting this pathway may provide a countermeasure against the damaging effects of IR exposure. PMID:24463449

  19. Notch Activity Modulates the Responsiveness of Neural Progenitors to Sonic Hedgehog Signaling

    PubMed Central

    Kong, Jennifer H.; Yang, Linlin; Dessaud, Eric; Chuang, Katherine; Moore, Destaye M.; Rohatgi, Rajat; Briscoe, James; Novitch, Bennett G.

    2015-01-01

    Summary Throughout the developing nervous system, neural stem and progenitor cells give rise to diverse classes of neurons and glia in a spatially and temporally coordinated manner. In the ventral spinal cord, much of this diversity emerges through the morphogen actions of Sonic hedgehog (Shh). Interpretation of the Shh gradient depends on both the amount of ligand and duration of exposure, but the mechanisms permitting prolonged responses to Shh are not well understood. We demonstrate that Notch signaling plays an essential role in this process, enabling neural progenitors to attain sufficiently high levels of Shh pathway activity needed to direct the ventral-most cell fates. Notch activity regulates subcellular localization of the Shh receptor Patched1, gating the translocation of the key effector Smoothened to primary cilia and its downstream signaling activities. These data reveal an unexpected role for Notch shaping the interpretation of the Shh morphogen gradient and influencing cell fate determination. PMID:25936505

  20. Loss of Notch3 Signaling in Vascular Smooth Muscle Cells Promotes Severe Heart Failure Upon Hypertension.

    PubMed

    Ragot, Hélène; Monfort, Astrid; Baudet, Mathilde; Azibani, Fériel; Fazal, Loubina; Merval, Régine; Polidano, Evelyne; Cohen-Solal, Alain; Delcayre, Claude; Vodovar, Nicolas; Chatziantoniou, Christos; Samuel, Jane-Lise

    2016-08-01

    Hypertension, which is a risk factor of heart failure, provokes adaptive changes at the vasculature and cardiac levels. Notch3 signaling plays an important role in resistance arteries by controlling the maturation of vascular smooth muscle cells. Notch3 deletion is protective in pulmonary hypertension while deleterious in arterial hypertension. Although this latter phenotype was attributed to renal and cardiac alterations, the underlying mechanisms remained unknown. To investigate the role of Notch3 signaling in the cardiac adaptation to hypertension, we used mice with either constitutive Notch3 or smooth muscle cell-specific conditional RBPJκ knockout. At baseline, both genotypes exhibited a cardiac arteriolar rarefaction associated with oxidative stress. In response to angiotensin II-induced hypertension, the heart of Notch3 knockout and SM-RBPJκ knockout mice did not adapt to pressure overload and developed heart failure, which could lead to an early and fatal acute decompensation of heart failure. This cardiac maladaptation was characterized by an absence of media hypertrophy of the media arteries, the transition of smooth muscle cells toward a synthetic phenotype, and an alteration of angiogenic pathways. A subset of mice exhibited an early fatal acute decompensated heart failure, in which the same alterations were observed, although in a more rapid timeframe. Altogether, these observations indicate that Notch3 plays a major role in coronary adaptation to pressure overload. These data also show that the hypertrophy of coronary arterial media on pressure overload is mandatory to initially maintain a normal cardiac function and is regulated by the Notch3/RBPJκ pathway. PMID:27296994

  1. Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling

    PubMed Central

    Bivik, Caroline; MacDonald, Ryan B.; Gunnar, Erika; Mazouni, Khalil; Schweisguth, Francois; Thor, Stefan

    2016-01-01

    The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems. PMID:27070787

  2. Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling.

    PubMed

    Bivik, Caroline; MacDonald, Ryan B; Gunnar, Erika; Mazouni, Khalil; Schweisguth, Francois; Thor, Stefan

    2016-04-01

    The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems. PMID:27070787

  3. An Obligatory Role of Mind Bomb-1 in Notch Signaling of Mammalian Development

    PubMed Central

    Im, Sun-Kyoung; Kim, Yoon-Young; Kim, Cheol-Hee; Suh, Pann-Ghill; Jan, Yuh Nung; Kong, Young-Yun

    2007-01-01

    Background The Notch signaling pathway is an evolutionarily conserved intercellular signaling module essential for cell fate specification that requires endocytosis of Notch ligands. Structurally distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), cooperatively regulate the endocytosis of Notch ligands in Drosophila. However, the respective roles of the mammalian E3 ubiquitin ligases, Neur1, Neur2, Mib1, and Mib2, in mammalian development are poorly understood. Methodology/Principal Findings Through extensive use of mammalian genetics, here we show that Neur1 and Neur2 double mutants and Mib2−/− mice were viable and grossly normal. In contrast, conditional inactivation of Mib1 in various tissues revealed the representative Notch phenotypes: defects of arterial specification as deltalike4 mutants, abnormal cerebellum and skin development as jagged1 conditional mutants, and syndactylism as jagged2 mutants. Conclusions/Significance Our data provide the first evidence that Mib1 is essential for Jagged as well as Deltalike ligand-mediated Notch signaling in mammalian development, while Neur1, Neur2, and Mib2 are dispensable. PMID:18043734

  4. Folic Acid Supplementation Stimulates Notch Signaling and Cell Proliferation in Embryonic Neural Stem Cells

    PubMed Central

    Liu, Huan; Huang, Guo-wei; Zhang, Xu-mei; Ren, Da-lin; X. Wilson, John

    2010-01-01

    The present study investigated the effect of folic acid supplementation on the Notch signaling pathway and cell proliferation in rat embryonic neural stem cells (NSCs). The NSCs were isolated from E14–16 rat brain and grown as neurospheres in serum-free suspension culture. Individual cultures were assigned to one of 3 treatment groups that differed according to the concentration of folic acid in the medium: Control (baseline folic acid concentration of 4 mg/l), low folic acid supplementation (4 mg/l above baseline, Folate-L) and high folic acid supplementation (40 mg/l above baseline, Folate-H). NSCs were identified by their expression of immunoreactive nestin and proliferating cells by incorporation of 5'bromo-2'deoxyuridine. Cell proliferation was also assessed by methyl thiazolyl tetrazolium assay. Notch signaling was analyzed by real-time PCR and western blot analyses of the expression of Notch1 and hairy and enhancer of split 5 (Hes5). Supplementation of NSCs with folic acid increased the mRNA and protein expression levels of Notch1 and Hes5. Folic acid supplementation also stimulated NSC proliferation dose-dependently. Embryonic NSCs respond to folic acid supplementation with increased Notch signaling and cell proliferation. This mechanism may mediate the effects of folic acid supplementation on neurogenesis in the embryonic nervous system. PMID:20838574

  5. The emerging role of Notch pathway in ageing: Focus on the related mechanisms in age-related diseases.

    PubMed

    Balistreri, Carmela Rita; Madonna, Rosalinda; Melino, Gerry; Caruso, Calogero

    2016-08-01

    Notch signaling is an evolutionarily conserved pathway, which is fundamental for the development of all tissues, organs and systems of human body. Recently, a considerable and still growing number of studies have highlighted the contribution of Notch signaling in various pathological processes of the adult life, such as age-related diseases. In particular, the Notch pathway has emerged as major player in the maintenance of tissue specific homeostasis, through the control of proliferation, migration, phenotypes and functions of tissue cells, as well as in the cross-talk between inflammatory cells and the innate immune system, and in onset of inflammatory age-related diseases. However, until now there is a confounding evidence about the related mechanisms. Here, we discuss mechanisms through which Notch signaling acts in a very complex network of pathways, where it seems to have the crucial role of hub. Thus, we stress the possibility to use Notch pathway, the related molecules and pathways constituting this network, both as innovative (predictive, diagnostic and prognostic) biomarkers and targets for personalised treatments for age-related diseases. PMID:27328278

  6. Competition between Jagged-Notch and Endothelin1 Signaling Selectively Restricts Cartilage Formation in the Zebrafish Upper Face

    PubMed Central

    Barske, Lindsey; Askary, Amjad; Zuniga, Elizabeth; Balczerski, Bartosz; Bump, Paul; Nichols, James T.; Crump, J. Gage

    2016-01-01

    The intricate shaping of the facial skeleton is essential for function of the vertebrate jaw and middle ear. While much has been learned about the signaling pathways and transcription factors that control facial patterning, the downstream cellular mechanisms dictating skeletal shapes have remained unclear. Here we present genetic evidence in zebrafish that three major signaling pathways − Jagged-Notch, Endothelin1 (Edn1), and Bmp − regulate the pattern of facial cartilage and bone formation by controlling the timing of cartilage differentiation along the dorsoventral axis of the pharyngeal arches. A genomic analysis of purified facial skeletal precursors in mutant and overexpression embryos revealed a core set of differentiation genes that were commonly repressed by Jagged-Notch and induced by Edn1. Further analysis of the pre-cartilage condensation gene barx1, as well as in vivo imaging of cartilage differentiation, revealed that cartilage forms first in regions of high Edn1 and low Jagged-Notch activity. Consistent with a role of Jagged-Notch signaling in restricting cartilage differentiation, loss of Notch pathway components resulted in expanded barx1 expression in the dorsal arches, with mutation of barx1 rescuing some aspects of dorsal skeletal patterning in jag1b mutants. We also identified prrx1a and prrx1b as negative Edn1 and positive Bmp targets that function in parallel to Jagged-Notch signaling to restrict the formation of dorsal barx1+ pre-cartilage condensations. Simultaneous loss of jag1b and prrx1a/b better rescued lower facial defects of edn1 mutants than loss of either pathway alone, showing that combined overactivation of Jagged-Notch and Bmp/Prrx1 pathways contribute to the absence of cartilage differentiation in the edn1 mutant lower face. These findings support a model in which Notch-mediated restriction of cartilage differentiation, particularly in the second pharyngeal arch, helps to establish a distinct skeletal pattern in the upper

  7. Notch signaling is essential for ventricular chamber development.

    PubMed

    Grego-Bessa, Joaquín; Luna-Zurita, Luis; del Monte, Gonzalo; Bolós, Victoria; Melgar, Pedro; Arandilla, Alejandro; Garratt, Alistair N; Zang, Heesuk; Mukouyama, Yoh-Suke; Chen, Hanying; Shou, Weinian; Ballestar, Esteban; Esteller, Manel; Rojas, Ana; Pérez-Pomares, José María; de la Pompa, José Luis

    2007-03-01

    Ventricular chamber morphogenesis, first manifested by trabeculae formation, is crucial for cardiac function and embryonic viability and depends on cellular interactions between the endocardium and myocardium. We show that ventricular Notch1 activity is highest at presumptive trabecular endocardium. RBPJk and Notch1 mutants show impaired trabeculation and marker expression, attenuated EphrinB2, NRG1, and BMP10 expression and signaling, and decreased myocardial proliferation. Functional and molecular analyses show that Notch inhibition prevents EphrinB2 expression, and that EphrinB2 is a direct Notch target acting upstream of NRG1 in the ventricles. However, BMP10 levels are found to be independent of both EphrinB2 and NRG1 during trabeculation. Accordingly, exogenous BMP10 rescues the myocardial proliferative defect of in vitro-cultured RBPJk mutants, while exogenous NRG1 rescues differentiation in parallel. We suggest that during trabeculation Notch independently regulates cardiomyocyte proliferation and differentiation, two exquisitely balanced processes whose perturbation may result in congenital heart disease.

  8. Notch Signaling is Essential for Ventricular Chamber Development

    PubMed Central

    Grego-Bessa, Joaquín; Luna-Zurita, Luis; Monte, Gonzalo del; Bolós, Victoria; Melgar, Pedro; Arandilla, Alejandro; Garratt, Alistair N.; Zang, Heesuk; Mukouyama, Yoh-suke; Chen, Hanying; Shou, Weinian; Ballestar, Esteban; Esteller, Manel; Rojas, Ana; Pérez-Pomares, José María; de la Pompa, José Luis

    2009-01-01

    Summary Ventricular chamber morphogenesis, first manifested by trabeculae formation, is crucial for cardiac function and embryonic viability and depends on cellular interactions between endocardium and myocardium. We show that ventricular Notch1 activity is highest at presumptive trabecular endocardium. RBPJk and Notch1 mutants show impaired trabeculation and marker expression, attenuated EphrinB2, NRG1 and BMP10 expression and signaling and decreased myocardial proliferation. Functional and molecular analyses show that Notch inhibition prevents EphrinB2 expression and that EphrinB2 is a direct Notch target acting upstream of NRG1 in the ventricles. However, BMP10 levels are found to be independent of both EphrinB2 and NRG1 during trabeculation. Accordingly, exogenous BMP10 rescues the myocardial proliferative defect of in vitro cultured RBPJk mutants, while exogenous NRG1 rescues differentiation in parallel. We suggest that during trabeculation Notch independently regulates cardiomyocyte proliferation and differentiation, two exquisitely balanced processes whose perturbation may result in congenital heart disease. PMID:17336907

  9. Notch-signalling is required for head regeneration and tentacle patterning in Hydra.

    PubMed

    Münder, Sandra; Tischer, Susanne; Grundhuber, Maresa; Büchels, Nathalie; Bruckmeier, Nadine; Eckert, Stefanie; Seefeldt, Carolin A; Prexl, Andrea; Käsbauer, Tina; Böttger, Angelika

    2013-11-01

    Local self-activation and long ranging inhibition provide a mechanism for setting up organising regions as signalling centres for the development of structures in the surrounding tissue. The adult hydra hypostome functions as head organiser. After hydra head removal it is newly formed and complete heads can be regenerated. The molecular components of this organising region involve Wnt-signalling and β-catenin. However, it is not known how correct patterning of hypostome and tentacles are achieved in the hydra head and whether other signals in addition to HyWnt3 are needed for re-establishing the new organiser after head removal. Here we show that Notch-signalling is required for re-establishing the organiser during regeneration and that this is due to its role in restricting tentacle activation. Blocking Notch-signalling leads to the formation of irregular head structures characterised by excess tentacle tissue and aberrant expression of genes that mark the tentacle boundaries. This indicates a role for Notch-signalling in defining the tentacle pattern in the hydra head. Moreover, lateral inhibition by HvNotch and its target HyHes are required for head regeneration and without this the formation of the β-catenin/Wnt dependent head organiser is impaired. Work on prebilaterian model organisms has shown that the Wnt-pathway is important for setting up signalling centres for axial patterning in early multicellular animals. Our data suggest that the integration of Wnt-signalling with Notch-Delta activity was also involved in the evolution of defined body plans in animals.

  10. Effects of S1 Cleavage on the Structure, Surface Export, and Signaling Activity of Human Notch1 and Notch2

    SciTech Connect

    Gordon, Wendy R.; Vardar-Ulu, Didem; L'Heureux, Sarah; Ashworth, Todd; Malecki, Michael J.; Sanchez-Irizarry, Cheryll; McArthur, Debbie G.; Histen, Gavin; Mitchell, Jennifer L.; Aster, Jon C.; Blacklow, Stephen C.

    2009-09-25

    Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia. The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity. S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or

  11. The many facets of Notch ligands

    PubMed Central

    D'souza, Brendan; Miyamoto, Alison; Weinmaster, Gerry

    2009-01-01

    The Notch signaling pathway regulates a diverse array of cell types and cellular processes and is tightly regulated by ligand binding. Both canonical and noncanonical Notch ligands have been identified that may account for some of the pleiotropic nature associated with Notch signaling. This review focuses on the molecular mechanisms by which Notch ligands function as signaling agonists and antagonists, and discusses different modes of activating ligands as well as findings that support intrinsic ligand signaling activity independent of Notch. Post-translational modification, proteolytic processing, endocytosis and membrane trafficking, as well as interactions with the actin cytoskeleton may contribute to the recently appreciated multi-functionality of Notch ligands. The regulation of Notch ligand expression by other signaling pathways provides a mechanism to coordinate Notch signaling with multiple cellular and developmental cues. The association of Notch ligands with inherited human disorders and cancer highlights the importance of understanding the molecular nature and activities intrinsic to Notch ligands. PMID:18758484

  12. Oscillatory Notch-pathway activity in a delay model of neuronal differentiation

    NASA Astrophysics Data System (ADS)

    Momiji, Hiroshi; Monk, Nicholas A. M.

    2009-08-01

    Lateral inhibition resulting from a double-negative feedback loop underlies the assignment of different fates to cells in many developmental processes. Previous studies have shown that the presence of time delays in models of lateral inhibition can result in significant oscillatory transients before patterned steady states are reached. We study the impact of local feedback loops in a model of lateral inhibition based on the Notch signaling pathway, elucidating the roles of intracellular and intercellular delays in controlling the overall system behavior. The model exhibits both in-phase and out-of-phase oscillatory modes and oscillation death. Interactions between oscillatory modes can generate complex behaviors such as intermittent oscillations. Our results provide a framework for exploring the recent observation of transient Notch-pathway oscillations during fate assignment in vertebrate neurogenesis.

  13. Cancer stem cell signaling pathways.

    PubMed

    Matsui, William H

    2016-09-01

    Tissue development and homeostasis are governed by the actions of stem cells. Multipotent cells are capable of self-renewal during the course of one's lifetime. The accurate and appropriate regulation of stem cell functions is absolutely critical for normal biological activity. Several key developmental or signaling pathways have been shown to play essential roles in this regulatory capacity. Specifically, the Janus-activated kinase/signal transducer and activator of transcription, Hedgehog, Wnt, Notch, phosphatidylinositol 3-kinase/phosphatase and tensin homolog, and nuclear factor-κB signaling pathways have all been shown experimentally to mediate various stem cell properties, such as self-renewal, cell fate decisions, survival, proliferation, and differentiation. Unsurprisingly, many of these crucial signaling pathways are dysregulated in cancer. Growing evidence suggests that overactive or abnormal signaling within and among these pathways may contribute to the survival of cancer stem cells (CSCs). CSCs are a relatively rare population of cancer cells capable of self-renewal, differentiation, and generation of serially transplantable heterogeneous tumors of several types of cancer. PMID:27611937

  14. The matricellular protein CCN3 regulates NOTCH1 signalling in chronic myeloid leukaemia.

    PubMed

    Suresh, Sukanya; McCallum, Lynn; Crawford, Lisa J; Lu, Wan Hua; Sharpe, Daniel J; Irvine, Alexandra E

    2013-11-01

    Deregulated NOTCH1 has been reported in lymphoid leukaemia, although its role in chronic myeloid leukaemia (CML) is not well established. We previously reported BCR-ABL down-regulation of a novel haematopoietic regulator, CCN3, in CML; CCN3 is a non-canonical NOTCH1 ligand. This study characterizes the NOTCH1–CCN3 signalling axis in CML. In K562 cells, BCR-ABL silencing reduced full-length NOTCH1 (NOTCH1-FL) and inhibited the cleavage of NOTCH1 intracellular domain (NOTCH1-ICD), resulting in decreased expression of the NOTCH1 targets c-MYC and HES1. K562 cells stably overexpressing CCN3 (K562/CCN3) or treated with recombinant CCN3(rCCN3) showed a significant reduction in NOTCH1 signalling (> 50% reduction in NOTCH1-ICD, p < 0.05).Gamma secretase inhibitor (GSI), which blocks NOTCH1 signalling, reduced K562/CCN3 colony formation but increased that of K562/control cells. GSI combined with either rCCN3 or imatinib reduced K562 colony formation with enhanced reduction of NOTCH1 signalling observed with combination treatments. We demonstrate an oncogenic role for NOTCH1 in CML and suggest that BCR-ABL disruption of NOTCH1–CCN3 signalling contributes to the pathogenesis of CML.

  15. Galectin-3 Inhibits Osteoblast Differentiation through Notch Signaling12

    PubMed Central

    Nakajima, Kosei; Kho, Dhong Hyo; Yanagawa, Takashi; Harazono, Yosuke; Gao, Xiaoge; Hogan, Victor; Raz, Avraham

    2014-01-01

    Patients with bone cancer metastasis suffer from unbearable pain and bone fractures due to bone remodeling. This is caused by tumor cells that disturb the bone microenvironment. Here, we have investigated the role of tumor-secreted sugar-binding protein, i.e., galectin-3, on osteoblast differentiation and report that it downregulates the expression of osteoblast differentiation markers, e.g., RUNX2, SP7, ALPL, COL1A1, IBSP, and BGLAP, of treated human fetal osteoblast (hFOB) cells. Co-culturing of hFOB cells with human breast cancer BT-549 and prostate cancer LNCaP cells harboring galectin-3 has resulted in inhibition of osteoblast differentiation by the secreted galectin-3 into culture medium. The inhibitory effect of galectin-3 was found to be through its binding to Notch1 in a sugar-dependent manner that has led to accelerated Notch1 cleavage and activation of Notch signaling. Taken together, our findings show that soluble galectin-3 in the bone microenvironment niche regulates bone remodeling through Notch signaling, suggesting a novel bone metastasis therapeutic target. PMID:25425968

  16. Faster embryonic segmentation through elevated Delta-Notch signalling

    PubMed Central

    Liao, Bo-Kai; Jörg, David J.; Oates, Andrew C.

    2016-01-01

    An important step in understanding biological rhythms is the control of period. A multicellular, rhythmic patterning system termed the segmentation clock is thought to govern the sequential production of the vertebrate embryo's body segments, the somites. Several genetic loss-of-function conditions, including the Delta-Notch intercellular signalling mutants, result in slower segmentation. Here, we generate DeltaD transgenic zebrafish lines with a range of copy numbers and correspondingly increased signalling levels, and observe faster segmentation. The highest-expressing line shows an altered oscillating gene expression wave pattern and shortened segmentation period, producing embryos with more, shorter body segments. Our results reveal surprising differences in how Notch signalling strength is quantitatively interpreted in different organ systems, and suggest a role for intercellular communication in regulating the output period of the segmentation clock by altering its spatial pattern. PMID:27302627

  17. Myeloid-Specific Blockade of Notch Signaling by RBP-J Knockout Attenuates Spinal Cord Injury Accompanied by Compromised Inflammation Response in Mice.

    PubMed

    Chen, Bei-Yu; Zheng, Min-Hua; Chen, Yan; Du, Yan-Ling; Sun, Xiao-Long; Zhang, Xing; Duan, Li; Gao, Fang; Liang, Liang; Qin, Hong-Yan; Luo, Zhuo-Jing; Han, Hua

    2015-12-01

    The outcome of spinal cord injury (SCI) is determined by both neural cell-intrinsic survival pathways and tissue microenvironment-derived signals. Macrophages dominating the inflammatory responses in SCI possess both destructive and reparative potentials, according to their activation status. Notch signaling is involved in both cell survival and macrophage-mediated inflammation, but a comprehensive role of Notch signaling in SCI has been elusive. In this study, we compared the effects of general Notch blockade by a pharmaceutical γ-secretase inhibitor (GSI) and myeloid-specific Notch signal disruption by recombination signal binding protein Jκ (RBP-J) knockout on SCI. The administration of Notch signal inhibitor GSI resulted in worsened hind limb locomotion and exacerbated inflammation. However, mice lacking RBP-J, the critical transcription factor mediating signals from all four mammalian Notch receptors, in myeloid lineage displayed promoted functional recovery, attenuated glial scar formation, improved neuronal survival and axon regrowth, and mitigated inflammatory response after SCI. These benefits were accompanied by enhanced AKT activation in the lesion area after SCI. These findings demonstrate that abrogating Notch signal in myeloid cells ameliorates inflammation response post-SCI and promotes functional recovery, but general pharmaceutical Notch interception has opposite effects. Therefore, clinical intervention of Notch signaling in SCI needs to pinpoint myeloid lineage to avoid the counteractive effects of global inhibition.

  18. Drosophila Epsin mediates a select endocytic pathway that DSL ligands must enter to activate Notch.

    PubMed

    Wang, Weidong; Struhl, Gary

    2004-11-01

    Recent findings suggest that Delta/Serrate/Lag2 (DSL) signals activate Notch by an unprecedented mechanism that requires the ligands to be endocytosed in signal-sending cells to activate the receptor in signal-receiving cells. Here, we show that cells devoid of Epsin, a conserved adaptor protein for Clathrin-mediated endocytosis, behave normally except that they cannot send DSL signals. Surprisingly, we find that Epsin is not required for bulk endocytosis of DSL proteins. Instead, Epsin appears to be essential for targeting DSL proteins to a special endocytic pathway that they must enter to acquire signaling activity. We present evidence that DSL proteins must be mono-ubiquitinated to be targeted by Epsin to this pathway. Furthermore, we show that the requirements for both Epsin and mono-ubiquitination can be bypassed by introducing the internalization signal that mediates endocytosis and recycling of the Low Density Lipoprotein (LDL) receptor. We propose that Epsin is essential for DSL signaling because it targets mono-ubiquitinated DSL proteins to an endocytic recycling compartment that they must enter to be converted into active ligands. Alternatively Epsin may be required to target mono-ubiquitinated DSL proteins to a particular subclass of coated pits that have special properties essential for Notch activation.

  19. Crucial role of Notch signaling in osteogenic differentiation of periodontal ligament stem cells in osteoporotic rats.

    PubMed

    Li, Ying; Li, S Q; Gao, Y M; Li, Jin; Zhang, Bin

    2014-06-01

    Estrogen deficiency-induced osteoporosis typically occurs in postmenopausal women and has been strongly associated with periodontal diseases. Periodontal ligament stem cells (PDLSCs) isolated from the periodontal ligament can differentiate into many types of specialized cells, including osteoblast-like cells that contribute to periodontal tissue repair. The Notch signaling pathway is highly conserved and associated with self-renewal potential and cell-fate determination. Recently, several studies have focused on the relationship between Notch signaling and osteogenic differentiation. However, the precise mechanisms underlying this relationship are largely unknown. We have successfully isolated PDLSCs from both ovariectomized (OVX) and sham-operated rats. Both the mRNA and protein levels of Notch1 and Jagged1 were upregulated when PDLSCs were cultured in osteogenic induction media. Mineralization assays showed decreased calcium deposits in OVX-PDLSCs treated with a γ-secretase inhibitor compared with control cells. Thus Notch signaling is important in maintaining the osteogenic differentiation of PDLSCs in osteoporotic rats, which help in the development of a potential therapeutic strategy for periodontal disease in postmenopausal women.

  20. Heat shock protein 70 (Hsp70) interacts with the Notch1 intracellular domain and contributes to the activity of Notch signaling in myelin-reactive CD4 T cells.

    PubMed

    Juryńczyk, Maciej; Lewkowicz, Przemysław; Domowicz, Małgorzata; Mycko, Marcin P; Selmaj, Krzysztof W

    2015-10-15

    Notch receptors (Notch1-4) are involved in the differentiation of CD4 T cells and the development of autoimmunity. Mechanisms regulating Notch signaling in CD4 T cells are not fully elucidated. In this study we investigated potential crosstalk between Notch pathway molecules and heat shock protein 70 (Hsp70), the major intracellular chaperone involved in the protein transport during immune responses and other stress conditions. Using Hsp70(-/-) mice we found that Hsp70 is critical for up-regulation of NICD1 and induction of Notch target genes in Jagged1- and Delta-like1-stimulated CD4 T cells. Co-immunoprecipitation analysis of wild-type CD4 T cells stimulated with either Jagged1 or Delta-like1 showed a direct interaction between NICD1 and Hsp70. Both molecules co-localized within the nucleus of CD4 T cells stimulated with Notch ligands. Molecular interaction and nuclear colocalization of NICD1 and Hsp70 were also detected in CD4 T cells reactive against myelin oligodendrocyte glycoprotein (MOG)35-55, which showed Hsp70-dependent up-regulation of both NICD1 and Notch target genes. In conclusion, we demonstrate for the first time that Hsp70 interacts with NICD1 and contributes to the activity of Notch signaling in CD4 T cells. Interaction between Hsp70 and NICD1 may represent a novel mechanism regulating Notch signaling in activated CD4 T cells.

  1. Canonical and non-canonical Notch ligands

    PubMed Central

    D’SOUZA, BRENDAN; MELOTY-KAPELLA, LAURENCE; WEINMASTER, GERRY

    2015-01-01

    Notch signaling induced by canonical Notch ligands is critical for normal embryonic development and tissue homeostasis through the regulation of a variety of cell fate decisions and cellular processes. Activation of Notch signaling is normally tightly controlled by direct interactions with ligand-expressing cells and dysregulated Notch signaling is associated with developmental abnormalities and cancer. While canonical Notch ligands are responsible for the majority of Notch signaling, a diverse group of structurally unrelated non-canonical ligands has also been identified that activate Notch and likely contribute to the pleiotropic effects of Notch signaling. Soluble forms of both canonical and non-canonical ligands have been isolated, some of which block Notch signaling and could serve as natural inhibitors of this pathway. Ligand activity can also be indirectly regulated by other signaling pathways at the level of ligand expression, serving to spatio-temporally compartmentalize Notch signaling activity and integrate Notch signaling into a molecular network that orchestrates developmental events. Here, we review the molecular mechanisms underlying the dual role of Notch ligands as activators and inhibitors of Notch signaling. Additionally, evidence that Notch ligands function independent of Notch are presented. We also discuss how ligand post-translational modification, endocytosis, proteolysis and spatio-temporal expression regulate their signaling activity. PMID:20816393

  2. Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy

    SciTech Connect

    Liu, Juntao; Mao, Zhangfan; Huang, Jie; Xie, Songping; Liu, Tianshu; Mao, Zhifu

    2014-02-21

    Highlights: • Notch signaling pathway members are expressed lower levels in CD133+ cells. • CD133+ cells are not as sensitive as CD133− cells to chemotherapy. • GSI could inhibit the growth of both CD133+ and CD133− cells. • Blockade of Notch signaling pathway enhanced the effect of chemotherapy with CDDP. • DAPT/CDDP co-therapy caused G2/M arrest and elimination in CD133+ cells. - Abstract: Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatments that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133− cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133− cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G{sub 2}/M phase, and there were half as many cells in S phase compared with the CD133− cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were

  3. Notch Signaling and Atoh1 Expression During Hair Cell Regeneration in the Mouse Utricle

    PubMed Central

    Wang, Guo-Peng; Chatterjee, Ishani; Batts, Shelley A.; Wong, Hiu Tung; Gong, Tzy-Wen; Gong, Shu-Sheng; Raphael, Yehoash

    2010-01-01

    The mammalian vestibular epithelium has a limited capacity for spontaneous hair cell regeneration. The mechanism underlying the regeneration is not well understood. Because the Notch signaling pathway mediates the formation of the sensory epithelial mosaic patterning during ear development, it may also play a role in hair cell regeneration in the mature mammalian vestibular epithelium after a lesion. To investigate the process of spontaneous regeneration in the vestibular epithelium vis-à-vis changes in Notch signaling, we induced a unilateral lesion by infusing streptomycin into the mouse posterior semicircular canal, and examined Notch signaling molecules and their mRNA expression levels by immunohistochemistry and quantitative real-time polymerase chain reaction (qRTPCR), respectively. We detected Jagged1 in supporting cells in both normal and lesioned utricles. Atoh1, a marker for early developing hair cells, was absent in the intact mature tissue, but re-appeared after the lesion. Many cells were either positive for both Atoh1 and myosin VIIa, or for one of them. qRTPCR data showed a post trauma decrease of Hes5 and an increase in Atoh1. Atoh1 up-regulation may either be a result of Hes5 down-regulation or mediated by another signaling pathway. PMID:20433915

  4. Notch signaling regulates venous arterialization during zebrafish fin regeneration

    PubMed Central

    Kametani, Yoshiko; Chi, Neil C.; Stainier, Didier Y.R.; Takada, Shinji

    2015-01-01

    In order to protect against blood pressure, a mature artery is supported by mural cells which include vascular smooth muscle cells and pericytes. To regenerate a functional vascular system, arteries should be properly reconstructed with mural cells although the mechanisms underlying artery reconstruction remain unclear. In this study, we examined the process of artery reconstruction during regeneration of the zebrafish caudal fin as a model to study arterial formation in an adult setting. During fin regeneration, the arteries and veins form a net-like vasculature called the vascular plexus, and this plexus undergoes remodeling to form a new artery and 2 flanking veins. We found that the new vascular plexus originates mainly from venous cells in the stump but very rarely from the arterial cells. Interestingly, these vein-derived cells contributed to the reconstructed arteries. This arterialization was dependent on Notch signaling, and further analysis revealed that Notch signaling was required for the initiation of arterial gene expression. In contrast, venous remodeling did not require Notch signaling. These results provide new insights towards understanding mechanisms of vascular regeneration and illustrate the utility of the adult zebrafish fin to study this process. PMID:25810153

  5. A Novel Transcriptional Factor Nkapl Is a Germ Cell-Specific Suppressor of Notch Signaling and Is Indispensable for Spermatogenesis

    PubMed Central

    Okuda, Hidenobu; Kiuchi, Hiroshi; Takao, Tetsuya; Miyagawa, Yasushi; Tsujimura, Akira; Nonomura, Norio; Miyata, Haruhiko; Okabe, Masaru; Ikawa, Masahito; Kawakami, Yoshitaka; Goshima, Naoki; Wada, Morimasa; Tanaka, Hiromitsu

    2015-01-01

    Spermatogenesis is an elaborately regulated system dedicated to the continuous production of spermatozoa via the genesis of spermatogonia. In this process, a variety of genes are expressed that are relevant to the differentiation of germ cells at each stage. Although Notch signaling plays a critical role in germ cell development in Drosophila and Caenorhabditis elegans, its function and importance for spermatogenesis in mammals is controversial. We report that Nkapl is a novel germ cell-specific transcriptional suppressor in Notch signaling. It is also associated with several molecules of the Notch corepressor complex such as CIR, HDAC3, and CSL. It was expressed robustly in spermatogonia and early spermatocytes after the age of 3 weeks. Nkapl-deleted mice showed complete arrest at the level of pachytene spermatocytes. In addition, apoptosis was observed in this cell type. Overexpression of NKAPL in germline stem cells demonstrated that Nkapl induced changes in spermatogonial stem cell (SSC) markers and the reduction of differentiation factors through the Notch signaling pathway, whereas testes with Nkapl deleted showed inverse changes in those markers and factors. Therefore, Nkapl is indispensable because aberrantly elevated Notch signaling has negative effects on spermatogenesis, affecting SSC maintenance and differentiation factors. Notch signaling should be properly regulated through the transcriptional factor Nkapl. PMID:25875095

  6. NOTCH Pathway Blockade Depletes CD133-Positive Glioblastoma Cells and Inhibits Growth of Tumor Neurospheres and Xenografts

    PubMed Central

    Fan, Xing; Khaki, Leila; Zhu, Thant S.; Soules, Mary E.; Talsma, Caroline E.; Gul, Naheed; Koh, Cheryl; Zhang, Jiangyang; Li, Yue-Ming; Maciaczyk, Jarek; Nikkhah, Guido; DiMeco, Francesco; Piccirillo, Sara; Vescovi, Angelo L.; Eberhart, Charles G.

    2010-01-01

    Cancer stem cells (CSCs) are thought to be critical for the engraftment and long-term growth of many tumors, including glioblastoma (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. It has been shown that the NOTCH signaling pathway regulates normal stem cells in the brain, and that GBMs contain stem-like cells with higher NOTCH activity. We therefore used low-passage and established GBM-derived neurosphere cultures to examine the overall requirement for NOTCH activity, and also examined the effects on tumor cells expressing stem cell markers. NOTCH blockade by γ-secretase inhibitors (GSIs) reduced neurosphere growth and clonogenicity in vitro, whereas expression of an active form of NOTCH2 increased tumor growth. The putative CSC markers CD133, NESTIN, BMI1, and OLIG2 were reduced following NOTCH blockade. When equal numbers of viable cells pretreated with either vehicle (dimethyl sulfoxide) or GSI were injected subcutaneously into nude mice, the former always formed tumors, whereas the latter did not. In vivo delivery of GSI by implantation of drug-impregnated polymer beads also effectively blocked tumor growth, and significantly prolonged survival, albeit in a relatively small cohort of animals. We found that NOTCH pathway inhibition appears to deplete stem-like cancer cells through reduced proliferation and increased apoptosis associated with decreased AKT and STAT3 phosphorylation. In summary, we demonstrate that NOTCH pathway blockade depletes stem-like cells in GBMs, suggesting that GSIs may be useful as chemotherapeutic reagents to target CSCs in malignant gliomas. PMID:19904829

  7. KCTD10 is involved in the cardiovascular system and Notch signaling during early embryonic development.

    PubMed

    Ren, Kaiqun; Yuan, Jing; Yang, Manjun; Gao, Xiang; Ding, Xiaofeng; Zhou, Jianlin; Hu, Xingwang; Cao, Jianguo; Deng, Xiyun; Xiang, Shuanglin; Zhang, Jian

    2014-01-01

    As a member of the polymerase delta-interacting protein 1 (PDIP1) gene family, potassium channel tetramerisation domain-containing 10 (KCTD10) interacts with proliferating cell nuclear antigen (PCNA) and polymerase δ, participates in DNA repair, DNA replication and cell-cycle control. In order to further investigate the physiological functions of KCTD10, we generated the KCTD10 knockout mice. The heterozygous KCTD10(+/-) mice were viable and fertile, while the homozygous KCTD10(-/-) mice showed delayed growth from E9.0, and died at approximately E10.5, which displayed severe defects in angiogenesis and heart development. Further study showed that VEGF induced the expression of KCTD10 in a time- and dose-dependent manner. Quantitative real-time PCR and western blotting results revealed that several key members in Notch signaling were up-regulated either in KCTD10-deficient embryos or in KCTD10-silenced HUVECs. Meanwhile, the endogenous immunoprecipitation (IP) analysis showed that KCTD10 interacted with Cullin3 and Notch1 simultaneously, by which mediating Notch1 proteolytic degradation. Our studies suggest that KCTD10 plays crucial roles in embryonic angiogenesis and heart development in mammalians by negatively regulating the Notch signaling pathway.

  8. Epithelial expression and chromosomal location of human TLE genes: Implications for notch signaling and neoplasia

    SciTech Connect

    Liu, Yanling; Dehni, Ghassan; Stifani, S.

    1996-01-01

    The TLE genes are the human homologues of Drosophila groucho, a member of the Notch signaling pathway. This pathway controls a number of different cell-fate choices in invertebrates and vertebrates. We are interested in investigating the functions of the TLE gene family during epithelial determination and carcinogenesis. We show that expression of individual TLE genes correlates with immature epithelial cells that are progressing toward their terminally differentiated state, suggesting a role during epithelial differentiation. In both normal tissues and conditions resulting from incorrect or incomplete maturation events, such as metaplastic and neoplastic transformations, TLE expression is elevated and coincides with Notch expression, implicating these molecules in the maintenance of the undifferentiated state in epithelial cells. We also show that TLE1 and TLE2 are organized in a tandem array at chromosomal location 19p13.3, while TLE3 maps to 15q22. 26 refs., 4 figs.

  9. Loss of TGF-β Adaptor β2SP Activates Notch Signaling and SOX9 Expression in Esophageal Adenocarcinoma

    PubMed Central

    Song, Shumei; Maru, Dipen M.; Ajani, Jaffer A.; Chan, Chia-Hsin; Honjo, Soichiro; Lin, Hui-Kuan; Correa, Arlene; Hofstetter, Wayne L.; Davila, Marta; Stroehlein, John; Mishra, Lopa

    2013-01-01

    TGF-β and Notch signaling pathways play important roles in regulating self-renewal of stem cells and gastrointestinal carcinogenesis. Loss of TGF-β signaling components activates Notch signaling in esophageal adenocarcinoma, but the basis for this effect has been unclear. Here we report that loss of TGF-β adapter β2SP (SPNB2) activates Notch signaling and its target SOX9 in primary fibroblasts or esophageal adenocarcinoma cells. Expression of the stem cell marker SOX9 was markedly higher in esophageal adenocarcinoma tumor tissues than normal tissues, and its higher nuclear staining in tumors correlated with poorer survival and lymph node invasion in esophageal adenocarcinoma patients. Downregulation of β2SP by lentivirus short hairpin RNA increased SOX9 transcription and expression, enhancing nuclear localization for both active Notch1 (intracellular Notch1, ICN1) and SOX9. In contrast, reintroduction into esophageal adenocarcinoma cells of β2SP and a dominant-negative mutant of the Notch coactivator mastermind-like (dnMAN) decreased SOX9 promoter activity. Tumor sphere formation and invasive capacity in vitro and tumor growth in vivo were increased in β2SP-silenced esophageal adenocarcinoma cells. Conversely, SOX9 silencing rescued the phenotype of esophageal adenocarcinoma cells with loss of β2SP. Interaction between Smad3 and ICN1 via Smad3 MH1 domain was also observed, with loss of β2SP increasing the binding between these proteins, inducing expression of Notch targets SOX9 and C-MYC, and decreasing expression of TGF-β targets p21(CDKN1A), p27 (CDKN1B), and E-cadherin. Taken together, our findings suggest that loss of β2SP switches TGF-β signaling from tumor suppression to tumor promotion by engaging Notch signaling and activating SOX9. PMID:23536563

  10. The transcription factor T-bet is essential for the development of NKp46+ innate lymphocytes via the Notch pathway.

    PubMed

    Rankin, Lucille C; Groom, Joanna R; Chopin, Michaël; Herold, Marco J; Walker, Jennifer A; Mielke, Lisa A; McKenzie, Andrew N J; Carotta, Sebastian; Nutt, Stephen L; Belz, Gabrielle T

    2013-04-01

    NKp46+ innate lymphoid cells (ILCs) serve important roles in regulating the intestinal microbiota and defense against pathogens. Whether NKp46+ ILCs arise directly from lymphoid tissue-inducer (LTi) cells or represent a separate lineage remains controversial. We report here that the transcription factor T-bet (encoded by Tbx21) was essential for the development of NKp46+ ILCs but not of LTi cells or nuocytes. Deficiency in interleukin 22 (IL-22)-producing NKp46+ ILCs resulted in greater susceptibility of Tbx21-/- mice to intestinal infection. Haploinsufficient T-bet expression resulted in lower expression of the signaling molecule Notch, and Notch signaling was necessary for the transition of LTi cells into NKp46+ ILCs. Furthermore, NKp46+ ILCs differentiated solely from the CD4- LTi population, not the CD4+ LTi population. Our results pinpoint the regulation of Notch signaling by T-bet as a distinct molecular pathway that guides the development of NKp46+ ILCs.

  11. The interplay of the Notch signaling in hepatic stellate cells and macrophages determines the fate of liver fibrogenesis.

    PubMed

    Bansal, Ruchi; van Baarlen, Joop; Storm, Gert; Prakash, Jai

    2015-01-01

    Hepatic stellate cells (HSCs) known as "master producers" and macrophages as "master regulators", are the key cell types that strongly contribute to the progression of liver fibrosis. Since Notch signaling regulates multiple cellular processes, we aimed to study the role of Notch signaling in HSCs differentiation and macrophages polarization and to evaluate its implication in liver fibrogenesis. Notch pathway components were found to be significantly upregulated in TGFβ-activated HSCs, inflammatory M1 macrophages, and in mouse and human fibrotic livers. Interestingly, inhibition of Notch using a selective γ-secretase inhibitor, Avagacestat, significantly inhibited TGFβ-induced HSC activation and contractility, and suppressed M1 macrophages. Additionally, Avagacestat inhibited M1 driven-fibroblasts activation and fibroblasts-driven M1 polarization (nitric oxide release) in fibroblasts and macrophages co-culture, and conditioned medium studies. In vivo, post-disease treatment with Avagacestat significantly attenuated fibrogenesis in CCl4-induced liver fibrosis mouse model. These effects were attributed to the reduction in HSCs activation, and inhibition of inflammatory M1 macrophages and upregulation of suppressive M2 macrophages. These findings suggest that Notch signaling plays a crucial role in HSC activation and M1/M2 polarization of macrophages in liver fibrosis. These results provide new insights for the development of novel therapies against liver fibrosis through modulation of Notch signaling.

  12. The interplay of the Notch signaling in hepatic stellate cells and macrophages determines the fate of liver fibrogenesis

    PubMed Central

    Bansal, Ruchi; van Baarlen, Joop; Storm, Gert; Prakash, Jai

    2015-01-01

    Hepatic stellate cells (HSCs) known as “master producers” and macrophages as “master regulators”, are the key cell types that strongly contribute to the progression of liver fibrosis. Since Notch signaling regulates multiple cellular processes, we aimed to study the role of Notch signaling in HSCs differentiation and macrophages polarization and to evaluate its implication in liver fibrogenesis. Notch pathway components were found to be significantly upregulated in TGFβ-activated HSCs, inflammatory M1 macrophages, and in mouse and human fibrotic livers. Interestingly, inhibition of Notch using a selective γ-secretase inhibitor, Avagacestat, significantly inhibited TGFβ-induced HSC activation and contractility, and suppressed M1 macrophages. Additionally, Avagacestat inhibited M1 driven-fibroblasts activation and fibroblasts-driven M1 polarization (nitric oxide release) in fibroblasts and macrophages co-culture, and conditioned medium studies. In vivo, post-disease treatment with Avagacestat significantly attenuated fibrogenesis in CCl4-induced liver fibrosis mouse model. These effects were attributed to the reduction in HSCs activation, and inhibition of inflammatory M1 macrophages and upregulation of suppressive M2 macrophages. These findings suggest that Notch signaling plays a crucial role in HSC activation and M1/M2 polarization of macrophages in liver fibrosis. These results provide new insights for the development of novel therapies against liver fibrosis through modulation of Notch signaling. PMID:26658360

  13. Nandrolone reduces activation of Notch signaling in denervated muscle associated with increased Numb expression

    SciTech Connect

    Liu, Xin-Hua; Yao, Shen; Qiao, Rui-Fang; Levine, Alice C.; Kirschenbaum, Alexander; Pan, Jiangping; Wu, Yong; Qin, Weiping; Bauman, William A.; Cardozo, Christopher P.

    2011-10-14

    Highlights: {yields} Nerve transection increased Notch signaling in paralyzed muscle. {yields} Nandrolone prevented denervation-induced Notch signaling. {yields} Nandrolone induced the expression of an inhibitor of the Notch signaling, Numb. {yields} Reduction of denervation-induced Notch signaling by nandrolone is likely through upregulation of Numb. -- Abstract: Nandrolone, an anabolic steroid, slows denervation-atrophy in rat muscle. The molecular mechanisms responsible for this effect are not well understood. Androgens and anabolic steroids activate Notch signaling in animal models of aging and thereby mitigate sarcopenia. To explore the molecular mechanisms by which nandrolone prevents denervation-atrophy, we investigated the effects of nandrolone on Notch signaling in denervated rat gastrocnemius muscle. Denervation significantly increased Notch activity reflected by elevated levels of nuclear Notch intracellular domain (NICD) and expression of Hey1 (a Notch target gene). Activation was greatest at 7 and 35 days after denervation but remained present at 56 days after denervation. Activation of Notch in denervated muscle was prevented by nandrolone associated with upregulated expression of Numb mRNA and protein. These data demonstrate that denervation activates Notch signaling, and that nandrolone abrogates this response associated with increased expression of Numb, suggesting a potential mechanism by which nandrolone reduces denervation-atrophy.

  14. Constitutive Notch Signaling Causes Abnormal Development of the Oviducts, Abnormal Angiogenesis, and Cyst Formation in Mouse Female Reproductive Tract.

    PubMed

    Ferguson, Lydia; Kaftanovskaya, Elena M; Manresa, Carmen; Barbara, Agustin M; Poppiti, Robert J; Tan, Yingchun; Agoulnik, Alexander I

    2016-03-01

    The Notch signaling pathway is critical for the differentiation of many tissues and organs in the embryo. To study the consequences of Notch1 gain-of-function signaling on female reproductive tract development, we used a cre-loxP strategy and Amhr2-cre transgene to generate mice with conditionally activated Notch1 (Rosa(Notch1)). The Amhr2-cre transgene is expressed in the mesenchyme of developing female reproductive tract and in granulosa cells in the ovary. Double transgenic Amhr2-cre, Rosa(Notch1) females were infertile, whereas control Rosa(Notch1) mice had normal fertility. All female reproductive organs in mutants showed hemorrhaging of blood vessels progressing with age. The mutant oviducts did not develop coiling, and were instead looped around the ovary. There were multiple blockages in the lumen along the oviduct length, creating a barrier for sperm or oocyte passage. Mutant females demonstrated inflamed uteri with increased vascularization and an influx of inflammatory cells. Additionally, older females developed ovarian, oviductal, and uterine cysts. The significant change in gene expression was detected in the mutant oviduct expression of Wnt4, essential for female reproductive tract development. Similar oviductal phenotypes have been detected previously in mice with activated Smo and in beta-catenin, Wnt4, Wnt7a, and Dicer conditional knockouts, indicating a common regulatory pathway disrupted by these genetic abnormalities. PMID:26843448

  15. Regulation of Notch signaling during T- and B-cell development by O-fucose glycans.

    PubMed

    Stanley, Pamela; Guidos, Cynthia J

    2009-07-01

    Notch signaling is required for the development of all T cells and marginal zone (MZ) B cells. Specific roles in T- and B-cell differentiation have been identified for different Notch receptors, the canonical Delta-like (Dll) and Jagged (Jag) Notch ligands, and downstream effectors of Notch signaling. Notch receptors and ligands are post-translationally modified by the addition of glycans to extracellular domain epidermal growth factor-like (EGF) repeats. The O-fucose glycans of Notch cell-autonomously modulate Notch-ligand interactions and the strength of Notch signaling. These glycans are initiated by protein O-fucosyltransferase 1 (Pofut1), and elongated by the transfer of N-acetylglucosamine (GlcNAc) to the fucose by beta1,3GlcNAc-transferases termed lunatic, manic, or radical fringe. This review discusses T- and B-cell development from progenitors deficient in O-fucose glycans. The combined data show that Lfng and Mfng regulate T-cell development by enhancing the interactions of Notch1 in T-cell progenitors with Dll4 on thymic epithelial cells. In the spleen, Lfng and Mfng cooperate to modify Notch2 in MZ B progenitors, enhancing their interaction with Dll1 on endothelial cells and regulating MZ B-cell production. Removal of O-fucose affects Notch signaling in myelopoiesis and lymphopoiesis, and the O-fucose glycan in the Notch1 ligand-binding domain is required for optimal T-cell development.

  16. Blockade of Jagged/Notch pathway abrogates transforming growth factor β2-induced epithelial-mesenchymal transition in human retinal pigment epithelium cells.

    PubMed

    Chen, X; Xiao, W; Liu, X; Zeng, M; Luo, L; Wu, M; Ye, S; Liu, Y

    2014-05-01

    The epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells plays a key role in proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), which lead to the loss of vision. The Jagged/Notch pathway has been reported to be essential in EMT during embryonic development, fibrotic diseases and cancer metastasis. However, the function of Jagged/Notch signaling in EMT of RPE cells is unknown. Thus, we hypothesized that a crosstalk between Notch and transforming growth factor β2 (TGF-β2) signaling could induce EMT in RPE cells, which subsequently contributes to PVR and PDR. Here, we demonstrate that Jagged-1/Notch pathway is involved in the TGF-β2-mediated EMT of human RPE cells. Blockade of Notch pathway with DAPT (a specific inhibitor of Notch receptor cleavage) and knockdown of Jagged-1 expression inhibited TGF-β2-induced EMT through regulating the expression of Snail, Slug and ZEB1. Besides the canonical Smad signaling pathway, the noncanonical PI3K/Akt and MAPK pathway also contributed to TGF-β2-induced up-regulation of Jagged-1 in RPE cells. Overexpression of Jagged-1 could mimic TGF-β2 induce EMT. Our data suggest that the Jagged-1/Notch signaling pathway plays a critical role in TGF-β2-induced EMT in human RPE cells, and may contribute to the development of PVR and PDR. Inhibition of the Jagged/Notch signaling pathway, therefore, may have therapeutic value in the prevention and treatment of PVR and PDR.

  17. TQ inhibits hepatocellular carcinoma growth in vitro and in vivo via repression of Notch signaling

    PubMed Central

    Ke, Xiquan; Zhao, Yan; Lu, Xinlan; Wang, Zhe; Liu, Yuanyuan; Ren, Mudan; Lu, Guifang; Zhang, Dan; Sun, Zhenguo; Xu, Zhipeng; Song, Jee Hoon; Cheng, Yulan; Meltzer, Stephen J.; He, Shuixiang

    2015-01-01

    Thymoquinone (TQ) has been reported to possess anti-tumor activity in various types of cancer. However, its effects and molecular mechanism of action in hepatocellular carcinoma (HCC) are still not completely understood. We observed that TQ inhibited tumor cell growth in vitro, where treatment with TQ arrested the cell cycle in G1 by upregulating p21 and downregulating cyclinD1 and CDK2 expression; moreover, TQ induced apoptosis by decreasing expression of Bcl-2 and increasing expression of Bax. Simultaneously, TQ demonstrated a suppressive impact on the Notch pathway, where overexpression of NICD1 reversed the inhibitory effect of TQ on cell proliferation, thereby attenuating the repressive effects of TQ on the Notch pathway, cyclinD1, CDK2 and Bcl-2, and also diminishing upregulation of p21 and Bax. In a xenograft model, TQ inhibited HCC growth in nude mice; this inhibitory effect in vivo, as well as of HCC cell growth in vitro, was associated with a discernible decline in NICD1 and Bcl-2 levels and a dramatic rise in p21 expression. In conclusion, TQ inhibits HCC cell growth by inducing cell cycle arrest and apoptosis, achieving these effects by repression of the Notch signaling pathway, suggesting that TQ represents a potential preventive or therapeutic agent in HCC patients. PMID:26416455

  18. Compensatory flux changes within an endocytic trafficking network maintain thermal robustness of Notch signaling.

    PubMed

    Shimizu, Hideyuki; Woodcock, Simon A; Wilkin, Marian B; Trubenová, Barbora; Monk, Nicholas A M; Baron, Martin

    2014-05-22

    Developmental signaling is remarkably robust to environmental variation, including temperature. For example, in ectothermic animals such as Drosophila, Notch signaling is maintained within functional limits across a wide temperature range. We combine experimental and computational approaches to show that temperature compensation of Notch signaling is achieved by an unexpected variety of endocytic-dependent routes to Notch activation which, when superimposed on ligand-induced activation, act as a robustness module. Thermal compensation arises through an altered balance of fluxes within competing trafficking routes, coupled with temperature-dependent ubiquitination of Notch. This flexible ensemble of trafficking routes supports Notch signaling at low temperature but can be switched to restrain Notch signaling at high temperature and thus compensates for the inherent temperature sensitivity of ligand-induced activation. The outcome is to extend the physiological range over which normal development can occur. Similar mechanisms may provide thermal robustness for other developmental signals.

  19. Compensatory Flux Changes within an Endocytic Trafficking Network Maintain Thermal Robustness of Notch Signaling

    PubMed Central

    Shimizu, Hideyuki; Woodcock, Simon A.; Wilkin, Marian B.; Trubenová, Barbora; Monk, Nicholas A.M.; Baron, Martin

    2014-01-01

    Summary Developmental signaling is remarkably robust to environmental variation, including temperature. For example, in ectothermic animals such as Drosophila, Notch signaling is maintained within functional limits across a wide temperature range. We combine experimental and computational approaches to show that temperature compensation of Notch signaling is achieved by an unexpected variety of endocytic-dependent routes to Notch activation which, when superimposed on ligand-induced activation, act as a robustness module. Thermal compensation arises through an altered balance of fluxes within competing trafficking routes, coupled with temperature-dependent ubiquitination of Notch. This flexible ensemble of trafficking routes supports Notch signaling at low temperature but can be switched to restrain Notch signaling at high temperature and thus compensates for the inherent temperature sensitivity of ligand-induced activation. The outcome is to extend the physiological range over which normal development can occur. Similar mechanisms may provide thermal robustness for other developmental signals. PMID:24855951

  20. Pre-clinical studies of Notch signaling inhibitor RO4929097 in inflammatory breast cancer cells.

    PubMed

    Debeb, Bisrat G; Cohen, Evan N; Boley, Kimberly; Freiter, Erik M; Li, Li; Robertson, Fredika M; Reuben, James M; Cristofanilli, Massimo; Buchholz, Thomas A; Woodward, Wendy A

    2012-07-01

    Basal breast cancer, common among patients presenting with inflammatory breast cancer (IBC), has been shown to be resistant to radiation and enriched in cancer stem cells. The Notch pathway plays an important role in self-renewal of breast cancer stem cells and contributes to inflammatory signaling which promotes the breast cancer stem cell phenotype. Herein, we inhibited Notch signaling using a gamma secretase inhibitor, RO4929097, in an in vitro model that enriches for cancer initiating cells (3D clonogenic assay) and conventional 2D clonogenic assay to compare the effect on radiosensitization of the SUM149 and SUM190 IBC cell lines. RO4929097 downregulated the Notch target genes Hes1, Hey1, and HeyL, and showed a significant reduction in anchorage independent growth in SUM190 and SUM149. However, the putative self-renewal assay mammosphere formation efficiency was increased with the drug. To assess radiosensitization of putative cancer stem cells, cells were exposed to increasing doses of radiation with or without 1 μM RO4929097 in their standard (2D) and self-renewal enriching (3D) culture conditions. In the conventional 2D clonogenic assay, RO4929097 significantly sensitized SUM190 cells to ionizing radiation and has a modest radiosensitization effect in SUM149 cells. In the 3D clonogenic assays, however, a radioprotective effect was seen in both SUM149 and SUM190 cells at higher doses. Both cell lines express IL-6 and IL-8 cytokines known to mediate the efficacy of Notch inhibition and to promote self-renewal of stem cells. We further showed that RO429097 inhibits normal T-cell synthesis of some inflammatory cytokines, including TNF-α, a potential mediator of IL-6 and IL-8 production in the microenvironment. These data suggest that additional targeting agents may be required to selectively target IBC stem cells through Notch inhibition, and that evaluation of microenvironmental influences may shed further light on the potential effects of this inhibitor.

  1. Notch signaling represses hypoxia-inducible factor-1α-induced activation of Wnt/β-catenin signaling in osteoblasts under cobalt-mimicked hypoxia

    PubMed Central

    LI, CHEN-TIAN; LIU, JIAN-XIU; YU, BO; LIU, RUI; DONG, CHAO; LI, SONG-JIAN

    2016-01-01

    The modification of Wnt and Notch signaling pathways by hypoxia, and its association with osteoblast proliferation and apoptosis remain to be fully elucidated. To investigate Wnt-Notch crosstalk, and its role in hypoxia-induced osteoblast proliferation and apoptosis regulation, the present study investigated the effects of cobalt-mimicked hypoxia on the mouse pre-osteoblast-like cell line, MC3T3-E1, when the Notch signals were repressed using a γ-secretase inhibitor DAPT. The data showed that the cobalt-mimicked hypoxia suppressed cell proliferation under normal conditions, but increased cell proliferation under conditions of Notch repression, in a concentration-dependent manner. The results of western blot and reverse transcription-quantitative polymerase chain reaction analyses showed that the cobalt treatment increased the levels of activated β-catenin protein and the expression levels of the target genes, axis inhibition protein 2 and myelocytomatosis oncogene, under DAPT-induced Notch repression. However, no significant changes were found in the expression levels of the Notch intracellular domain protein or the Notch target gene, hes1. In a β-catenin gene-knockdown experiment, the proliferation of the MC3T3-E1 cells under hypoxia were decreased by DAPT treatment, and knockdown of the expression of hypoxia-inducible factor-1α (HIF-1α) suppressed the cobalt-induced increase in Wnt target gene levels. No significant difference in cell proliferation rate was found following DAPT treatment when the expression of HIF-1α was knocked down. The results of the present study showed the opposing effects of Wnt and Notch signaling under cobalt-mimicked hypoxia, which were partially regulated by HIF-1α, The results also showed that osteoblast proliferation was dependent on Wnt-Notch signal crosstalk. PMID:27220406

  2. Notch1-mediated signaling regulates proliferation of porcine satellite cells (PSCs).

    PubMed

    Qin, Lili; Xu, Jian; Wu, Zhenfang; Zhang, Zhe; Li, Jiaqi; Wang, Chong; Long, Qiaoming

    2013-02-01

    Notch signaling is an evolutionarily conserved cell-cell communication mechanism involved in the regulation of cell proliferation, differentiation and fate decisions of mammalian cells. In the present study, we investigated the possible requirement for Notch signaling in the proliferation and differentiation of porcine satellite cells. We show that Notch1, 2 and 3 are expressed in cultured porcine satellite cells. Knock-down of NOTCH1, but not NOTCH2 and NOTCH3, decreases the proliferation of porcine satellite cells. In contrast, enhancement of NOTCH1 expression via treatment of porcine satellite cells with recombinant NF-κB increases the proliferation of porcine satellite cells. The alteration of porcine satellite cell proliferation is associated with significant changes in the expression of cell cycle related genes (cyclin B1, D1, D2, E1 and p21), myogenic regulatory factors (MyoD and myogenin) and the Notch effector Hes5. In addition, alteration of Notch1 expression in porcine satellite cells causes changes in the expression of GSK3β-3. Taken together, these findings suggest that of the four notch-related genes, Notch1is likely to be required for regulating the proliferation and therefore the maintenance of porcine satellite cells in vivo, and do so through activation of the Notch effector gene Hes5. PMID:23160004

  3. Notch1-mediated signaling regulates proliferation of porcine satellite cells (PSCs).

    PubMed

    Qin, Lili; Xu, Jian; Wu, Zhenfang; Zhang, Zhe; Li, Jiaqi; Wang, Chong; Long, Qiaoming

    2013-02-01

    Notch signaling is an evolutionarily conserved cell-cell communication mechanism involved in the regulation of cell proliferation, differentiation and fate decisions of mammalian cells. In the present study, we investigated the possible requirement for Notch signaling in the proliferation and differentiation of porcine satellite cells. We show that Notch1, 2 and 3 are expressed in cultured porcine satellite cells. Knock-down of NOTCH1, but not NOTCH2 and NOTCH3, decreases the proliferation of porcine satellite cells. In contrast, enhancement of NOTCH1 expression via treatment of porcine satellite cells with recombinant NF-κB increases the proliferation of porcine satellite cells. The alteration of porcine satellite cell proliferation is associated with significant changes in the expression of cell cycle related genes (cyclin B1, D1, D2, E1 and p21), myogenic regulatory factors (MyoD and myogenin) and the Notch effector Hes5. In addition, alteration of Notch1 expression in porcine satellite cells causes changes in the expression of GSK3β-3. Taken together, these findings suggest that of the four notch-related genes, Notch1is likely to be required for regulating the proliferation and therefore the maintenance of porcine satellite cells in vivo, and do so through activation of the Notch effector gene Hes5.

  4. A Screen for Modifiers of Notch Signaling Uncovers Amun, a Protein With a Critical Role in Sensory Organ Development

    PubMed Central

    Shalaby, Nevine A.; Parks, Annette L.; Morreale, Eric J.; Osswalt, Marisa C.; Pfau, Kristen M.; Pierce, Eric L.; Muskavitch, Marc A. T.

    2009-01-01

    Notch signaling is an evolutionarily conserved pathway essential for many cell fate specification events during metazoan development. We conducted a large-scale transposon-based screen in the developing Drosophila eye to identify genes involved in Notch signaling. We screened 10,447 transposon lines from the Exelixis collection for modifiers of cell fate alterations caused by overexpression of the Notch ligand Delta and identified 170 distinct modifier lines that may affect up to 274 genes. These include genes known to function in Notch signaling, as well as a large group of characterized and uncharacterized genes that have not been implicated in Notch pathway function. We further analyze a gene that we have named Amun and show that it encodes a protein that localizes to the nucleus and contains a putative DNA glycosylase domain. Genetic and molecular analyses of Amun show that altered levels of Amun function interfere with cell fate specification during eye and sensory organ development. Overexpression of Amun decreases expression of the proneural transcription factor Achaete, and sensory organ loss caused by Amun overexpression can be rescued by coexpression of Achaete. Taken together, our data suggest that Amun acts as a transcriptional regulator that can affect cell fate specification by controlling Achaete levels. PMID:19448274

  5. Gene expression analysis reveals that Delta/Notch signalling is not involved in onychophoran segmentation.

    PubMed

    Janssen, Ralf; Budd, Graham E

    2016-03-01

    Delta/Notch (Dl/N) signalling is involved in the gene regulatory network underlying the segmentation process in vertebrates and possibly also in annelids and arthropods, leading to the hypothesis that segmentation may have evolved in the last common ancestor of bilaterian animals. Because of seemingly contradicting results within the well-studied arthropods, however, the role and origin of Dl/N signalling in segmentation generally is still unclear. In this study, we investigate core components of Dl/N signalling by means of gene expression analysis in the onychophoran Euperipatoides kanangrensis, a close relative to the arthropods. We find that neither Delta or Notch nor any other investigated components of its signalling pathway are likely to be involved in segment addition in onychophorans. We instead suggest that Dl/N signalling may be involved in posterior elongation, another conserved function of these genes. We suggest further that the posterior elongation network, rather than classic Dl/N signalling, may be in the control of the highly conserved segment polarity gene network and the lower-level pair-rule gene network in onychophorans. Consequently, we believe that the pair-rule gene network and its interaction with Dl/N signalling may have evolved within the arthropod lineage and that Dl/N signalling has thus likely been recruited independently for segment addition in different phyla. PMID:26935716

  6. Gene expression analysis reveals that Delta/Notch signalling is not involved in onychophoran segmentation.

    PubMed

    Janssen, Ralf; Budd, Graham E

    2016-03-01

    Delta/Notch (Dl/N) signalling is involved in the gene regulatory network underlying the segmentation process in vertebrates and possibly also in annelids and arthropods, leading to the hypothesis that segmentation may have evolved in the last common ancestor of bilaterian animals. Because of seemingly contradicting results within the well-studied arthropods, however, the role and origin of Dl/N signalling in segmentation generally is still unclear. In this study, we investigate core components of Dl/N signalling by means of gene expression analysis in the onychophoran Euperipatoides kanangrensis, a close relative to the arthropods. We find that neither Delta or Notch nor any other investigated components of its signalling pathway are likely to be involved in segment addition in onychophorans. We instead suggest that Dl/N signalling may be involved in posterior elongation, another conserved function of these genes. We suggest further that the posterior elongation network, rather than classic Dl/N signalling, may be in the control of the highly conserved segment polarity gene network and the lower-level pair-rule gene network in onychophorans. Consequently, we believe that the pair-rule gene network and its interaction with Dl/N signalling may have evolved within the arthropod lineage and that Dl/N signalling has thus likely been recruited independently for segment addition in different phyla.

  7. Regulation of differentiation flux by Notch signalling influences the number of dopaminergic neurons in the adult brain

    PubMed Central

    Trujillo-Paredes, Niurka; Valencia, Concepción; Guerrero-Flores, Gilda; Arzate, Dulce-María; Baizabal, José-Manuel; Guerra-Crespo, Magdalena; Fuentes-Hernández, Ayari; Zea-Armenta, Iván; Covarrubias, Luis

    2016-01-01

    ABSTRACT Notch signalling is a well-established pathway that regulates neurogenesis. However, little is known about the role of Notch signalling in specific neuronal differentiation. Using Dll1 null mice, we found that Notch signalling has no function in the specification of mesencephalic dopaminergic neural precursor cells (NPCs), but plays an important role in regulating their expansion and differentiation into neurons. Premature neuronal differentiation was observed in mesencephalons of Dll1-deficient mice or after treatment with a Notch signalling inhibitor. Coupling between neurogenesis and dopaminergic differentiation was indicated from the coincident emergence of neuronal and dopaminergic markers. Early in differentiation, decreasing Notch signalling caused a reduction in NPCs and an increase in dopaminergic neurons in association with dynamic changes in the proportion of sequentially-linked dopaminergic NPCs (Msx1/2+, Ngn2+, Nurr1+). These effects in differentiation caused a significant reduction in the number of dopaminergic neurons produced. Accordingly, Dll1 haploinsufficient adult mice, in comparison with their wild-type littermates, have a consistent reduction in neuronal density that was particularly evident in the substantia nigra pars compacta. Our results are in agreement with a mathematical model based on a Dll1-mediated regulatory feedback loop between early progenitors and their dividing precursors that controls the emergence and number of dopaminergic neurons. PMID:26912775

  8. Notch pathway is activated in cell culture and mouse models of mutant SOD1-related familial amyotrophic lateral sclerosis, with suppression of its activation as an additional mechanism of neuroprotection for lithium and valproate.

    PubMed

    Wang, S-Y; Ren, M; Jiang, H-Z; Wang, J; Jiang, H-Q; Yin, X; Qi, Y; Wang, X-D; Dong, G-T; Wang, T-H; Yang, Y-Q; Feng, H-L

    2015-08-20

    Amyotrophic lateral sclerosis (ALS) is an idiopathic and lethal neurodegenerative disease that currently has no effective treatment. A recent study found that the Notch signaling pathway was up-regulated in a TAR DNA-binding protein-43 (TDP-43) Drosophila model of ALS. Notch signaling acts as a master regulator in the central nervous system. However, the mechanisms by which Notch participates in the pathogenesis of ALS have not been completely elucidated. Recent studies have shown that the mood stabilizers lithium and valproic acid (VPA) are able to regulate Notch signaling. Our study sought to confirm the relationship between the Notch pathway and ALS and whether the Notch pathway contributes to the neuroprotective effects of lithium and VPA in ALS. We found that the Notch pathway was activated in in vitro and in vivo models of ALS, and suppression of Notch activation with a Notch signaling inhibitor, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) and Notch1 siRNA significantly reduced neuronal apoptotic signaling, as evidenced by the up-regulation of Bcl-2 as well as the down-regulation of Bax and cytochrome c. We also found that lithium and VPA suppressed the Notch activation associated with the superoxide dismutase-1 (SOD1) mutation, and the combination of lithium and VPA produced a more robust effect than either agent alone. Our findings indicate that the Notch pathway plays a critical role in ALS, and the neuroprotective effects of lithium and VPA against mutant SOD1-mediated neuronal damage are at least partially dependent on their suppression of Notch activation.

  9. γ-Secretase inhibitor DAPT attenuates intimal hyperplasia of vein grafts by inhibition of Notch1 signaling.

    PubMed

    Xiao, Yong Guang; Wang, Wei; Gong, Dan; Mao, Zhi Fu

    2014-06-01

    The proliferation and high plasticity of vascular smooth muscle cells (vSMCs) are the major reasons for restenosis of vein grafts. N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), specific inhibitor of γ-secretase, has been shown to regulate vSMC proliferation and differentiation through the Notch signaling pathway, but the pathophysiological importance of these findings in venous grafts has not yet been determined. A rat vein graft model was employed wherein the left jugular vein was surgically interposed into the left common carotid artery. Daily subcutaneous injections of DAPT or placebo (DMSO) were administered postoperatively (control animals received no treatment). We showed that DAPT can inhibit restenosis of vein grafts by inhibiting vSMC proliferation and increasing apoptosis in vivo. Notch1 signaling was highly active during the development of intima thickening. By blocking the Notch signaling pathway, the γ-secretase inhibitor DAPT can significantly attenuated intima thickening. These changes in vein grafts coincided with enhanced binding of myocardin to the smooth muscle-specific protein SM22 and smooth muscle myosin heavy chain at the promoters of vSMC differentiation-specific genes. These studies showed that DAPT can restore the vSMC phenotype and inhibit vSMC proliferation through suppression of the Notch1 signaling pathway, and thus opens a new avenue for the treatment of restenosis in vein grafts. PMID:24751889

  10. UBR-5, a Conserved HECT-Type E3 Ubiquitin Ligase, Negatively Regulates Notch-Type Signaling in Caenorhabditis elegans

    PubMed Central

    Safdar, Komal; Gu, Anniya; Xu, Xia; Au, Vinci; Taylor, Jon; Flibotte, Stephane; Moerman, Donald G.; Maine, Eleanor M.

    2016-01-01

    Notch-type signaling mediates cell−cell interactions important for animal development. In humans, reduced or inappropriate Notch signaling activity is associated with various developmental defects and disease states, including cancers. Caenorhabditis elegans expresses two Notch-type receptors, GLP-1 and LIN-12. GLP-1 mediates several cell-signaling events in the embryo and promotes germline proliferation in the developing and adult gonad. LIN-12 acts redundantly with GLP-1 in certain inductive events in the embryo and mediates several cell−cell interactions during larval development. Recovery of genetic suppressors and enhancers of glp-1 or lin-12 loss- or gain-of-function mutations has identified numerous regulators of GLP-1 and LIN-12 signaling activity. Here, we report the molecular identification of sog-1, a gene identified in screens for recessive suppressors of conditional glp-1 loss-of-function mutations. The sog-1 gene encodes UBR-5, the sole C. elegans member of the UBR5/Hyd family of HECT-type E3 ubiquitin ligases. Molecular and genetic analyses indicate that the loss of ubr-5 function suppresses defects caused by reduced signaling via GLP-1 or LIN-12. In contrast, ubr-5 mutations do not suppress embryonic or larval lethality associated with mutations in a downstream transcription factor, LAG-1. In the gonad, ubr-5 acts in the receiving cells (germ cells) to limit GLP-1 signaling activity. SEL-10 is the F-box component of SCFSEL-10 E3 ubiquitin–ligase complex that promotes turnover of Notch intracellular domain. UBR-5 acts redundantly with SEL-10 to limit Notch signaling in certain tissues. We hypothesize that UBR-5 activity limits Notch-type signaling by promoting turnover of receptor or limiting its interaction with pathway components. PMID:27185398

  11. Role of Notch Signaling in the Maintenance of Human Mesenchymal Stem Cells Under Hypoxic Conditions

    PubMed Central

    Moriyama, Mariko; Isshi, Haruki; Ishihara, Shin; Okura, Hanayuki; Ichinose, Akihiro; Ozawa, Toshiyuki; Matsuyama, Akifumi; Hayakawa, Takao

    2014-01-01

    Human adipose tissue-derived multilineage progenitor cells (hADMPCs) are attractive for cell therapy and tissue engineering because of their multipotency and ease of isolation without serial ethical issues. However, their limited in vitro lifespan in culture systems hinders their therapeutic application. Some somatic stem cells, including hADMPCs, are known to be localized in hypoxic regions; thus, hypoxia may be beneficial for ex vivo culture of these stem cells. These cells exhibit a high level of glycolytic metabolism in the presence of high oxygen levels and further increase their glycolysis rate under hypoxia. However, the physiological role of glycolytic activation and its regulatory mechanisms are still incompletely understood. Here, we show that Notch signaling is required for glycolysis regulation under hypoxic conditions. Our results demonstrate that 5% O2 dramatically increased the glycolysis rate, improved the proliferation efficiency, prevented senescence, and maintained the multipotency of hADMPCs. Intriguingly, these effects were not mediated by hypoxia-inducible factor (HIF), but rather by the Notch signaling pathway. Five percent O2 significantly increased the level of activated Notch1 and expression of its downstream gene, HES1. Furthermore, 5% O2 markedly increased glucose consumption and lactate production of hADMPCs, which decreased back to normoxic levels on treatment with a γ-secretase inhibitor. We also found that HES1 was involved in induction of GLUT3, TPI, and PGK1 in addition to reduction of TIGAR and SCO2 expression. These results clearly suggest that Notch signaling regulates glycolysis under hypoxic conditions and, thus, likely affects the cell lifespan via glycolysis. PMID:24878247

  12. Notch1 signaling regulates chondrogenic lineage determination through Sox9 activation.

    PubMed

    Haller, R; Schwanbeck, R; Martini, S; Bernoth, K; Kramer, J; Just, U; Rohwedel, J

    2012-03-01

    Notch signaling is involved in several cell lineage determination processes during embryonic development. Recently, we have shown that Sox9 is most likely a primary target gene of Notch1 signaling in embryonic stem cells (ESCs). By using our in vitro differentiation protocol for chondrogenesis from ESCs through embryoid bodies (EBs) together with our tamoxifen-inducible system to activate Notch1, we analyzed the function of Notch signaling and its induction of Sox9 during EB differentiation towards the chondrogenic lineage. Temporary activation of Notch1 during early stages of EB, when lineage determination occurs, was accompanied by rapid and transient Sox9 upregulation and resulted in induction of chondrogenic differentiation during later stages of EB cultivation. Using siRNA targeting Sox9, we knocked down and adjusted this early Notch1-induced Sox9 expression peak to non-induced levels, which led to reversion of Notch1-induced chondrogenic differentiation. In contrast, continuous Notch1 activation during EB cultivation resulted in complete inhibition of chondrogenic differentiation. Furthermore, a reduction and delay of cardiac differentiation observed in EBs after early Notch1 activation was not reversed by siRNA-mediated Sox9 knockdown. Our data indicate that Notch1 signaling has an important role during early stages of chondrogenic lineage determination by regulation of Sox9 expression. PMID:21869831

  13. Critical Roles of Notch and Wnt/β-Catenin Pathways in the Regulation of Hyperplasia and/or Colitis in Response to Bacterial Infection

    PubMed Central

    Ahmed, Ishfaq; Chandrakesan, Parthasarathy; Tawfik, Ossama; Xia, Lijun; Anant, Shrikant

    2012-01-01

    Notch and Wnt/β-catenin signals play essential roles in intestinal development and homeostasis. Citrobacter rodentium induces transmissible murine colonic hyperplasia (TMCH) and various degrees of inflammation, depending upon the genetic background. We aimed at delineating the role of the Notch and Wnt/β-catenin pathways in the regulation of colonic crypt hyperplasia and/or colitis following C. rodentium infection. During TMCH, relative levels of the Notch intracellular domain (NICD) increased significantly, along with increases in Jagged-1 and Hes-1 coinciding with the progression and regression phases of hyperplasia. Blocking of Notch signaling with dibenzazepine (DBZ) for 5 days before the onset of hyperplasia also blocked Wnt/β-catenin signaling. Targeting the Notch pathway for 5 days after the onset of hyperplasia failed to inhibit Wnt/β-catenin-regulated crypt hyperplasia. Chronic DBZ administration for 10 days blocked both Notch and Wnt signaling, disrupted the intestinal barrier, and induced colitis. Core-3−/− mice, which are defective in mucin secretion and are susceptible to experimental triggers of colitis, also exhibited significant colitis in response to C. rodentium plus DBZ. Chronic DBZ administration in these mice did not result in depletion of the putative stem cell marker doublecortin-like kinase-1 (DCLK1) in the crypts. Dietary bael (Aegle marmelos) extract (4%) and curcumin (4%) restored signaling via the Notch and Wnt/β-catenin pathways, thereby promoting crypt regeneration, and also replenished the mucus layer, leading to amelioration of C. rodentium- and DBZ-induced colitis in NIH:Swiss mice. Thus, the balancing act between cell proliferation and mucus production to restore barrier integrity seems to depend upon the interplay between the Wnt/β-catenin and Notch pathways in the TMCH model. PMID:22710872

  14. A role for the primary cilium in Notch signaling and epidermal differentiation during skin development.

    PubMed

    Ezratty, Ellen J; Stokes, Nicole; Chai, Sophia; Shah, Alok S; Williams, Scott E; Fuchs, Elaine

    2011-06-24

    Ciliogenesis precedes lineage-determining signaling in skin development. To understand why, we performed shRNA-mediated knockdown of seven intraflagellar transport proteins (IFTs) and conditional ablation of Ift-88 and Kif3a during embryogenesis. In both cultured keratinocytes and embryonic epidermis, all of these eliminated cilia, and many (not Kif3a) caused hyperproliferation. Surprisingly and independent of proliferation, ciliary mutants displayed defects in Notch signaling and commitment of progenitors to differentiate. Notch receptors and Notch-processing enzymes colocalized with cilia in wild-type epidermal cells. Moreover, differentiation defects in ciliary mutants were cell autonomous and rescued by activated Notch (NICD). By contrast, Shh signaling was neither operative nor required for epidermal ciliogenesis, Notch signaling, or differentiation. Rather, Shh signaling defects in ciliary mutants occurred later, arresting hair follicle morphogenesis in the skin. These findings unveil temporally and spatially distinct functions for primary cilia at the nexus of signaling, proliferation, and differentiation.

  15. Decreased Satellite Cell Number and Function in Humans and Mice With Type 1 Diabetes Is the Result of Altered Notch Signaling.

    PubMed

    D'Souza, Donna M; Zhou, Sarah; Rebalka, Irena A; MacDonald, Blair; Moradi, Jasmin; Krause, Matthew P; Al-Sajee, Dhuha; Punthakee, Zubin; Tarnopolsky, Mark A; Hawke, Thomas J

    2016-10-01

    Type 1 diabetes (T1D) negatively influences skeletal muscle health; however, its effect on muscle satellite cells (SCs) remains largely unknown. SCs from samples from rodents (Akita) and human subjects with T1D were examined to discern differences in SC density and functionality compared with samples from their respective control subjects. Examination of the Notch pathway was undertaken to investigate its role in changes to SC functionality. Compared with controls, Akita mice demonstrated increased muscle damage after eccentric exercise along with a decline in SC density and myogenic capacity. Quantification of components of the Notch signaling pathway revealed a persistent activation of Notch signaling in Akita SCs, which could be reversed with the Notch inhibitor DAPT. Similar to Akita samples, skeletal muscle from human subjects with T1D displayed a significant reduction in SC content, and the Notch ligand, DLL1, was significantly increased compared with control subjects, supporting the dysregulated Notch pathway observed in Akita muscles. These data indicate that persistent activation in Notch signaling impairs SC functionality in the T1D muscle, resulting in a decline in SC content. Given the vital role played by the SC in muscle growth and maintenance, these findings suggest that impairments in SC capacities play a primary role in the skeletal muscle myopathy that characterizes T1D. PMID:27335233

  16. Decreased Satellite Cell Number and Function in Humans and Mice With Type 1 Diabetes Is the Result of Altered Notch Signaling.

    PubMed

    D'Souza, Donna M; Zhou, Sarah; Rebalka, Irena A; MacDonald, Blair; Moradi, Jasmin; Krause, Matthew P; Al-Sajee, Dhuha; Punthakee, Zubin; Tarnopolsky, Mark A; Hawke, Thomas J

    2016-10-01

    Type 1 diabetes (T1D) negatively influences skeletal muscle health; however, its effect on muscle satellite cells (SCs) remains largely unknown. SCs from samples from rodents (Akita) and human subjects with T1D were examined to discern differences in SC density and functionality compared with samples from their respective control subjects. Examination of the Notch pathway was undertaken to investigate its role in changes to SC functionality. Compared with controls, Akita mice demonstrated increased muscle damage after eccentric exercise along with a decline in SC density and myogenic capacity. Quantification of components of the Notch signaling pathway revealed a persistent activation of Notch signaling in Akita SCs, which could be reversed with the Notch inhibitor DAPT. Similar to Akita samples, skeletal muscle from human subjects with T1D displayed a significant reduction in SC content, and the Notch ligand, DLL1, was significantly increased compared with control subjects, supporting the dysregulated Notch pathway observed in Akita muscles. These data indicate that persistent activation in Notch signaling impairs SC functionality in the T1D muscle, resulting in a decline in SC content. Given the vital role played by the SC in muscle growth and maintenance, these findings suggest that impairments in SC capacities play a primary role in the skeletal muscle myopathy that characterizes T1D.

  17. Coordination of insulin and Notch pathway activities by microRNA miR-305 mediates adaptive homeostasis in the intestinal stem cells of the Drosophila gut.

    PubMed

    Foronda, David; Weng, Ruifen; Verma, Pushpa; Chen, Ya-Wen; Cohen, Stephen M

    2014-11-01

    Homeostasis of the intestine is maintained by dynamic regulation of a pool of intestinal stem cells. The balance between stem cell self-renewal and differentiation is regulated by the Notch and insulin signaling pathways. Dependence on the insulin pathway places the stem cell pool under nutritional control, allowing gut homeostasis to adapt to environmental conditions. Here we present evidence that miR-305 is required for adaptive homeostasis of the gut. miR-305 regulates the Notch and insulin pathways in the intestinal stem cells. Notably, miR-305 expression in the stem cells is itself under nutritional control via the insulin pathway. This link places regulation of Notch pathway activity under nutritional control. These findings provide a mechanism through which the insulin pathway controls the balance between stem cell self-renewal and differentiation that is required for adaptive homeostasis in the gut in response to changing environmental conditions.

  18. Notch signaling controls chondrocyte hypertrophy via indirect regulation of Sox9

    PubMed Central

    Kohn, Anat; Rutkowski, Timothy P; Liu, Zhaoyang; Mirando, Anthony J; Zuscik, Michael J; O’Keefe, Regis J; Hilton, Matthew J

    2015-01-01

    RBPjk-dependent Notch signaling regulates both the onset of chondrocyte hypertrophy and the progression to terminal chondrocyte maturation during endochondral ossification. It has been suggested that Notch signaling can regulate Sox9 transcription, although how this occurs at the molecular level in chondrocytes and whether this transcriptional regulation mediates Notch control of chondrocyte hypertrophy and cartilage development is unknown or controversial. Here we have provided conclusive genetic evidence linking RBPjk-dependent Notch signaling to the regulation of Sox9 expression and chondrocyte hypertrophy by examining tissue-specific Rbpjk mutant (Prx1Cre;Rbpjkf/f), Rbpjk mutant/Sox9 haploinsufficient (Prx1Cre;Rbpjkf/f;Sox9f/+), and control embryos for alterations in SOX9 expression and chondrocyte hypertrophy during cartilage development. These studies demonstrate that Notch signaling regulates the onset of chondrocyte maturation in a SOX9-dependent manner, while Notch-mediated regulation of terminal chondrocyte maturation likely functions independently of SOX9. Furthermore, our in vitro molecular analyses of the Sox9 promoter and Notch-mediated regulation of Sox9 gene expression in chondrogenic cells identified the ability of Notch to induce Sox9 expression directly in the acute setting, but suppresses Sox9 transcription with prolonged Notch signaling that requires protein synthesis of secondary effectors. PMID:26558140

  19. Antagonism of notch signaling activity by members of a novel protein family encoded by the bearded and enhancer of split gene complexes.

    PubMed

    Lai, E C; Bodner, R; Kavaler, J; Freschi, G; Posakony, J W

    2000-01-01

    Cell-cell signaling through the Notch receptor is a principal mechanism underlying cell fate specification in a variety of developmental processes in metazoans, such as neurogenesis. In this report we describe our investigation of seven members of a novel gene family in Drosophila with important connections to Notch signaling. These genes all encode small proteins containing predicted basic amphipathic (&agr;)-helical domains in their amino-terminal regions, as described originally for Bearded; accordingly, we refer to them as Bearded family genes. Five members of the Bearded family are located in a newly discovered gene complex, the Bearded Complex; two others reside in the previously identified Enhancer of split Complex. All members of this family contain, in their proximal upstream regions, at least one high-affinity binding site for the Notch-activated transcription factor Suppressor of Hairless, suggesting that all are directly regulated by the Notch pathway. Consistent with this, we show that Bearded family genes are expressed in a variety of territories in imaginal tissue that correspond to sites of active Notch signaling. We demonstrate that overexpression of any family member antagonizes the activity of the Notch pathway in multiple cell fate decisions during adult sensory organ development. These results suggest that Bearded family genes encode a novel class of effectors or modulators of Notch signaling.

  20. Mef2 Interacts with the Notch Pathway during Adult Muscle Development in Drosophila melanogaster

    PubMed Central

    Caine, Charlotte; Kasherov, Petar; Silber, Joël; Lalouette, Alexis

    2014-01-01

    Myogenesis of indirect flight muscles (IFMs) in Drosophila melanogaster follows a well-defined cellular developmental scheme. During embryogenesis, a set of cells, the Adult Muscle Precursors (AMPs), are specified. These cells will become proliferating myoblasts during the larval stages which will then give rise to the adult IFMs. Although the cellular aspect of this developmental process is well studied, the molecular biology behind the different stages is still under investigation. In particular, the interactions required during the transition from proliferating myoblasts to differentiated myoblasts ready to fuse to the muscle fiber. It has been previously shown that the Notch pathway is active in proliferating myoblasts, and that this pathway is inhibited in developing muscle fibers. Furthermore, the Myocyte Enhancing Factor 2 (Mef2), Vestigial (Vg) and Scalloped (Sd) transcription factors are necessary for IFM development and that Vg is required for Notch pathway repression in differentiating fibers. Here we examine the interactions between Notch and Mef2 and mechanisms by which the Notch pathway is inhibited during differentiation. We show that Mef2 is capable of inhibiting the Notch pathway in non myogenic cells. A previous screen for Mef2 potential targets identified Delta a component of the Notch pathway. Dl is expressed in Mef2 and Sd-positive developing fibers. Our results show that Mef2 and possibly Sd regulate a Dl enhancer specifically expressed in the developing IFMs and that Mef2 is required for Dl expression in developing IFMs. PMID:25247309

  1. Ilyanassa Notch signaling implicated in dynamic signaling between all three germ layers.

    PubMed

    Gharbiah, Maey; Nakamoto, Ayaki; Johnson, Adam B; Lambert, J David; Nagy, Lisa M

    2014-01-01

    Two cells (3D and 4d) in the mud snail Ilyanassa obsoleta function to induce proper cell fate. In this study, we provide support for the hypothesis that Notch signaling in Ilyanassa obsoleta functions in inductive signaling at multiple developmental stages. The expression patterns of Notch, Delta and Suppressor of Hairless (SuH) are consistent with a function for Notch signaling in endoderm formation, the function of 3D/4d and the sublineages of 4d. Veligers treated with DAPT show a range of defects that include a loss of endodermal structures, and varying degrees of loss of targets of 4d inductive signaling. Veligers that result from injection of Ilyanassa Delta siRNAi in general mimic the defects observed in the DAPT treated larvae. The most severe DAPT phenotypes mimic early ablations of 4d. However, the early specification of 4d itself appears normal and MAPK activation in both 3D/4d and the micromeres, which are known to activate MAPK as a result of 3D/4d induction, are normal in DAPT treated larvae. Treating larvae at successively later timepoints with DAPT suggests that Notch/Delta signaling is not only required during early 4d inductive signaling, but during subsequent stages of cell fate determination as well. Based on our results, combined with previous reports implicating the endoderm in maintaining induced fate specification in Ilyanassa, we propose a speculative model that Notch signaling is required to specify endoderm fates and 4d sublineages, as well as to maintain cell fates induced by 4d.

  2. Celery Seed Extract Blocks Peroxide Injury in Macrophages via Notch1/NF-κB Pathway.

    PubMed

    Si, Yanhong; Guo, Shoudong; Fang, Yongqi; Qin, Shucun; Li, Furong; Zhang, Ying; Jiao, Peng; Zhang, Chunduo; Gao, Linlin

    2015-01-01

    Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and injury is one of the major atherogenic factors. This study is aimed to investigate the protective effect of celery seed extract (CSE) on ox-LDL-induced injury of macrophages and the underlying signaling pathway. RAW264.7 macrophages were pre-incubated with CSE for 24 h, followed by stimulation with ox-LDL. Oil red O staining and enzymatic colorimetry indicated CSE significantly lessened lipid droplets and total cholesterol (TC) content in ox-LDL-injured macrophages. ELISA revealed that CSE decreased the secretion of inflammatory cytokine TNF-α and IL-6 by 12-27% and 5-15% respectively. MTT assay showed CSE promoted cell viability by 16-40%. Cell apoptosis was also analyzed by flow cytometry and laser scanning confocal microscope and the data indicated CSE inhibited ox-LDL-induced apoptosis of macrophages. Meanwhile, western blot analysis showed CSE suppressed NF-κBp65 and notch1 protein expressions stimulated by ox-LDL in macrophages. These results suggest that CSE inhibits ox-LDL-induced macrophages injury via notch1/NF-κB pathway. PMID:25916469

  3. Notch pathway activation contributes to inhibition of C2C12 myoblast differentiation by ethanol.

    PubMed

    Arya, Michelle A; Tai, Albert K; Wooten, Eric C; Parkin, Christopher D; Kudryavtseva, Elena; Huggins, Gordon S

    2013-01-01

    The loss of muscle mass in alcoholic myopathy may reflect alcohol inhibition of myogenic cell differentiation into myotubes. Here, using a high content imaging system we show that ethanol inhibits C2C12 myoblast differentiation by reducing myogenic fusion, creating smaller and less complex myotubes compared with controls. Ethanol administration during C2C12 differentiation reduced MyoD and myogenin expression, and microarray analysis identified ethanol activation of the Notch signaling pathway target genes Hes1 and Hey1. A reporter plasmid regulated by the Hes1 proximal promoter was activated by alcohol treatment in C2C12 cells. Treatment of differentiating C2C12 cells with a gamma secretase inhibitor (GSI) abrogated induction of Hes1. On a morphological level GSI treatment completely rescued myogenic fusion defects and partially restored other myotube parameters in response to alcohol. We conclude that alcohol inhibits C2C12 myoblast differentiation and the inhibition of myogenic fusion is mediated by Notch pathway activation.

  4. Celery Seed Extract Blocks Peroxide Injury in Macrophages via Notch1/NF-κB Pathway.

    PubMed

    Si, Yanhong; Guo, Shoudong; Fang, Yongqi; Qin, Shucun; Li, Furong; Zhang, Ying; Jiao, Peng; Zhang, Chunduo; Gao, Linlin

    2015-01-01

    Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and injury is one of the major atherogenic factors. This study is aimed to investigate the protective effect of celery seed extract (CSE) on ox-LDL-induced injury of macrophages and the underlying signaling pathway. RAW264.7 macrophages were pre-incubated with CSE for 24 h, followed by stimulation with ox-LDL. Oil red O staining and enzymatic colorimetry indicated CSE significantly lessened lipid droplets and total cholesterol (TC) content in ox-LDL-injured macrophages. ELISA revealed that CSE decreased the secretion of inflammatory cytokine TNF-α and IL-6 by 12-27% and 5-15% respectively. MTT assay showed CSE promoted cell viability by 16-40%. Cell apoptosis was also analyzed by flow cytometry and laser scanning confocal microscope and the data indicated CSE inhibited ox-LDL-induced apoptosis of macrophages. Meanwhile, western blot analysis showed CSE suppressed NF-κBp65 and notch1 protein expressions stimulated by ox-LDL in macrophages. These results suggest that CSE inhibits ox-LDL-induced macrophages injury via notch1/NF-κB pathway.

  5. Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update

    PubMed Central

    Miele, Lucio; Harris, Pamela Jo; Jeong, Woondong; Bando, Hideaki; Kahn, Michael; Yang, Sherry X.

    2015-01-01

    During the past decade, cancer stem cells (CSCs) have been increasingly identified in many malignancies. Although the origin and plasticity of these cells remain controversial, tumour heterogeneity and the presence of small populations of cells with stem-like characteristics is established in most malignancies. CSCs display many features of embryonic or tissue stem cells, and typically demonstrate persistent activation of one or more highly conserved signal transduction pathways involved in development and tissue homeostasis, including the Notch, Hedgehog (HH), and Wnt pathways. CSCs generally have slow growth rates and are resistant to chemotherapy and/or radiotherapy. Thus, new treatment strategies targeting these pathways to control stem-cell replication, survival and differentiation are under development. Herein, we provide an update on the latest advances in the clinical development of such approaches, and discuss strategies for overcoming CSC-associated primary or acquired resistance to cancer treatment. Given the crosstalk between the different embryonic developmental signalling pathways, as well as other pathways, designing clinical trials that target CSCs with rational combinations of agents to inhibit possible compensatory escape mechanisms could be of particular importance. We also share our views on the future directions for targeting CSCs to advance the clinical development of these classes of agents. PMID:25850553

  6. A novel non-canonical Notch signaling regulates expression of synaptic vesicle proteins in excitatory neurons

    PubMed Central

    Hayashi, Yukari; Nishimune, Hiroshi; Hozumi, Katsuto; Saga, Yumiko; Harada, Akihiro; Yuzaki, Michisuke; Iwatsubo, Takeshi; Kopan, Raphael; Tomita, Taisuke

    2016-01-01

    Notch signaling plays crucial roles for cellular differentiation during development through γ-secretase-dependent intramembrane proteolysis followed by transcription of target genes. Although recent studies implicate that Notch regulates synaptic plasticity or cognitive performance, the molecular mechanism how Notch works in mature neurons remains uncertain. Here we demonstrate that a novel Notch signaling is involved in expression of synaptic proteins in postmitotic neurons. Levels of several synaptic vesicle proteins including synaptophysin 1 and VGLUT1 were increased when neurons were cocultured with Notch ligands-expressing NIH3T3 cells. Neuron-specific deletion of Notch genes decreased these proteins, suggesting that Notch signaling maintains the expression of synaptic vesicle proteins in a cell-autonomous manner. Unexpectedly, cGMP-dependent protein kinase (PKG) inhibitor, but not γ-secretase inhibitor, abolished the elevation of synaptic vesicle proteins, suggesting that generation of Notch intracellular domain is dispensable for this function. These data uncover a ligand-dependent, but γ-secretase-independent, non-canonical Notch signaling involved in presynaptic protein expression in postmitotic neurons. PMID:27040987

  7. Epithelial Notch signaling regulates lung alveolar morphogenesis and airway epithelial integrity

    PubMed Central

    Tsao, Po-Nien; Matsuoka, Chisa; Wei, Shu-Chen; Sato, Atsuyasu; Sato, Susumu; Hasegawa, Koichi; Chen, Hung-kuan; Ling, Thai-Yen; Mori, Munemasa; Cardoso, Wellington V.; Morimoto, Mitsuru

    2016-01-01

    Abnormal enlargement of the alveolar spaces is a hallmark of conditions such as chronic obstructive pulmonary disease and bronchopulmonary dysplasia. Notch signaling is crucial for differentiation and regeneration and repair of the airway epithelium. However, how Notch influences the alveolar compartment and integrates this process with airway development remains little understood. Here we report a prominent role of Notch signaling in the epithelial–mesenchymal interactions that lead to alveolar formation in the developing lung. We found that alveolar type II cells are major sites of Notch2 activation and show by Notch2-specific epithelial deletion (Notch2cNull) a unique contribution of this receptor to alveologenesis. Epithelial Notch2 was required for type II cell induction of the PDGF-A ligand and subsequent paracrine activation of PDGF receptor-α signaling in alveolar myofibroblast progenitors. Moreover, Notch2 was crucial in maintaining the integrity of the epithelial and smooth muscle layers of the distal conducting airways. Our data suggest that epithelial Notch signaling regulates multiple aspects of postnatal development in the distal lung and may represent a potential target for intervention in pulmonary diseases. PMID:27364009

  8. High throughput kinase inhibitor screens reveal TRB3 and MAPK-ERK/TGFβ pathways as fundamental Notch regulators in breast cancer.

    PubMed

    Izrailit, Julia; Berman, Hal K; Datti, Alessandro; Wrana, Jeffrey L; Reedijk, Michael

    2013-01-29

    Expression of the Notch ligand Jagged 1 (JAG1) and Notch activation promote poor-prognosis in breast cancer. We used high throughput screens to identify elements responsible for Notch activation in this context. Chemical kinase inhibitor and kinase-specific small interfering RNA libraries were screened in a breast cancer cell line engineered to report Notch. Pathway analyses revealed MAPK-ERK signaling to be the predominant JAG1/Notch regulator and this was supported by gene set enrichment analyses in 51 breast cancer cell lines. In accordance with the chemical screen, kinome small interfering RNA high throughput screens identified Tribbles homolog 3 (TRB3), a known regulator of MAPK-ERK, among the most significant hits. We demonstrate that TRB3 is a master regulator of Notch through the MAPK-ERK and TGFβ pathways. Complementary in vitro and in vivo studies underscore the importance of TRB3 for tumor growth. These data demonstrate a dominant role for TRB3 and MAPK-ERK/TGFβ pathways as Notch regulators in breast cancer, establishing TRB3 as a potential therapeutic target.

  9. Expression pattern of the Hedgehog signaling pathway in pituitary adenomas.

    PubMed

    Yavropoulou, Maria P; Maladaki, Anna; Topouridou, Konstantina; Kotoula, Vasiliki; Poulios, Chris; Daskalaki, Emily; Foroglou, Nikolaos; Karkavelas, George; Yovos, John G

    2016-01-12

    Several studies have demonstrated the role of Wnt and Notch signaling in the pathogenesis of pituitary adenomas, but data are scarce regarding the role of Hedgehog signaling. In this study we investigated the differential expression of gene targets of the Hedgehog signaling pathway. Formalin-fixed, paraffin-embedded specimens from adult patients who underwent transphenoidal resection and normal human pituitary tissues that were obtained from autopsies were used. Clinical information and data from pre-operative MRI scan (extracellular tumor extension, tumor size, displacement of the optic chiasm) were retrieved from the Hospital's database. We used a customized RT(2) Profiler PCR Array, to investigate the expression of genes related to Notch and Hedgehog signaling pathways (PTCH1, PTCH2, GLI1, GLI3, NOTCH3, JAG1, HES1, and HIP). A total of 52 pituitary adenomas (32 non-functioning adenomas, 15 somatotropinomas and 5 prolactinomas) were used in the final analysis. In non-functioning pituitary adenomas there was a significant decrease (approximately 75%) in expression of all Hedgehog related genes that were tested, while Notch3 and Jagged-1 expression was found significantly increased, compared with normal pituitary tissue controls. In contrast, somatotropinomas demonstrated a significant increase in expression of all Hedgehog related genes and a decrease in the expression of Notch3 and Jagged-1. There was no significant difference in the expression of Hedgehog and Notch related genes between prolactinomas and healthy pituitary tissues. Hedgehog signalling appears to be activated in somatotropinomas but not in non-functioning pituitary adenomas in contrast to the expression pattern of Notch signalling pathway. PMID:26620835

  10. Structure and Function of the CSL-KyoT2 Corepressor Complex – a Negative Regulator of Notch Signaling

    PubMed Central

    Collins, Kelly J.; Yuan, Zhenyu; Kovall, Rhett A.

    2013-01-01

    Summary Notch refers to a highly conserved cell-to-cell signaling pathway with essential roles in embryonic development and tissue maintenance. Dysfunctional signaling causes human disease, highlighting the importance of pathway regulation. Notch signaling ultimately results in the activation of target genes, which is regulated by the nuclear effector CSL. CSL dually functions as an activator and repressor of transcription through differential interactions with coactivator or corepressor proteins, respectively. While the structures of CSL-coactivator complexes have been determined, the structures of CSL-corepressor complexes are unknown. Here, using a combination of structural, biophysical, and cellular approaches, we characterize the structure and function of CSL in complex with the corepressor KyoT2. Collectively, our studies provide molecular insights into how KyoT2 binds CSL with high affinity and competes with coactivators, such as Notch, for binding CSL. These studies are important for understanding how CSL functions as both an activator and repressor of transcription of Notch target genes. PMID:24290140

  11. Inhibition of Notch signaling reduces the number of surviving Dclk1+ reserve crypt epithelial stem cells following radiation injury.

    PubMed

    Qu, Dongfeng; May, Randal; Sureban, Sripathi M; Weygant, Nathaniel; Chandrakesan, Parthasarathy; Ali, Naushad; Li, Linheng; Barrett, Terrence; Houchen, Courtney W

    2014-03-01

    We have previously reported that doublecortin-like kinase 1 (Dclk1) is a putative intestinal stem cell (ISC) marker. In this report, we evaluated the use of Dclk1 as a marker of surviving ISCs in response to treatment with high-dose total body irradiation (TBI). Both apoptotic and mitotic Dclk1(+) cells were observed 24 h post-TBI associated with a corresponding loss of intestinal crypts observed at 84 h post-TBI. Although the Notch signaling pathway plays an important role in regulating proliferation and lineage commitment within the intestine, its role in ISC function in response to severe genotoxic injury is not yet fully understood. We employed the microcolony assay to functionally assess the effects of Notch inhibition with difluorophenacetyl-l-alanyl-S-phenylglycine t-butyl ester (DAPT) on intestinal crypt stem cell survival following severe (>8 Gy) radiation injury. Following treatment with DAPT, we observed a nearly 50% reduction in the number of surviving Dclk1(+) crypt epithelial cells at 24 h after TBI and similar reduction in the number of surviving small intestinal crypts at 84 h. These data indicate that inhibition of Notch signaling decreases ISC survival following radiation injury, suggesting that the Notch signaling pathway plays an important role in ISC-mediated crypt regeneration. These results also suggest that crypt epithelial cell Dclk1 expression can be used as one potential marker to evaluate the early survival of ISCs following severe radiation injury.

  12. Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron

    PubMed Central

    Lindström, Nils O; Lawrence, Melanie L; Burn, Sally F; Johansson, Jeanette A; Bakker, Elvira RM; Ridgway, Rachel A; Chang, C-Hong; Karolak, Michele J; Oxburgh, Leif; Headon, Denis J; Sansom, Owen J; Smits, Ron; Davies, Jamie A; Hohenstein, Peter

    2015-01-01

    The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in β-catenin activity along the axis of the nephron tubule. By modifying β-catenin activity, we force cells within nephrons to differentiate according to the imposed β-catenin activity level, thereby causing spatial shifts in nephron segments. The β-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises β-catenin activity and promotes segment identities associated with low β-catenin activity. β-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating β-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning. DOI: http://dx.doi.org/10.7554/eLife.04000.001 PMID:25647637

  13. Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron.

    PubMed

    Lindström, Nils O; Lawrence, Melanie L; Burn, Sally F; Johansson, Jeanette A; Bakker, Elvira R M; Ridgway, Rachel A; Chang, C-Hong; Karolak, Michele J; Oxburgh, Leif; Headon, Denis J; Sansom, Owen J; Smits, Ron; Davies, Jamie A; Hohenstein, Peter

    2015-02-03

    The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in β-catenin activity along the axis of the nephron tubule. By modifying β-catenin activity, we force cells within nephrons to differentiate according to the imposed β-catenin activity level, thereby causing spatial shifts in nephron segments. The β-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises β-catenin activity and promotes segment identities associated with low β-catenin activity. β-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating β-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning.

  14. Notch Signaling Activation in Cervical Cancer Cells Induces Cell Growth Arrest with the Involvement of the Nuclear Receptor NR4A2

    PubMed Central

    Sun, Lichun; Liu, Mingqiu; Sun, Guang-Chun; Yang, Xu; Qian, Qingqing; Feng, Shuyu; Mackey, L. Vienna; Coy, David H.

    2016-01-01

    Cervical cancer is a second leading cancer death in women world-wide, with most cases in less developed countries. Notch signaling is highly conserved with its involvement in many cancers. In the present study, we established stable cervical cell lines with Notch activation and inactivation and found that Notch activation played a suppressive role in cervical cancer cells. Meanwhile, the transient overexpression of the active intracellular domain of all four Notch receptors (ICN1, 2, 3, and 4) also induced the suppression of cervical cancer Hela cell growth. ICN1 also induced cell cycle arrest at phase G1. Notch1 signaling activation affected the expression of serial genes, especially the genes associated with cAMP signaling, with an increase of genes like THBS1, VCL, p63, c-Myc and SCG2, a decrease of genes like NR4A2, PCK2 and BCL-2. Particularly, The nuclear receptor NR4A2 was observed to induce cell proliferation via MTT assay and reduce cell apoptosis via FACS assay. Furthermore, NR4A2's activation could reverse ICN1-induced suppression of cell growth while erasing ICN1-induced increase of tumor suppressor p63. These findings support that Notch signaling mediates cervical cancer cell growth suppression with the involvement of nuclear receptor NR4A2. Notably, Notch/NR4A2/p63 signaling cascade possibly is a new signling pathway undisclosed. PMID:27471554

  15. Notch Signaling Activation in Cervical Cancer Cells Induces Cell Growth Arrest with the Involvement of the Nuclear Receptor NR4A2.

    PubMed

    Sun, Lichun; Liu, Mingqiu; Sun, Guang-Chun; Yang, Xu; Qian, Qingqing; Feng, Shuyu; Mackey, L Vienna; Coy, David H

    2016-01-01

    Cervical cancer is a second leading cancer death in women world-wide, with most cases in less developed countries. Notch signaling is highly conserved with its involvement in many cancers. In the present study, we established stable cervical cell lines with Notch activation and inactivation and found that Notch activation played a suppressive role in cervical cancer cells. Meanwhile, the transient overexpression of the active intracellular domain of all four Notch receptors (ICN1, 2, 3, and 4) also induced the suppression of cervical cancer Hela cell growth. ICN1 also induced cell cycle arrest at phase G1. Notch1 signaling activation affected the expression of serial genes, especially the genes associated with cAMP signaling, with an increase of genes like THBS1, VCL, p63, c-Myc and SCG2, a decrease of genes like NR4A2, PCK2 and BCL-2. Particularly, The nuclear receptor NR4A2 was observed to induce cell proliferation via MTT assay and reduce cell apoptosis via FACS assay. Furthermore, NR4A2's activation could reverse ICN1-induced suppression of cell growth while erasing ICN1-induced increase of tumor suppressor p63. These findings support that Notch signaling mediates cervical cancer cell growth suppression with the involvement of nuclear receptor NR4A2. Notably, Notch/NR4A2/p63 signaling cascade possibly is a new signling pathway undisclosed. PMID:27471554

  16. Notch Signaling Induces Rapid Degradation of Achaete-Scute Homolog 1

    PubMed Central

    Sriuranpong, Virote; Borges, Michael W.; Strock, Christopher L.; Nakakura, Eric K.; Watkins, D. Neil; Blaumueller, Christine M.; Nelkin, Barry D.; Ball, Douglas W.

    2002-01-01

    In neural development, Notch signaling plays a key role in restricting neuronal differentiation, promoting the maintenance of progenitor cells. Classically, Notch signaling causes transactivation of Hairy-enhancer of Split (HES) genes which leads to transcriptional repression of neural determination and differentiation genes. We now report that in addition to its known transcriptional mechanism, Notch signaling also leads to rapid degradation of the basic helix-loop-helix (bHLH) transcription factor human achaete-scute homolog 1 (hASH1). Using recombinant adenoviruses expressing active Notch1 in small-cell lung cancer cells, we showed that the initial appearance of Notch1 coincided with the loss of hASH1 protein, preceding the full decay of hASH1 mRNA. Overexpression of HES1 alone was capable of down-regulating hASH1 mRNA but could not replicate the acute reduction of hASH1 protein induced by Notch1. When adenoviral hASH1 was coinfected with Notch1, we still observed a dramatic and abrupt loss of the exogenous hASH1 protein, despite high levels of ongoing hASH1 RNA expression. Notch1 treatment decreased the apparent half-life of the adenoviral hASH1 protein and increased the fraction of hASH1 which was polyubiquitinylated. The proteasome inhibitor MG132 reversed the Notch1-induced degradation. The Notch RAM domain was dispensable but a lack of the OPA and PEST domains inactivated this Notch1 action. Overexpression of the hASH1-dimerizing partner E12 could protect hASH1 from degradation. This novel function of activated Notch to rapidly degrade a class II bHLH protein may prove to be important in many contexts in development and in cancer. PMID:11940670

  17. Notch signaling induces rapid degradation of achaete-scute homolog 1.

    PubMed

    Sriuranpong, Virote; Borges, Michael W; Strock, Christopher L; Nakakura, Eric K; Watkins, D Neil; Blaumueller, Christine M; Nelkin, Barry D; Ball, Douglas W

    2002-05-01

    In neural development, Notch signaling plays a key role in restricting neuronal differentiation, promoting the maintenance of progenitor cells. Classically, Notch signaling causes transactivation of Hairy-enhancer of Split (HES) genes which leads to transcriptional repression of neural determination and differentiation genes. We now report that in addition to its known transcriptional mechanism, Notch signaling also leads to rapid degradation of the basic helix-loop-helix (bHLH) transcription factor human achaete-scute homolog 1 (hASH1). Using recombinant adenoviruses expressing active Notch1 in small-cell lung cancer cells, we showed that the initial appearance of Notch1 coincided with the loss of hASH1 protein, preceding the full decay of hASH1 mRNA. Overexpression of HES1 alone was capable of down-regulating hASH1 mRNA but could not replicate the acute reduction of hASH1 protein induced by Notch1. When adenoviral hASH1 was coinfected with Notch1, we still observed a dramatic and abrupt loss of the exogenous hASH1 protein, despite high levels of ongoing hASH1 RNA expression. Notch1 treatment decreased the apparent half-life of the adenoviral hASH1 protein and increased the fraction of hASH1 which was polyubiquitinylated. The proteasome inhibitor MG132 reversed the Notch1-induced degradation. The Notch RAM domain was dispensable but a lack of the OPA and PEST domains inactivated this Notch1 action. Overexpression of the hASH1-dimerizing partner E12 could protect hASH1 from degradation. This novel function of activated Notch to rapidly degrade a class II bHLH protein may prove to be important in many contexts in development and in cancer.

  18. Niclosamide inhibits colon cancer progression through downregulation of the Notch pathway and upregulation of the tumor suppressor miR-200 family

    PubMed Central

    Suliman, Mohammed A.; Zhang, Zhenxing; Na, Heya; Ribeiro, Ailton L.L.; Zhang, Yu; Niang, Bachir; Hamid, Abdu Salim; Zhang, Hua; Xu, Lijie; Zuo, Yunfei

    2016-01-01

    Colorectal cancer (CRC) is among the most frequent causes of cancer-related deaths worldwide. Thus, there is a need for the development of new therapeutic approaches for the treatment of CRC. Accumulating evidence has revealed that niclosamide, an anthelminthic drug, exerts antitumor activity in several types of cancer, including colon cancer. However, the underlying molecular mechanisms responsible for the effects of this drug remain elusive. Previous studies have shown that the aberrant Notch signaling pathway contributes to the carcinogenesis of colon cancer. Herein, we examined the effects of niclosamide on the growth, migration and apoptosis of colon cancer cells, and the role of the Notch signaling pathway. By performing MTT, wound-healing and Transwell migration assays, we observed that niclosamide suppressed the growth and migration of colon cancer cells, and flow cytometry demonstrated that cell apoptosis was induced. This was associated with the decreased protein expression of Notch1, Notch2, Notch3 and Hey1, and the increased expression of the tumor suppressor microRNA (miR or miRNA)-200 family members (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) that are typically downregulated in colon cancer. Collectively, these findings demonstrate that niclosamide potentially inhibits the progression of colon cancer by downregulating Notch signaling and by upregulating the miR-200 family members. PMID:27460529

  19. Generation of late-born neurons in the ventral spinal cord requires the coordination of retinoic acid and Notch signaling.

    PubMed

    Ryu, Jae-Ho; Kong, Hee Jeong; Park, Jung Youn; Lim, Kyung-Eun; An, Cheul Min; Lee, Jehee; Yeo, Sang-Yeob

    2015-08-18

    Neural progenitor cells generate various types of neurons and glia in a tightly regulated manner. During primary neurogenesis, retinoic acid (RA) acts earlier than Notch signaling and regulates differentiation and proliferation by upregulating proneural and neurogenic genes in the neural plate. However, the relationship between Notch signaling and the retinoid pathway during late neurogenesis remains unclear. We investigated the role of Mindbomb (Mib)-mediated Notch signaling in the differentiation of neural progenitors during late neurogenesis by overexpressing Mib and administering RA to Tg[hsp70-Mib:EGFP]. The majority of cells in the p3 domain differentiated into GABAergic Kolmer-Agduhr (KA) cells in Tg[hsp70-mib:EGFP] embryos heat-shocked during late neurogenesis, whereas these phenotypes were suppressed by exogenous RA. Our observations suggest that Mib-mediated Notch signaling plays a critical role in the temporal differentiation of neural progenitors, and that the generation of late-born KA″ cells is regulated by the interplay between Mib and RA. PMID:26151587

  20. Nox2 contributes to the arterial endothelial specification of mouse induced pluripotent stem cells by upregulating Notch signaling

    PubMed Central

    Kang, Xueling; Wei, Xiangxiang; Wang, Xinhong; Jiang, Li; Niu, Cong; Zhang, Jianyi; Chen, Sifeng; Meng, Dan

    2016-01-01

    Reactive oxygen species (ROS) have a crucial role in stem-cell differentiation; however, the mechanisms by which ROS regulate the differentiation of stem cells into endothelial cells (ECs) are unknown. Here, we determine the role of ROS produced by NADPH oxidase 2 (Nox2) in the endothelial-lineage specification of mouse induced-pluripotent stem cells (miPSCs). When wild-type (WT) and Nox2-knockout (Nox2−/−) miPSCs were differentiated into ECs (miPSC-ECs), the expression of endothelial markers, arterial endothelial markers, pro-angiogenic cytokines, and Notch pathway components was suppressed in the Nox2−/− cells but increased in both WT and Nox2−/− miPSCs when Nox2 expression was upregulated. Higher levels of Nox2 expression increased Notch signaling and arterial EC differentiation, and this increase was abolished by the inhibition of ROS generation or by the silencing of Notch1 expression. Nox2 deficiency was associated with declines in the survival and angiogenic potency of miPSC-ECs, and capillary and arterial density were lower in the ischemic limbs of mice after treatment with Nox2−/− miPSC-ECs than WT miPSC-EC treatment. Taken together, these observations indicate that Nox2-mediated ROS production promotes arterial EC specification in differentiating miPSCs by activating the Notch signaling pathway and contributes to the angiogenic potency of transplanted miPSC-derived ECs. PMID:27642005

  1. Nox2 contributes to the arterial endothelial specification of mouse induced pluripotent stem cells by upregulating Notch signaling.

    PubMed

    Kang, Xueling; Wei, Xiangxiang; Wang, Xinhong; Jiang, Li; Niu, Cong; Zhang, Jianyi; Chen, Sifeng; Meng, Dan

    2016-01-01

    Reactive oxygen species (ROS) have a crucial role in stem-cell differentiation; however, the mechanisms by which ROS regulate the differentiation of stem cells into endothelial cells (ECs) are unknown. Here, we determine the role of ROS produced by NADPH oxidase 2 (Nox2) in the endothelial-lineage specification of mouse induced-pluripotent stem cells (miPSCs). When wild-type (WT) and Nox2-knockout (Nox2(-/-)) miPSCs were differentiated into ECs (miPSC-ECs), the expression of endothelial markers, arterial endothelial markers, pro-angiogenic cytokines, and Notch pathway components was suppressed in the Nox2(-/-) cells but increased in both WT and Nox2(-/-) miPSCs when Nox2 expression was upregulated. Higher levels of Nox2 expression increased Notch signaling and arterial EC differentiation, and this increase was abolished by the inhibition of ROS generation or by the silencing of Notch1 expression. Nox2 deficiency was associated with declines in the survival and angiogenic potency of miPSC-ECs, and capillary and arterial density were lower in the ischemic limbs of mice after treatment with Nox2(-/-) miPSC-ECs than WT miPSC-EC treatment. Taken together, these observations indicate that Nox2-mediated ROS production promotes arterial EC specification in differentiating miPSCs by activating the Notch signaling pathway and contributes to the angiogenic potency of transplanted miPSC-derived ECs. PMID:27642005

  2. Regeneration of sensory hair cells requires localized interactions between the Notch and Wnt pathways

    PubMed Central

    Romero-Carvajal, Andrés; Acedo, Joaquín Navajas; Jiang, Linjia; Kozlovskaja-Gumbrienė, Agnė; Alexander, Richard; Li, Hua; Piotrowski, Tatjana

    2015-01-01

    Summary In vertebrates, mechano-electrical transduction of sound is accomplished by sensory hair cells. While mammalian hair cells are not replaced when lost, in fish they constantly renew and regenerate after injury. In vivo tracking and cell fate analyses of all dividing cells during lateral line hair cell regeneration revealed that support and hair cell progenitors localize to distinct tissue compartments. Importantly, we find that the balance between self-renewal and differentiation in these compartments is controlled by spatially restricted Notch signaling and its inhibition of Wnt-induced proliferation. The ability to simultaneously study and manipulate individual cell behaviors and multiple pathways in vivo, transforms the lateral line into a powerful paradigm to mechanistically dissect sensory organ regeneration. The striking similarities to other vertebrate stem cell compartments uniquely place zebrafish to help elucidate why mammals possess such low capacity to regenerate hair cells. PMID:26190147

  3. Regulation of Notch Signaling by an Evolutionary Conserved DEAD Box RNA Helicase, Maheshvara in Drosophila melanogaster.

    PubMed

    Surabhi, Satya; Tripathi, Bipin K; Maurya, Bhawana; Bhaskar, Pradeep K; Mukherjee, Ashim; Mutsuddi, Mousumi

    2015-11-01

    Notch signaling is an evolutionary conserved process that influences cell fate determination, cell proliferation, and cell death in a context-dependent manner. Notch signaling is fine-tuned at multiple levels and misregulation of Notch has been implicated in a variety of human diseases. We have characterized maheshvara (mahe), a novel gene in Drosophila melanogaster that encodes a putative DEAD box protein that is highly conserved across taxa and belongs to the largest group of RNA helicase. A dynamic pattern of mahe expression along with the maternal accumulation of its transcripts is seen during early stages of embryogenesis. In addition, a strong expression is also seen in the developing nervous system. Ectopic expression of mahe in a wide range of tissues during development results in a variety of defects, many of which resemble a typical Notch loss-of-function phenotype. We illustrate that ectopic expression of mahe in the wing imaginal discs leads to loss of Notch targets, Cut and Wingless. Interestingly, Notch protein levels are also lowered, whereas no obvious change is seen in the levels of Notch transcripts. In addition, mahe overexpression can significantly rescue ectopic Notch-mediated proliferation of eye tissue. Further, we illustrate that mahe genetically interacts with Notch and its cytoplasmic regulator deltex in trans-heterozygous combination. Coexpression of Deltex and Mahe at the dorso-ventral boundary results in a wing-nicking phenotype and a more pronounced loss of Notch target Cut. Taken together we report identification of a novel evolutionary conserved RNA helicase mahe, which plays a vital role in regulation of Notch signaling.

  4. Cisplatin selects for stem-like cells in osteosarcoma by activating Notch signaling

    PubMed Central

    Yang, Jian; Gao, Tian; Simões, Bruno M.; Eyre, Rachel; Guo, Weichun; Clarke, Robert B.

    2016-01-01

    Notch signaling regulates normal stem cells and is also thought to regulate cancer stem cells (CSCs). Recent data indicate that Notch signaling plays a role in the development and progression of osteosarcoma, however the regulation of Notch in chemo-resistant stem-like cells has not yet been fully elucidated. In this study we generated cisplatin-resistant osteosarcoma cells by treating them with sub-lethal dose of cisplatin, sufficient to induce DNA damage responses. Cisplatin-resistant osteosarcoma cells exhibited lower proliferation, enhanced spheroid formation and more mesenchymal characteristics than cisplatin-sensitive cells, were enriched for Stro-1+/CD117+ cells and showed increased expression of stem cell-related genes. A similar effect was observed in vivo, and in addition in vivo tumorigenicity was enhanced during serial transplantation. Using several publicly available datasets, we identified that Notch expression was closely associated with osteosarcoma stem cells and chemotherapy resistance. We confirmed that cisplatin-induced enrichment of osteosarcoma stem cells was mediated through Notch signaling in vitro, and immunohistochemistry showed that cleaved Notch1 (NICD1) positive cells were significantly increased in a relapsed xenograft which had received cisplatin treatment. Furthermore, pretreatment with a γ-secretase inhibitor (GSI) to prevent Notch signalling inhibited cisplatin-enriched osteosarcoma stem cell activity in vitro, including Stro-1+/CD117+ double positive cells and spheroid formation capacity. The Notch inhibitor DAPT also prevented tumor recurrence in resistant xenograft tumors. Overall, our results show that cisplatin induces the enrichment of osteosarcoma stem-like cells through Notch signaling, and targeted inactivation of Notch may be useful for the elimination of CSCs and overcoming drug resistance. PMID:27102300

  5. Notch signaling: from stem cell expansion to improving cord blood transplantation.

    PubMed

    Mayani, Hector

    2010-08-01

    Ex vivo expansion of hematopoietic stem and progenitor cells has been a major goal for experimental hematologists and stem cell biologists during the last two decades. The clinical implications of such a procedure are obvious, considering the increasing interest in cell therapy protocols. This is particularly true in the setting of cord blood transplants, in which increased numbers of such primitive cells are needed. The study analyzed in this article indicates that by stimulating the Notch signal transduction pathway in primitive cord blood cells it is possible to significantly increase the numbers of both hematopoietic stem and progenitor cells. Furthermore, infusion of such expanded cells in patients receiving a cord blood transplant results in a significant reduction in the time to myeloid engraftment. The relevance of this study is twofold--on the one hand, it shows that the Notch pathway is involved in the expansion capacity of primitive hematopoietic cells in culture, and on the other hand, it indicates that ex vivo-expanded stem/progenitor cells can have a role in hematopoietic transplantation settings. PMID:21083031

  6. Canonical Notch signaling plays an instructive role in auditory supporting cell development

    PubMed Central

    Campbell, Dean P.; Chrysostomou, Elena; Doetzlhofer, Angelika

    2016-01-01

    The auditory sensory epithelium, composed of mechano-sensory hair cells (HCs) and highly specialized glial-like supporting cells (SCs), is critical for our ability to detect sound. SCs provide structural and functional support to HCs and play an essential role in cochlear development, homeostasis and repair. Despite their importance, however, surprisingly little is known about the molecular mechanisms guiding SC differentiation. Here, we provide evidence that in addition to its well-characterized inhibitory function, canonical Notch signaling plays a positive, instructive role in the differentiation of SCs. Using γ-secretase inhibitor DAPT to acutely block canonical Notch signaling, we identified a cohort of Notch-regulated SC-specific genes, with diverse functions in cell signaling, cell differentiation, neuronal innervation and synaptogenesis. We validated the newly identified Notch-regulated genes in vivo using genetic gain (Emx2Cre/+; Rosa26N1ICD/+) and loss-of-function approaches (Emx2Cre/+; Rosa26DnMAML1/+). Furthermore, we demonstrate that Notch over-activation in the differentiating murine cochlea (Emx2Cre/+; Rosa26N1ICD/+) actively promotes a SC-specific gene expression program. Finally, we show that outer SCs –so called Deiters’ cells are selectively lost by prolonged reduction (Emx2Cre/+; Rosa26DnMAML1/+/+) or abolishment of canonical Notch signaling (Fgfr3-iCreER; Rbpj−/Δ), indicating a critical role for Notch signaling in Deiters’ cell development. PMID:26786414

  7. Notch signaling is required for the formation of mesangial cells from a stromal mesenchyme precursor during kidney development

    PubMed Central

    Boyle, Scott C.; Liu, Zhenyi; Kopan, Raphael

    2014-01-01

    Mesangial cells are specialized pericyte/smooth muscle cells that surround and constrain the vascular network within the glomerulus of the kidney. They are derived from the stromal mesenchyme, a progenitor population distinct from nephron stem cells. Whether mesangial cells have a distinct origin from vascular smooth muscle cells (VSMCs) and the pathways that govern their specification are unknown. Here we show that Notch signaling in stromal progenitors is essential for mesangial cell formation but is dispensable for the smooth muscle and interstitial cell lineages. Deletion of RBPjk, the common DNA-binding partner of all active Notch receptors, with Foxd1tgCre results in glomerular aneurysm and perinatal death from kidney failure. This defect occurs early in glomerular development as stromal-derived, desmin-positive cells fail to coalesce near forming nephrons and thus do not invade the vascular cleft of the S-shaped body. This is in contrast to other mutants in which the loss of the mesangium was due to migration defects, and suggests that loss of Notch signaling results in a failure to specify this population from the stroma. Interestingly, Pdgfrb-positive VSMCs do not enter the vascular cleft and cannot rescue the mesangial deficiency. Notch1 and Notch2 act redundantly through γ-secretase and RBPjk in this process, as individual mutants have mesangial cells at birth. Together, these data demonstrate a unique origin of mesangial cells and demonstrate a novel, redundant function for Notch receptors in mesangial cell specification, proliferation or survival during kidney development. PMID:24353058

  8. NOTCH, ASCL1, p53 and RB alterations define an alternative pathway driving neuroendocrine and small cell lung carcinomas.

    PubMed

    Meder, Lydia; König, Katharina; Ozretić, Luka; Schultheis, Anne M; Ueckeroth, Frank; Ade, Carsten P; Albus, Kerstin; Boehm, Diana; Rommerscheidt-Fuss, Ursula; Florin, Alexandra; Buhl, Theresa; Hartmann, Wolfgang; Wolf, Jürgen; Merkelbach-Bruse, Sabine; Eilers, Martin; Perner, Sven; Heukamp, Lukas C; Buettner, Reinhard

    2016-02-15

    Small cell lung cancers (SCLCs) and extrapulmonary small cell cancers (SCCs) are very aggressive tumors arising de novo as primary small cell cancer with characteristic genetic lesions in RB1 and TP53. Based on murine models, neuroendocrine stem cells of the terminal bronchioli have been postulated as the cellular origin of primary SCLC. However, both in lung and many other organs, combined small cell/non-small cell tumors and secondary transitions from non-small cell carcinomas upon cancer therapy to neuroendocrine and small cell tumors occur. We define features of "small cell-ness" based on neuroendocrine markers, characteristic RB1 and TP53 mutations and small cell morphology. Furthermore, here we identify a pathway driving the pathogenesis of secondary SCLC involving inactivating NOTCH mutations, activation of the NOTCH target ASCL1 and canonical WNT-signaling in the context of mutual bi-allelic RB1 and TP53 lesions. Additionally, we explored ASCL1 dependent RB inactivation by phosphorylation, which is reversible by CDK5 inhibition. We experimentally verify the NOTCH-ASCL1-RB-p53 signaling axis in vitro and validate its activation by genetic alterations in vivo. We analyzed clinical tumor samples including SCLC, SCC and pulmonary large cell neuroendocrine carcinomas and adenocarcinomas using amplicon-based Next Generation Sequencing, immunohistochemistry and fluorescence in situ hybridization. In conclusion, we identified a novel pathway underlying rare secondary SCLC which may drive small cell carcinomas in organs other than lung, as well. PMID:26340530

  9. Inhibition of Notch signaling promotes browning of white adipose tissue and ameliorates obesity.

    PubMed

    Bi, Pengpeng; Shan, Tizhong; Liu, Weiyi; Yue, Feng; Yang, Xin; Liang, Xin-Rong; Wang, Jinghua; Li, Jie; Carlesso, Nadia; Liu, Xiaoqi; Kuang, Shihuan

    2014-08-01

    Beige adipocytes in white adipose tissue (WAT) are similar to classical brown adipocytes in that they can burn lipids to produce heat. Thus, an increase in beige adipocyte content in WAT browning would raise energy expenditure and reduce adiposity. Here we report that adipose-specific inactivation of Notch1 or its signaling mediator Rbpj in mice results in browning of WAT and elevated expression of uncoupling protein 1 (Ucp1), a key regulator of thermogenesis. Consequently, as compared to wild-type mice, Notch mutants exhibit elevated energy expenditure, better glucose tolerance and improved insulin sensitivity and are more resistant to high fat diet-induced obesity. By contrast, adipose-specific activation of Notch1 leads to the opposite phenotypes. At the molecular level, constitutive activation of Notch signaling inhibits, whereas Notch inhibition induces, Ppargc1a and Prdm16 transcription in white adipocytes. Notably, pharmacological inhibition of Notch signaling in obese mice ameliorates obesity, reduces blood glucose and increases Ucp1 expression in white fat. Therefore, Notch signaling may be therapeutically targeted to treat obesity and type 2 diabetes.

  10. Notch signaling and proneural genes work together to control the neural building blocks for the initial scaffold in the hypothalamus

    PubMed Central

    Ware, Michelle; Hamdi-Rozé, Houda; Dupé, Valérie

    2014-01-01

    The vertebrate embryonic prosencephalon gives rise to the hypothalamus, which plays essential roles in sensory information processing as well as control of physiological homeostasis and behavior. While patterning of the hypothalamus has received much attention, initial neurogenesis in the developing hypothalamus has mostly been neglected. The first differentiating progenitor cells of the hypothalamus will give rise to neurons that form the nucleus of the tract of the postoptic commissure (nTPOC) and the nucleus of the mammillotegmental tract (nMTT). The formation of these neuronal populations has to be highly controlled both spatially and temporally as these tracts will form part of the ventral longitudinal tract (VLT) and act as a scaffold for later, follower axons. This review will cumulate and summarize the existing data available describing initial neurogenesis in the vertebrate hypothalamus. It is well-known that the Notch signaling pathway through the inhibition of proneural genes is a key regulator of neurogenesis in the vertebrate central nervous system. It has only recently been proposed that loss of Notch signaling in the developing chick embryo causes an increase in the number of neurons in the hypothalamus, highlighting an early function of the Notch pathway during hypothalamus formation. Further analysis in the chick and mouse hypothalamus confirms the expression of Notch components and Ascl1 before the appearance of the first differentiated neurons. Many newly identified proneural target genes were also found to be expressed during neuronal differentiation in the hypothalamus. Given the critical role that hypothalamic neural circuitry plays in maintaining homeostasis, it is particularly important to establish the targets downstream of this Notch/proneural network. PMID:25520625

  11. Aging of signal transduction pathways, and pathology

    PubMed Central

    Carlson, Morgan E.; Silva, Haroldo S.; Conboy, Irina M.

    2008-01-01

    The major cell signaling pathways, and their specific mechanisms of transduction, have been a subject of investigation for many years. As our understanding of these pathways advances, we find that they are evolutionarily well-conserved not only individually, but also at the level of their crosstalk and signal integration. Productive interactions within the key signal transduction networks determine success in embryonic organogenesis, and postnatal tissue repair throughout adulthood. However, aside from clues revealed through examining age-related degenerative diseases, much remains uncertain about imbalances within these pathways during normal aging. Further, little is known about the molecular mechanisms by which alterations in the major cell signal transduction networks cause age-related pathologies. The aim of this review is to describe the complex interplay between the Notch, TGFβ, WNT, RTK-Ras and Hh signaling pathways, with a specific focus on the changes introduced within these networks by the aging process, and those typical of age-associated human pathologies. PMID:18474281

  12. Immunohistochemical expression of aberrant Notch-1 signaling in vitiligo: an implication for pathogenesis.

    PubMed

    Seleit, Iman; Bakry, Ola Ahmed; Abdou, Asmaa Gaber; Dawoud, Noha Mohammed

    2014-06-01

    The etiopathogenetic mechanisms leading to pigment loss in vitiligo are not fully understood. Notch signaling is required for development and maintenance of melanocyte lineage and acts as a key component among keratinocyte-melanocyte interactions. The current study aimed to investigate the possible role of Notch signaling and its effect on the whole melanocyte lineage in vitiligo and correlating it with the different clinicopathologic parameters. Using immunohistochemical technique, Notch-1 expression was evaluated in 50 lesional and 20 perilesional biopsies of patients with vitiligo in comparison with 20 normal skin biopsies as a control group. Lesional biopsies were stained with human melanoma black-45 and tyrosinase-related protein-2 to demonstrate the melanocyte lineage. Membranous and/or nuclear expression of Notch-1 was in favor of control and perilesional skin, whereas cytoplasmic expression appeared only in vitiliginous lesions (P < .05). Membranous and/or nuclear expression of Notch-1 was significantly associated with epidermal human melanoma black-45 positivity (P = .01) and percentage of expression in both epidermis (P = .02) and hair follicles (P = .03) of lesional skin. Cytoplasmic pattern of Notch-1 expression in epidermis was significantly found in lesions with white hair (P = .04) and in cases with marked keratinocyte vacuolization (P = .03). Segmental and acrofacial vitiligo were associated with mild to moderate Notch-1 intensity, whereas generalized vitiligo was associated with strong intensity of expression (P = .02). In conclusion, Notch-1 signaling is inactivated in vitiligo with consequent loss of epidermal and/or follicular active melanocytes. Aberrant Notch signaling in vitiliginous white hair and acral and segmental vitiligo may be the cause of their treatment resistance.

  13. Effects of Linker Length and Transient Secondary Structure Elements in the Intrinsically Disordered Notch RAM Region on Notch Signaling.

    PubMed

    Sherry, Kathryn P; Johnson, Scott E; Hatem, Christine L; Majumdar, Ananya; Barrick, Doug

    2015-11-01

    Formation of the bivalent interaction between the Notch intracellular domain (NICD) and the transcription factor CBF-1/RBP-j, Su(H), Lag-1 (CSL) is a key event in Notch signaling because it switches Notch-responsive genes from a repressed state to an activated state. Interaction of the intrinsically disordered RBP-j-associated molecule (RAM) region of NICD with CSL is thought to both disrupt binding of corepressor proteins to CSL and anchor NICD to CSL, promoting interaction of the ankyrin domain of NICD with CSL through an effective concentration mechanism. To quantify the role of disorder in the RAM linker region on the effective concentration enhancement of Notch transcriptional activation, we measured the effects of linker length variation on activation. The resulting activation profile has general features of a worm-like chain model for effective concentration. However, deviations from the model for short sequence deletions suggest that RAM contains sequence-specific structural elements that may be important for activation. Structural characterization of the RAM linker with sedimentation velocity analytical ultracentrifugation and NMR spectroscopy reveals that the linker is compact and contains three transient helices and two extended and dynamic regions. To test if these secondary structure elements are important for activation, we made sequence substitutions to change the secondary structure propensities of these elements and measured transcriptional activation of the resulting variants. Substitutions to two of these nonrandom elements (helix 2, extended region 1) have effects on activation, but these effects do not depend on the nature of the substituting residues. Thus, the primary sequences of these elements, but not their secondary structures, are influencing signaling.

  14. Role of stromal cell-mediated Notch signaling in CLL resistance to chemotherapy

    PubMed Central

    Kamdje, A H Nwabo; Bassi, G; Pacelli, L; Malpeli, G; Amati, E; Nichele, I; Pizzolo, G; Krampera, M

    2012-01-01

    Stromal cells are essential components of the bone marrow (BM) microenvironment that regulate and support the survival of different tumors, including chronic lymphocytic leukemia (CLL). In this study, we investigated the role of Notch signaling in the promotion of survival and chemoresistance of human CLL cells in coculture with human BM-mesenchymal stromal cells (hBM-MSCs) of both autologous and allogeneic origin. The presence of BM-MSCs rescued CLL cells from apoptosis both spontaneously and following induction with various drugs, including Fludarabine, Cyclophosphamide, Bendamustine, Prednisone and Hydrocortisone. The treatment with a combination of anti-Notch-1, Notch-2 and Notch-4 antibodies or γ-secretase inhibitor XII (GSI XII) reverted this protective effect by day 3, even in presence of the above-mentioned drugs. Overall, our findings show that stromal cell-mediated Notch-1, Notch-2 and Notch-4 signaling has a role in CLL survival and resistance to chemotherapy. Therefore, its blocking could be an additional tool to overcome drug resistance and improve the therapeutic strategies for CLL. PMID:22829975

  15. Notch Activation of Ca2+ Signaling in the Development of Hypoxic Pulmonary Vasoconstriction and Pulmonary Hypertension

    PubMed Central

    Smith, Kimberly A.; Voiriot, Guillaume; Tang, Haiyang; Fraidenburg, Dustin R.; Song, Shanshan; Yamamura, Hisao; Yamamura, Aya; Guo, Qiang; Wan, Jun; Pohl, Nicole M.; Tauseef, Mohammad; Bodmer, Rolf; Ocorr, Karen; Thistlethwaite, Patricia A.; Haddad, Gabriel G.; Powell, Frank L.; Makino, Ayako; Mehta, Dolly

    2015-01-01

    Hypoxic pulmonary vasoconstriction (HPV) is an important physiological response that optimizes the ventilation/perfusion ratio. Chronic hypoxia causes vascular remodeling, which is central to the pathogenesis of hypoxia-induced pulmonary hypertension (HPH). We have previously shown that Notch3 is up-regulated in HPH and that activation of Notch signaling enhances store-operated Ca2+ entry (SOCE), an important mechanism that contributes to pulmonary arterial smooth muscle cell (PASMC) proliferation and contraction. Here, we investigate the role of Notch signaling in HPV and hypoxia-induced enhancement of SOCE. We examined SOCE in human PASMCs exposed to hypoxia and pulmonary arterial pressure in mice using the isolated perfused/ventilated lung method. Wild-type and canonical transient receptor potential (TRPC) 6−/− mice were exposed to chronic hypoxia to induce HPH. Inhibition of Notch signaling with a γ-secretase inhibitor attenuates hypoxia-enhanced SOCE in PASMCs and hypoxia-induced increase in pulmonary arterial pressure. Our results demonstrate that hypoxia activates Notch signaling and up-regulates TRPC6 channels. Additionally, treatment with a Notch ligand can mimic hypoxic responses. Finally, inhibition of TRPC6, either pharmacologically or genetically, attenuates HPV, hypoxia-enhanced SOCE, and the development of HPH. These results demonstrate that hypoxia-induced activation of Notch signaling mediates HPV and the development of HPH via functional activation and up-regulation of TRPC6 channels. Understanding the molecular mechanisms that regulate cytosolic free Ca2+ concentration and PASMC proliferation is critical to elucidation of the pathogenesis of HPH. Targeting Notch regulation of TRPC6 will be beneficial in the development of novel therapies for pulmonary hypertension associated with hypoxia. PMID:25569851

  16. Prevention against diffuse spinal cord astrocytoma: can the Notch pathway be a novel treatment target?

    PubMed Central

    Sun, Jian-jun; Wang, Zhen-yu; Li, Ling-song; Yu, Hai-yan; Xu, Yong-sheng; Wu, Hai-bo; Luo, Yi; Liu, Bin; Zheng, Mei; Mao, Jin-long; Lou, Xiao-hui

    2015-01-01

    This study was designed to investigate whether the Notch pathway is involved in the development of diffuse spinal cord astrocytomas. BALB/c nude mice received injections of CD133+ and CD133− cell suspensions prepared using human recurrent diffuse spinal cord astrocytoma tissue through administration into the right parietal lobe. After 7–11 weeks, magnetic resonance imaging was performed weekly. Xenografts were observed on the surfaces of the brains of mice receiving the CD133+ cell suspension, and Notch-immunopositive expression was observed in the xenografts. By contrast, no xenografts appeared in the identical position on the surfaces of the brains of mice receiving the CD133− cell suspension, and Notch-immunopositive expression was hardly detected either. Hematoxylin-eosin staining and immunohistochemical staining revealed xenografts on the convex surfaces of the brains of mice that underwent CD133+ astrocytoma transplantation. Some sporadic astroglioma cells showed pseudopodium-like structures, which extended into the cerebral white matter. However, it should be emphasized that the subcortex xenograft with Notch-immunopositive expression was found in the fourth mouse received injection of CD133− astrocytoma cells. However, these findings suggest that the Notch pathway plays an important role in the formation of astrocytomas, and can be considered a novel treatment target for diffuse spinal cord astrocytoma. PMID:25883623

  17. A Disintegrin and Metalloproteinase10 (ADAM10) Regulates NOTCH Signaling during Early Retinal Development

    PubMed Central

    Toonen, Joseph A.; Ronchetti, Adam; Sidjanin, D. J.

    2016-01-01

    ADAM10 and ADAM17 are two closely related members of the ADAM (a disintegrin and metalloprotease) family of membrane-bound sheddases, which proteolytically cleave surface membrane proteins. Both ADAM10 and ADAM17 have been implicated in the proteolytic cleavage of NOTCH receptors and as such regulators of NOTCH signaling. During retinal development, NOTCH signaling facilitates retinal neurogenesis by maintaining progenitor cells in a proliferative state and by mediating retinal cell fates. However, the roles of ADAM10 and ADAM17 in the retina are not well defined. In this study, we set out to clarify the roles of ADAM10 and ADAM17 during early retinal development. The retinal phenotype of conditionally abated Adam17 retinae (Adam17 CKO) did not differ from the controls whereas conditionally ablated Adam10 retinae (Adam10 CKO) exhibited abnormal morphogenesis characterized by the formation of rosettes and a loss of retinal laminae phenotypically similar to morphological abnormalities identified in mice with retinal NOTCH signaling deficiency. Additionally, Adam10 CKO retinae exhibited abnormal neurogenesis characterized by fewer proliferating progenitor cells and greater differentiation of early photoreceptors and retinal ganglion cells. Moreover, constitutive activation of the NOTCH1-intracellular domain (N1-ICD) rescued Adam10 CKO abnormal neurogenesis, as well as abnormal retinal morphology by maintaining retinal cells in the progenitor state. Collectively these findings provide in vivo genetic evidence that ADAM10, and not ADAM17, is indispensable for proper retinal development as a regulator of NOTCH signaling. PMID:27224017

  18. Notch2 receptor signaling controls functional differentiation of dendritic cells in the spleen and intestine.

    PubMed

    Lewis, Kanako L; Caton, Michele L; Bogunovic, Milena; Greter, Melanie; Grajkowska, Lucja T; Ng, Dennis; Klinakis, Apostolos; Charo, Israel F; Jung, Steffen; Gommerman, Jennifer L; Ivanov, Ivaylo I; Liu, Kang; Merad, Miriam; Reizis, Boris

    2011-11-23

    Dendritic cells (DCs) in tissues and lymphoid organs comprise distinct functional subsets that differentiate in situ from circulating progenitors. Tissue-specific signals that regulate DC subset differentiation are poorly understood. We report that DC-specific deletion of the Notch2 receptor caused a reduction of DC populations in the spleen. Within the splenic CD11b(+) DC subset, Notch signaling blockade ablated a distinct population marked by high expression of the adhesion molecule Esam. The Notch-dependent Esam(hi) DC subset required lymphotoxin beta receptor signaling, proliferated in situ, and facilitated CD4(+) T cell priming. The Notch-independent Esam(lo) DCs expressed monocyte-related genes and showed superior cytokine responses. In addition, Notch2 deletion led to the loss of CD11b(+)CD103(+) DCs in the intestinal lamina propria and to a corresponding decrease of IL-17-producing CD4(+) T cells in the intestine. Thus, Notch2 is a common differentiation signal for T cell-priming CD11b(+) DC subsets in the spleen and intestine.

  19. Gamma-secretase Inhibitor Prevents Proliferation and Migration of Ductus Arteriosus Smooth Muscle Cells through the Notch3-HES1/2/5 Pathway

    PubMed Central

    Wu, Jiunn-Ren; Yeh, Jwu-Lai; Liou, Shu-Fen; Dai, Zen-Kong; Wu, Bin-Nan; Hsu, Jong-Hau

    2016-01-01

    Patent ductus arteriosus (PDA) can cause morbidity and mortality in neonates. Vascular remodeling, characterized by proliferation and migration of smooth muscle cells (SMCs), is an essential process for postnatal DA closure. Notch signaling is an important mediator of vascular remodelling but its role in DA is unkonwn. We investigated the effects and underlying mechanisms of γ-secretase inhibitor DAPT, a Notch signaling inhibitor on angiotensin II (Ang II)-induced proliferation and migration of DASMCs. Proliferation and migration of DASMCs cultured from neonatal Wistar rats were induced by Ang II, with or without DAPT pre-treatment. In addition, potential underlying mechanisms including cell cycle progression, Ca2+ influx, reactive oxygen species (ROS) production, signal transduction of MAPK and Akt, and Notch receptor with its target gene pathway were examined. We found that DAPT inhibited Ang II-induced DASMCs proliferation and migration dose dependently. DAPT also arrested the cell cycle progression in the G0/G1-phase, and attenuated calcium overload and ROS production caused by Ang II. Moreover, DAPT inhibited nuclear translocation of Notch3 receptor intracellular domain, with decreased expression of its down-stream genes including HES1, HES2 and HES5. Finally, Ang II-activated ERK1/2, JNK and Akt were also counteracted by DAPT. In conclusion, DAPT inhibits Ang II-induced DASMCs proliferation and migration. These effects are potentially mediated by decreased calcium influx, reduced ROS production, and down-regulation of ERK1/2, JNK and Akt, through the Notch3-HES1/2/5 pathway. Therefore, Notch signaling has a role in DA remodeling and may provide a target pathway for therapeutic intervention of PDA. PMID:27570480

  20. Gamma-secretase Inhibitor Prevents Proliferation and Migration of Ductus Arteriosus Smooth Muscle Cells through the Notch3-HES1/2/5 Pathway.

    PubMed

    Wu, Jiunn-Ren; Yeh, Jwu-Lai; Liou, Shu-Fen; Dai, Zen-Kong; Wu, Bin-Nan; Hsu, Jong-Hau

    2016-01-01

    Patent ductus arteriosus (PDA) can cause morbidity and mortality in neonates. Vascular remodeling, characterized by proliferation and migration of smooth muscle cells (SMCs), is an essential process for postnatal DA closure. Notch signaling is an important mediator of vascular remodelling but its role in DA is unkonwn. We investigated the effects and underlying mechanisms of γ-secretase inhibitor DAPT, a Notch signaling inhibitor on angiotensin II (Ang II)-induced proliferation and migration of DASMCs. Proliferation and migration of DASMCs cultured from neonatal Wistar rats were induced by Ang II, with or without DAPT pre-treatment. In addition, potential underlying mechanisms including cell cycle progression, Ca(2+) influx, reactive oxygen species (ROS) production, signal transduction of MAPK and Akt, and Notch receptor with its target gene pathway were examined. We found that DAPT inhibited Ang II-induced DASMCs proliferation and migration dose dependently. DAPT also arrested the cell cycle progression in the G0/G1-phase, and attenuated calcium overload and ROS production caused by Ang II. Moreover, DAPT inhibited nuclear translocation of Notch3 receptor intracellular domain, with decreased expression of its down-stream genes including HES1, HES2 and HES5. Finally, Ang II-activated ERK1/2, JNK and Akt were also counteracted by DAPT. In conclusion, DAPT inhibits Ang II-induced DASMCs proliferation and migration. These effects are potentially mediated by decreased calcium influx, reduced ROS production, and down-regulation of ERK1/2, JNK and Akt, through the Notch3-HES1/2/5 pathway. Therefore, Notch signaling has a role in DA remodeling and may provide a target pathway for therapeutic intervention of PDA. PMID:27570480

  1. Genetic amplification of the NOTCH modulator LNX2 upregulates the WNT/β-catenin pathway in colorectal cancer

    PubMed Central

    Camps, Jordi; Pitt, Jason J.; Emons, Georg; Hummon, Amanda B.; Case, Chanelle M.; Grade, Marian; Jones, Tamara L.; Nguyen, Quang T.; Ghadimi, B. Michael; Beissbarth, Tim; Difilippantonio, Michael J.; Caplen, Natasha J.; Ried, Thomas

    2016-01-01

    Chromosomal copy number alterations (aneuploidy) define the genomic landscape of most cancer cells, but identification of the oncogenic drivers behind these imbalances remains an unfinished task. In this study, we conducted a systematic analysis of colorectal carcinomas that integrated genomic copy number changes and gene expression profiles. This analysis revealed 44 highly overexpressed genes mapping to localized amplicons on chromosome 13, gains of which occur often in colorectal cancers. RNAi-mediated silencing identified eight candidates whose loss of function reduced cell viability 20% or more in colorectal cancer cell lines. The functional space of the genes NUPL1, LNX2, POLR1D, POMP, SLC7A1, DIS3, KLF5, and GPR180 was established by global expression profiling after RNAi exposure. One candidate, LNX2, not previously known as an oncogene, was involved in regulating NOTCH signaling. Silencing LNX2 reduced NOTCH levels but also downregulated the transcription factor TCF7L2 and markedly reduced WNT signaling. LNX2 overexpression and chromosome 13 amplification therefore constitutively activates the WNT pathway, offering evidence of an aberrant NOTCH-WNT axis in colorectal cancer. PMID:23319804

  2. TNFα and Endothelial Cells Modulate Notch Signaling in the Bone Marrow Microenvironment during Inflammation

    PubMed Central

    Fernandez, Luis; Rodriguez, Sonia; Huang, Hui; Chora, Angelo; Mumaw, Christin; Cruz, Eugenia; Pollok, Karen; Cristina, Filipa; Price, Joanne E.; Ferkowicz, Michael J.; Scadden, David T.; Clauss, Matthias; Cardoso, Angelo A.; Carlesso, Nadia

    2009-01-01

    Objective Homeostasis of the hematopoietic compartment is challenged and maintained during conditions of stress by mechanisms that are poorly defined. To understand how the bone marrow (BM) microenvironment influences hematopoiesis, we explored the role of Notch signaling and bone marrow endothelial cells in providing microenvironmental cues to hematopoietic cells in the presence of inflammatory stimuli. Methods The human BM endothelial cell line BMEC and primary human BM endothelial cells were analyzed for expression of Notch ligands and the ability to expand hematopoietic progenitors in an in vitro co-culture system. In vivo experiments were carried out to identify modulation of Notch signaling in BM endothelial and hematopoietic cells in mice challenged with TNFα or LPS, or in Tie2-tmTNFα transgenic mice characterized by constitutive TNFα activation. Results BM endothelial cells were found to express Jagged ligands and to greatly support progenitor’s colony-forming ability. This effect was markedly decreased by Notch antagonists and augmented by increasing levels of Jagged2. Physiologic upregulation of Jagged2 expression on BMEC was observed upon TNFα activation. Injection of TNFα or LPS upregulated 3 to 4 fold Jagged2 expression on murine BM endothelial cells in vivo and resulted in increased Notch activation on murine hematopoietic stem/progenitor cells. Similarly, constitutive activation of endothelial cells in Tie2-tmTNFα mice was characterized by increased expression of Jagged2 and by augmented Notch activation on hematopoietic stem/progenitor cells. Conclusions Our results provide the first evidence that BM endothelial cells promote expansion of hematopoietic progenitor cells by a Notch-dependent mechanism and that TNFα and LPS can modulate the levels of Notch ligand expression and Notch activation in the bone marrow microenvironment in vivo. PMID:18439488

  3. Exploring the potential relationship between Notch pathway genes expression and their promoter methylation in mice hippocampal neurogenesis.

    PubMed

    Zhang, Zhen; Gao, Feng; Kang, Xiaokui; Li, Jia; Zhang, Litong; Dong, Wentao; Jin, Zhangning; Li, Fan; Gao, Nannan; Cai, Xinwang; Yang, Shuyuan; Zhang, Jianning; Ren, Xinliang; Yang, Xinyu

    2015-04-01

    The Notch pathway is a highly conserved pathway that regulates hippocampal neurogenesis during embryonic development and adulthood. It has become apparent that intracellular epigenetic modification including DNA methylation is deeply involved in fate specification of neural stem cells (NSCs). However, it is still unclear whether the Notch pathway regulates hippocampal neurogenesis by changing the Notch genes' DNA methylation status. Here, we present the evidence from DNA methylation profiling of Notch1, Hes1 and Ngn2 promoters during neurogenesis in the dentate gyrus (DG) of postnatal, adult and traumatic brains. We observed the expression of Notch1, Hes1 and Ngn2 in hippocampal DG with qPCR, Western blot and immunofluorescence staining. In addition, we investigated the methylation status of Notch pathway genes using the bisulfite sequencing PCR (BSP) method. The number of Notch1 or Hes1 (+) and BrdU (+) cells decreased in the subgranular zone (SGZ) of the DG in the hippocampus following TBI. Nevertheless, the number of Ngn2-positive cells in the DG of injured mice was markedly higher than in the DG of non-TBI mice. Accordingly, the DNA methylation level of the three gene promoters changed with their expression in the DG. These findings suggest that the strict spatio-temporal expression of Notch effector genes plays an important role during hippocampal neurogenesis and suggests the possibility that Notch1, Hes1 and Ngn2 were regulated by changing some specific CpG sites of their promoters to further orchestrate neurogenesis in vivo.

  4. The interplay between DSL proteins and ubiquitin ligases in Notch signaling.

    PubMed

    Pitsouli, Chrysoula; Delidakis, Christos

    2005-09-01

    Lateral inhibition is a pattern refining process that generates single neural precursors from a field of equipotent cells and is mediated via Notch signaling. Of the two Notch ligands Delta and Serrate, only the former was thought to participate in this process. We now show that macrochaete lateral inhibition involves both Delta and Serrate. In this context, Serrate interacts with Neuralized, a ubiquitin ligase that was heretofore thought to act only on Delta. Neuralized physically associates with Serrate and stimulates its endocytosis and signaling activity. We also characterize a mutation in mib1, a Drosophila homolog of mind bomb, another Delta-targeting ubiquitin ligase from zebrafish. Mib1 affects the signaling activity of Delta and Serrate in both lateral inhibition and wing dorsoventral boundary formation. Simultaneous absence of neuralized and mib1 completely abolishes Notch signaling in both aforementioned contexts, making it likely that ubiquitination is a prerequisite for Delta/Serrate signaling.

  5. Jagged-Notch signaling ensures dorsal skeletal identity in the vertebrate face

    PubMed Central

    Zuniga, Elizabeth; Stellabotte, Frank; Crump, J. Gage

    2010-01-01

    The development of the vertebrate face relies on the regionalization of neural crest-derived skeletal precursors along the dorsoventral (DV) axis. Here we show that Jagged-Notch signaling ensures dorsal identity within the hyoid and mandibular components of the facial skeleton by repressing ventral fates. In a genetic screen in zebrafish, we identified a loss-of-function mutation in jagged 1b (jag1b) that results in dorsal expansion of ventral gene expression and partial transformation of the dorsal hyoid skeleton to a ventral morphology. Conversely, misexpression of human jagged 1 (JAG1) represses ventral gene expression and dorsalizes the ventral hyoid and mandibular skeletons. We further show that jag1b is expressed specifically in dorsal skeletal precursors, where it acts through the Notch2 receptor to activate hey1 expression. Whereas Jagged-Notch positive feedback propagates jag1b expression throughout the dorsal domain, Endothelin 1 (Edn1) inhibits jag1b and hey1 expression in the ventral domain. Strikingly, reduction of Jag1b or Notch2 function partially rescues the ventral defects of edn1 mutants, indicating that Edn1 promotes facial skeleton development in part by inhibiting Jagged-Notch signaling in ventral skeletal precursors. Together, these results indicate a novel function of Jagged-Notch signaling in ensuring dorsal identity within broad fields of facial skeletal precursors. PMID:20431122

  6. The Notch pathway regulates both the proliferation and differentiation of follicular cells in the panoistic ovary of Blattella germanica.

    PubMed

    Irles, Paula; Elshaer, Nashwa; Piulachs, Maria-Dolors

    2016-01-01

    The Notch pathway is an essential regulator of cell proliferation and differentiation during development. Its involvement in insect oogenesis has been examined in insect species with meroistic ovaries, and it is known to play a fundamental role in cell fate decisions and the induction of the mitosis-to-endocycle switch in follicular cells (FCs). This work reports the functions of the main components of the Notch pathway (Notch and its ligands Delta and Serrate) during oogenesis in Blattella germanica, a phylogenetically basal species with panoistic ovary. As is revealed by RNAi-based analyses, Notch and Delta were found to contribute towards maintaining the FCs in an immature, non-apoptotic state. This ancestral function of Notch appears in opposition to the induction of transition from mitosis to endocycle that Notch exerts in Drosophila melanogaster, a change in the Notch function that might be in agreement with the evolution of the insect ovary types. Notch was also shown to play an active role in inducing ovarian follicle elongation via the regulation of the cytoskeleton. In addition, Delta and Notch interactions were seen to determine the differentiation of the posterior population of FCs. Serrate levels were found to be Notch-dependent and are involved in the control of the FC programme, although they would appear to play no crucial role in panoistic ovary oogenesis. PMID:26763344

  7. The Notch pathway regulates both the proliferation and differentiation of follicular cells in the panoistic ovary of Blattella germanica

    PubMed Central

    Irles, Paula; Elshaer, Nashwa; Piulachs, Maria-Dolors

    2016-01-01

    The Notch pathway is an essential regulator of cell proliferation and differentiation during development. Its involvement in insect oogenesis has been examined in insect species with meroistic ovaries, and it is known to play a fundamental role in cell fate decisions and the induction of the mitosis-to-endocycle switch in follicular cells (FCs). This work reports the functions of the main components of the Notch pathway (Notch and its ligands Delta and Serrate) during oogenesis in Blattella germanica, a phylogenetically basal species with panoistic ovary. As is revealed by RNAi-based analyses, Notch and Delta were found to contribute towards maintaining the FCs in an immature, non-apoptotic state. This ancestral function of Notch appears in opposition to the induction of transition from mitosis to endocycle that Notch exerts in Drosophila melanogaster, a change in the Notch function that might be in agreement with the evolution of the insect ovary types. Notch was also shown to play an active role in inducing ovarian follicle elongation via the regulation of the cytoskeleton. In addition, Delta and Notch interactions were seen to determine the differentiation of the posterior population of FCs. Serrate levels were found to be Notch-dependent and are involved in the control of the FC programme, although they would appear to play no crucial role in panoistic ovary oogenesis. PMID:26763344

  8. The Notch pathway regulates both the proliferation and differentiation of follicular cells in the panoistic ovary of Blattella germanica.

    PubMed

    Irles, Paula; Elshaer, Nashwa; Piulachs, Maria-Dolors

    2016-01-01

    The Notch pathway is an essential regulator of cell proliferation and differentiation during development. Its involvement in insect oogenesis has been examined in insect species with meroistic ovaries, and it is known to play a fundamental role in cell fate decisions and the induction of the mitosis-to-endocycle switch in follicular cells (FCs). This work reports the functions of the main components of the Notch pathway (Notch and its ligands Delta and Serrate) during oogenesis in Blattella germanica, a phylogenetically basal species with panoistic ovary. As is revealed by RNAi-based analyses, Notch and Delta were found to contribute towards maintaining the FCs in an immature, non-apoptotic state. This ancestral function of Notch appears in opposition to the induction of transition from mitosis to endocycle that Notch exerts in Drosophila melanogaster, a change in the Notch function that might be in agreement with the evolution of the insect ovary types. Notch was also shown to play an active role in inducing ovarian follicle elongation via the regulation of the cytoskeleton. In addition, Delta and Notch interactions were seen to determine the differentiation of the posterior population of FCs. Serrate levels were found to be Notch-dependent and are involved in the control of the FC programme, although they would appear to play no crucial role in panoistic ovary oogenesis.

  9. EZH2 expands breast stem cells through activation of NOTCH1 signaling.

    PubMed

    Gonzalez, Maria E; Moore, Heather M; Li, Xin; Toy, Kathy A; Huang, Wei; Sabel, Michael S; Kidwell, Kelley M; Kleer, Celina G

    2014-02-25

    Breast cancer is the second-leading cause of cancer-related deaths in women, but the details of how it begins remain elusive. Increasing evidence supports the association of aggressive triple-negative (TN) breast cancer with heightened expression of the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2) and increased tumor-initiating cells (TICs). However, mechanistic links between EZH2 and TICs are unclear, and direct demonstration of a tumorigenic function of EZH2 in vivo is lacking. Here, we identify an unrecognized EZH2/NOTCH1 axis that controls breast TICs in TN breast carcinomas. EZH2 overexpression increases NOTCH1 expression and signaling, and inhibition of NOTCH1 activity prevents EZH2-mediated stem cell expansion in nontumorigenic breast cells. We uncover a unique role of EZH2 in activating, rather than repressing, NOTCH1 signaling through binding to the NOTCH1 promoter in TN breast cancer cells. EZH2 binding is independent of its catalytic histone H3 lysine 27 methyltransferase activity and of the Polycomb Repressive Complex 2 but corresponds instead to transcriptional activation marks. In vivo, EZH2 knockdown decreases the onset and volume of xenografts derived from TN breast TICs. Conversely, transgenic EZH2 overexpression accelerates mammary tumor initiation and increases NOTCH1 activation in mouse mammary tumor virus-neu mice. Consonant with these findings, in clinical samples, high levels of EZH2 are significantly associated with activated NOTCH1 protein and increased TICs in TN invasive carcinomas. These data reveal a functional and mechanistic link between EZH2 levels, NOTCH1 signaling activation, and TICs, and provide previously unidentified evidence that EZH2 enhances breast cancer initiation. PMID:24516139

  10. EZH2 expands breast stem cells through activation of NOTCH1 signaling

    PubMed Central

    Gonzalez, Maria E.; Moore, Heather M.; Li, Xin; Toy, Kathy A.; Huang, Wei; Sabel, Michael S.; Kidwell, Kelley M.; Kleer, Celina G.

    2014-01-01

    Breast cancer is the second-leading cause of cancer-related deaths in women, but the details of how it begins remain elusive. Increasing evidence supports the association of aggressive triple-negative (TN) breast cancer with heightened expression of the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2) and increased tumor-initiating cells (TICs). However, mechanistic links between EZH2 and TICs are unclear, and direct demonstration of a tumorigenic function of EZH2 in vivo is lacking. Here, we identify an unrecognized EZH2/NOTCH1 axis that controls breast TICs in TN breast carcinomas. EZH2 overexpression increases NOTCH1 expression and signaling, and inhibition of NOTCH1 activity prevents EZH2-mediated stem cell expansion in nontumorigenic breast cells. We uncover a unique role of EZH2 in activating, rather than repressing, NOTCH1 signaling through binding to the NOTCH1 promoter in TN breast cancer cells. EZH2 binding is independent of its catalytic histone H3 lysine 27 methyltransferase activity and of the Polycomb Repressive Complex 2 but corresponds instead to transcriptional activation marks. In vivo, EZH2 knockdown decreases the onset and volume of xenografts derived from TN breast TICs. Conversely, transgenic EZH2 overexpression accelerates mammary tumor initiation and increases NOTCH1 activation in mouse mammary tumor virus-neu mice. Consonant with these findings, in clinical samples, high levels of EZH2 are significantly associated with activated NOTCH1 protein and increased TICs in TN invasive carcinomas. These data reveal a functional and mechanistic link between EZH2 levels, NOTCH1 signaling activation, and TICs, and provide previously unidentified evidence that EZH2 enhances breast cancer initiation. PMID:24516139

  11. Notch-regulated miR-223 targets the aryl hydrocarbon receptor pathway and increases cytokine production in macrophages from rheumatoid arthritis patients

    PubMed Central

    Ogando, Jesús; Tardáguila, Manuel; Díaz-Alderete, Andrea; Usategui, Alicia; Miranda-Ramos, Vanessa; Martínez-Herrera, Dannys Jorge; de la Fuente, Lorena; García-León, María J.; Moreno, María C.; Escudero, Sara; Cañete, Juan D.; Toribio, María L.; Cases, Ildefonso; Pascual-Montano, Alberto; Pablos, José Luis; Mañes, Santos

    2016-01-01

    Evidence links aryl hydrocarbon receptor (AHR) activation to rheumatoid arthritis (RA) pathogenesis, although results are inconsistent. AHR agonists inhibit pro-inflammatory cytokine expression in macrophages, pivotal cells in RA aetiopathogenesis, which hints at specific circuits that regulate the AHR pathway in RA macrophages. We compared microRNA (miR) expression in CD14+ cells from patients with active RA or with osteoarthritis (OA). Seven miR were downregulated and one (miR-223) upregulated in RA compared to OA cells. miR-223 upregulation correlated with reduced Notch3 and Notch effector expression in RA patients. Overexpression of the Notch-induced repressor HEY-1 and co-culture of healthy donor monocytes with Notch ligand-expressing cells showed direct Notch-mediated downregulation of miR-223. Bioinformatics predicted the AHR regulator ARNT (AHR nuclear translocator) as a miR-223 target. Pre-miR-223 overexpression silenced ARNT 3’UTR-driven reporter expression, reduced ARNT (but not AHR) protein levels and prevented AHR/ARNT-mediated inhibition of pro-inflammatory cytokine expression. miR-223 counteracted AHR/ARNT-induced Notch3 upregulation in monocytes. Levels of ARNT and of CYP1B1, an AHR/ARNT signalling effector, were reduced in RA compared to OA synovial tissue, which correlated with miR-223 levels. Our results associate Notch signalling to miR-223 downregulation in RA macrophages, and identify miR-223 as a negative regulator of the AHR/ARNT pathway through ARNT targeting. PMID:26838552

  12. Relaxin Prevents Cardiac Fibroblast-Myofibroblast Transition via Notch-1-Mediated Inhibition of TGF-β/Smad3 Signaling

    PubMed Central

    Sassoli, Chiara; Chellini, Flaminia; Pini, Alessandro; Tani, Alessia; Nistri, Silvia; Nosi, Daniele; Zecchi-Orlandini, Sandra; Bani, Daniele; Formigli, Lucia

    2013-01-01

    The hormone relaxin (RLX) is produced by the heart and has beneficial actions on the cardiovascular system. We previously demonstrated that RLX stimulates mouse neonatal cardiomyocyte growth, suggesting its involvement in endogenous mechanisms of myocardial histogenesis and regeneration. In the present study, we extended the experimentation by evaluating the effects of RLX on primary cultures of neonatal cardiac stromal cells. RLX inhibited TGF-β1-induced fibroblast-myofibroblast transition, as judged by its ability to down-regulate α-smooth muscle actin and type I collagen expression. We also found that the hormone up-regulated metalloprotease (MMP)-2 and MMP-9 expression and downregulated the tissue inhibitor of metalloproteinases (TIMP)-2 in TGF-β1-stimulated cells. Interestingly, the effects of RLX on cardiac fibroblasts involved the activation of Notch-1 pathway. Indeed, Notch-1 expression was significantly decreased in TGF-β1-stimulatedfibroblasts as compared to the unstimulated controls; this reduction was prevented by the addition of RLX to TGF-β1-stimulated cells. Moreover, pharmacological inhibition of endogenous Notch-1 signaling by N-3,5-difluorophenyl acetyl-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), a γ-secretase specific inhibitor, as well as the silencing of Notch-1 ligand, Jagged-1, potentiated TGF-β1-induced myofibroblast differentiation and abrogated the inhibitory effects of RLX. Interestingly, RLX and Notch-1 exerted their inhibitory effects by interfering with TGF-β1 signaling, since the addition of RLX to TGF-β1-stimulated cells caused a significant decrease in Smad3 phosphorylation, a typical downstream event of TGF-β1 receptor activation, while the treatment with a prevented this effect. These data suggest that Notch signaling can down-regulate TGF-β1/Smad3-induced fibroblast-myofibroblast transition and that RLX could exert its well known anti-fibrotic action through the up-regulation of this pathway. In conclusion

  13. PLCε knockdown inhibits prostate cancer cell proliferation via suppression of Notch signalling and nuclear translocation of the androgen receptor.

    PubMed

    Wang, Yin; Wu, Xiaohou; Ou, Liping; Yang, Xue; Wang, Xiaorong; Tang, Min; Chen, E; Luo, Chunli

    2015-06-28

    Phospholipase Cε (PLCε), a key regulator of diverse cellular functions, has been implicated in various malignancies. Indeed, PLCε functions include cell proliferation, apoptosis and malignant transformation. Here, we show that PLCε expression is elevated in prostate cancer (PCa) tissues compared to benign prostate tissues. Furthermore, PLCε depletion using an adenovirally delivered shRNA significantly decreased cell growth and colony formation, arresting the PC3 and LNCaP cell lines in the S phase of the cell cycle. We also observed that PLCε was significantly correlated with Notch1 and androgen receptor (AR). Additionally, we demonstrate that the activation of both the Notch and AR signalling pathways is involved in PLCε-mediated oncogenic effects in PCa. Our findings suggest that PLCε is a putative oncogene and prognostic marker, potentially representing a novel therapeutic target for PCa.

  14. Notch Signaling Mediates the Age-Associated Decrease in Adhesion of Germline Stem Cells to the Niche

    PubMed Central

    Tseng, Chen-Yuan; Kao, Shih-Han; Wan, Chih-Ling; Cho, Yueh; Tung, Shu-Yun; Hsu, Hwei-Jan

    2014-01-01

    Stem cells have an innate ability to occupy their stem cell niche, which in turn, is optimized to house stem cells. Organ aging is associated with reduced stem cell occupancy in the niche, but the mechanisms involved are poorly understood. Here, we report that Notch signaling is increased with age in Drosophila female germline stem cells (GSCs), and this results in their removal from the niche. Clonal analysis revealed that GSCs with low levels of Notch signaling exhibit increased adhesiveness to the niche, thereby out-competing their neighbors with higher levels of Notch; adhesiveness is altered through regulation of E-cadherin expression. Experimental enhancement of Notch signaling in GSCs hastens their age-dependent loss from the niche, and such loss is at least partially mediated by Sex lethal. However, disruption of Notch signaling in GSCs does not delay GSC loss during aging, and nor does it affect BMP signaling, which promotes self-renewal of GSCs. Finally, we show that in contrast to GSCs, Notch activation in the niche (which maintains niche integrity, and thus mediates GSC retention) is reduced with age, indicating that Notch signaling regulates GSC niche occupancy both intrinsically and extrinsically. Our findings expose a novel role of Notch signaling in controlling GSC-niche adhesion in response to aging, and are also of relevance to metastatic cancer cells, in which Notch signaling suppresses cell adhesion. PMID:25521289

  15. Systematic identification of signaling pathways with potential to confer anticancer drug resistance.

    PubMed

    Martz, Colin A; Ottina, Kathleen A; Singleton, Katherine R; Jasper, Jeff S; Wardell, Suzanne E; Peraza-Penton, Ashley; Anderson, Grace R; Winter, Peter S; Wang, Tim; Alley, Holly M; Kwong, Lawrence N; Cooper, Zachary A; Tetzlaff, Michael; Chen, Pei-Ling; Rathmell, Jeffrey C; Flaherty, Keith T; Wargo, Jennifer A; McDonnell, Donald P; Sabatini, David M; Wood, Kris C

    2014-12-23

    Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant complementary DNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS-MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)-mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially passaged breast cancer xenografts in mice, and treating mice with a γ-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAF(V600E) melanoma cells in culture, and the abundance of Notch1 pathway markers was increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts. PMID:25538079

  16. Decidual vascular endothelial cells promote maternal-fetal immune tolerance by inducing regulatory T cells through canonical Notch1 signaling.

    PubMed

    Yao, Yanyi; Song, Jieping; Wang, Weipeng; Liu, Nian

    2016-05-01

    Adaptation of the maternal immune response to accommodate the semiallogeneic fetus is necessary for pregnancy success. However, the mechanisms by which the fetus avoids rejection despite expression of paternal alloantigens remain incompletely understood. Regulatory T cells (Treg cells) are pivotal for maintaining immune homeostasis, preventing autoimmune disease and fetus rejection. In this study, we found that maternal decidual vascular endothelial cells (DVECs) sustained Foxp3 expression in resting Treg cells in vitro. Moreover, under in vitro Treg cell induction condition with agonistic antibodies and transforming growth factor (TGF)-β, DVECs promoted Treg cell differentiation from non-Treg conventional T cells. Consistent with the promotion of Treg cell maintenance and differentiation, Treg cell-associated gene expression such as TGF-β, Epstein-Barr-induced gene-3, CD39 and glucocorticoid-induced tumor necrosis factor receptor was also increased in the presence of DVECs. Further study revealed that DVECs expressed Notch ligands such as Jagged-1, Delta-like protein 1 (DLL-1) and DLL-4, while Treg cells expressed Notch1 on their surface. The effects of DVECs on Treg cells was inhibited by siRNA-induced knockdown of expression of Jagged-1 and DLL-1 in DVECs. Downregulation of Notch1 in Treg cells using lentiviral shRNA transduction decreased Foxp3 expression in Treg cells. Adoptive transfer of Notch1-deficient Treg cells increased abortion rate in a murine semiallogeneic pregnancy model. Taken together, our study suggests that maternal DVECs are able to maintain decidual Treg cell identity and promote Treg cell differentiation through activation of Notch1 signal pathway in Treg cells and subsequently inhibit the immune response against semiallogeneic fetuses and preventing spontaneous abortion. PMID:26714886

  17. Atractylenolide I-mediated Notch pathway inhibition attenuates gastric cancer stem cell traits

    SciTech Connect

    Ma, Li; Mao, Rurong; Shen, Ke; Zheng, Yuanhong; Li, Yueqi; Liu, Jianwen; Ni, Lei

    2014-07-18

    Highlights: • This paper supports the anti-tumor effects of AT-I on gastric cancer in vitro. • AT-I attenuates gastric cancer stem cell traits. • It is the systematic study regarding AT-I suppression of Notch pathway in GC and GCSLCs. - Abstract: Atractylenolide I (AT-I), one of the main naturally occurring compounds of Rhizoma Atractylodis Macrocephalae, has remarkable anti-cancer effects on various cancers. However, its effects on the treatment of gastric cancer remain unclear. Via multiple cellular and molecular approaches, we demonstrated that AT-I could potently inhibit cancer cell proliferation and induce apoptosis through inactivating Notch pathway. AT-I treatment led to the reduction of expressions of Notch1, Jagged1, and its downstream Hes1/ Hey1. Our results showed that AT-I inhibited the self-renewal capacity of gastric stem-like cells (GCSLCs) by suppression of their sphere formation capacity and cell viability. AT-I attenuated gastric cancer stem cell (GCSC) traits partly through inactivating Notch1, leading to reducing the expressions of its downstream target Hes1, Hey1 and CD44 in vitro. Collectively, our results suggest that AT-I might develop as a potential therapeutic drug for the treatment of gastric cancer.

  18. Thiocoraline activates the Notch pathway in carcinoids and reduces tumor progression in vivo

    PubMed Central

    Wyche, Thomas P.; Dammalapati, Ajitha; Cho, Hyunah; Harrison, April D.; Kwon, Glen S.; Chen, Herbert; Bugni, Tim S.; Jaskula-Sztul, Renata

    2014-01-01

    Carcinoids are slow-growing neuroendocrine tumors (NETs) that are characterized by hormone overproduction; surgery is currently the only option for treatment. Activation of the Notch pathway has previously been shown to have a role in tumor suppression in NETs. The marine-derived thiodepsipeptide thiocoraline was investigated in vitro in two carcinoid cell lines (BON and H727). Carcinoid cells treated with nanomolar concentrations of thiocoraline resulted in a decrease in cell proliferation and an alteration of malignant phenotype evidenced by decrease of NET markers, ASCL-1, CgA, and NSE. Western blot analysis demonstrated the activation of Notch1 on the protein level in BON cells. Additionally, thiocoraline activated downstream Notch targets HES1, HES5, and HEY2. Thiocoraline effectively suppressed carcinoid cell growth by promoting cell cycle arrest in BON and H727 cells. An in vivo study demonstrated that thiocoraline, formulated with polymeric micelles, slowed carcinoid tumor progression. Thus, the therapeutic potential of thiocoraline, which induced activation of the Notch pathway, in carcinoid tumors was demonstrated. PMID:25412645

  19. NACK is an integral component of the Notch transcriptional activation complex and is critical for development and tumorigenesis.

    PubMed

    Weaver, Kelly L; Alves-Guerra, Marie-Clotilde; Jin, Ke; Wang, Zhiqiang; Han, Xiaoqing; Ranganathan, Prathibha; Zhu, Xiaoxia; DaSilva, Thiago; Liu, Wei; Ratti, Francesca; Demarest, Renee M; Tzimas, Cristos; Rice, Meghan; Vasquez-Del Carpio, Rodrigo; Dahmane, Nadia; Robbins, David J; Capobianco, Anthony J

    2014-09-01

    The Notch signaling pathway governs many distinct cellular processes by regulating transcriptional programs. The transcriptional response initiated by Notch is highly cell context dependent, indicating that multiple factors influence Notch target gene selection and activity. However, the mechanism by which Notch drives target gene transcription is not well understood. Herein, we identify and characterize a novel Notch-interacting protein, Notch activation complex kinase (NACK), which acts as a Notch transcriptional coactivator. We show that NACK associates with the Notch transcriptional activation complex on DNA, mediates Notch transcriptional activity, and is required for Notch-mediated tumorigenesis. We demonstrate that Notch1 and NACK are coexpressed during mouse development and that homozygous loss of NACK is embryonic lethal. Finally, we show that NACK is also a Notch target gene, establishing a feed-forward loop. Thus, our data indicate that NACK is a key component of the Notch transcriptional complex and is an essential regulator of Notch-mediated tumorigenesis and development.

  20. Notch receptors in human choroid plexus tumors.

    PubMed

    Beschorner, R; Waidelich, J; Trautmann, K; Psaras, T; Schittenhelm, J

    2013-08-01

    Notch signaling plays a role in development and formation of the normal choroid plexus (nCP), and in formation of various tumors in humans. Activation of Notch3 has been reported to promote tumor growth in invasive gliomas and to initiate formation of choroid plexus tumors (CPT) in mice. We investigated the expression of all currently known Notch receptors (Notch 1-4) in 55 samples of nCP and 88 CPT, including 61 choroid plexus papillomas (CPP), 22 atypical CPP and 5 choroid plexus carcinomas by immunohistochemistry. Notch expression was semiquantitatively evaluated separately for membranous/cytoplasmic and for nuclear staining. In addition, we examined Her2 expression (EGFR2, Her2/neu, ErbB2, CD340) because of its functional link to Notch signaling. All samples were negative for Notch3. Membranous/cytoplasmic expression of Notch1 (p<0.0001) and Notch4 (p=0.046) was significantly higher, whereas Notch2 expression was significantly lower (p<0.0001) in nCP compared to CPT. Nuclear expression of Notch1, -2 and -4 was significantly higher in CPT compared to nCP (p<0.0001 each). Expression of Notch2 and Notch4 showed a shift from a prevailing membranous/cytoplasmic expression in nCP to a predominant nuclear expression in CPT. Her2 was weakly expressed in 42/84 CPT but only in 2/53 nCP (p=0.0001) and positively correlated with nuclear expression of Notch1, -2 and 4 in CPT. In summary, a shift between membranous/cytoplasmic (non-canonical signaling pathway) and nuclear expression (canonical signaling pathway) of Notch1, -2 and -4 and upregulation of Her2 indicate neoplastic transformation in human CP and may reveal new therapeutic approaches.

  1. Notch signaling deficiency underlies age-dependent depletion of satellite cells in muscular dystrophy.

    PubMed

    Jiang, Chunhui; Wen, Yefei; Kuroda, Kazuki; Hannon, Kevin; Rudnicki, Michael A; Kuang, Shihuan

    2014-08-01

    Duchenne muscular dystrophy (DMD) is a devastating disease characterized by muscle wasting, loss of mobility and death in early adulthood. Satellite cells are muscle-resident stem cells responsible for the repair and regeneration of damaged muscles. One pathological feature of DMD is the progressive depletion of satellite cells, leading to the failure of muscle repair. Here, we attempted to explore the molecular mechanisms underlying satellite cell ablation in the dystrophin mutant mdx mouse, a well-established model for DMD. Initial muscle degeneration activates satellite cells, resulting in increased satellite cell number in young mdx mice. This is followed by rapid loss of satellite cells with age due to the reduced self-renewal ability of mdx satellite cells. In addition, satellite cell composition is altered even in young mdx mice, with significant reductions in the abundance of non-committed (Pax7+ and Myf5-) satellite cells. Using a Notch-reporter mouse, we found that the mdx satellite cells have reduced activation of Notch signaling, which has been shown to be necessary to maintain satellite cell quiescence and self-renewal. Concomitantly, the expression of Notch1, Notch3, Jag1, Hey1 and HeyL are reduced in the mdx primary myoblast. Finally, we established a mouse model to constitutively activate Notch signaling in satellite cells, and show that Notch activation is sufficient to rescue the self-renewal deficiencies of mdx satellite cells. These results demonstrate that Notch signaling is essential for maintaining the satellite cell pool and that its deficiency leads to depletion of satellite cells in DMD.

  2. NOTCH, ASCL1, p53 and RB alterations define an alternative pathway driving neuroendocrine and small cell lung carcinomas

    PubMed Central

    Meder, Lydia; König, Katharina; Ozretić, Luka; Schultheis, Anne M.; Ueckeroth, Frank; Ade, Carsten P.; Albus, Kerstin; Boehm, Diana; Rommerscheidt‐Fuss, Ursula; Florin, Alexandra; Buhl, Theresa; Hartmann, Wolfgang; Wolf, Jürgen; Merkelbach‐Bruse, Sabine; Eilers, Martin; Perner, Sven; Heukamp, Lukas C.

    2015-01-01

    Small cell lung cancers (SCLCs) and extrapulmonary small cell cancers (SCCs) are very aggressive tumors arising de novo as primary small cell cancer with characteristic genetic lesions in RB1 and TP53. Based on murine models, neuroendocrine stem cells of the terminal bronchioli have been postulated as the cellular origin of primary SCLC. However, both in lung and many other organs, combined small cell/non‐small cell tumors and secondary transitions from non‐small cell carcinomas upon cancer therapy to neuroendocrine and small cell tumors occur. We define features of “small cell‐ness” based on neuroendocrine markers, characteristic RB1 and TP53 mutations and small cell morphology. Furthermore, here we identify a pathway driving the pathogenesis of secondary SCLC involving inactivating NOTCH mutations, activation of the NOTCH target ASCL1 and canonical WNT‐signaling in the context of mutual bi‐allelic RB1 and TP53 lesions. Additionaly, we explored ASCL1 dependent RB inactivation by phosphorylation, which is reversible by CDK5 inhibition. We experimentally verify the NOTCH‐ASCL1‐RB‐p53 signaling axis in vitro and validate its activation by genetic alterations in vivo. We analyzed clinical tumor samples including SCLC, SCC and pulmonary large cell neuroendocrine carcinomas and adenocarcinomas using amplicon‐based Next Generation Sequencing, immunohistochemistry and fluorescence in situ hybridization. In conclusion, we identified a novel pathway underlying rare secondary SCLC which may drive small cell carcinomas in organs other than lung, as well. PMID:26340530

  3. BLOS2 negatively regulates Notch signaling during neural and hematopoietic stem and progenitor cell development

    PubMed Central

    Zhou, Wenwen; He, Qiuping; Zhang, Chunxia; He, Xin; Cui, Zongbin; Liu, Feng; Li, Wei

    2016-01-01

    Notch signaling plays a crucial role in controling the proliferation and differentiation of stem and progenitor cells during embryogenesis or organogenesis, but its regulation is incompletely understood. BLOS2, encoded by the Bloc1s2 gene, is a shared subunit of two lysosomal trafficking complexes, biogenesis of lysosome-related organelles complex-1 (BLOC-1) and BLOC-1-related complex (BORC). Bloc1s2−/− mice were embryonic lethal and exhibited defects in cortical development and hematopoiesis. Loss of BLOS2 resulted in elevated Notch signaling, which consequently increased the proliferation of neural progenitor cells and inhibited neuronal differentiation in cortices. Likewise, ablation of bloc1s2 in zebrafish or mice led to increased hematopoietic stem and progenitor cell production in the aorta-gonad-mesonephros region. BLOS2 physically interacted with Notch1 in endo-lysosomal trafficking of Notch1. Our findings suggest that BLOS2 is a novel negative player in regulating Notch signaling through lysosomal trafficking to control multiple stem and progenitor cell homeostasis in vertebrates. DOI: http://dx.doi.org/10.7554/eLife.18108.001 PMID:27719760

  4. Demonstration of optical steganography transmission using temporal phase coded optical signals with spectral notch filtering.

    PubMed

    Hong, Xuezhi; Wang, Dawei; Xu, Lei; He, Sailing

    2010-06-01

    A novel approach is proposed and experimentally demonstrated for optical steganography transmission in WDM networks using temporal phase coded optical signals with spectral notch filtering. A temporal phase coded stealth channel is temporally and spectrally overlaid onto a public WDM channel. Direct detection of the public channel is achieved in the presence of the stealth channel. The interference from the public channel is suppressed by spectral notching before the detection of the optical stealth signal. The approach is shown to have good compatibility and robustness to the existing WDM network for optical steganography transmission.

  5. The clerodane diterpene casearin J induces apoptosis of T-ALL cells through SERCA inhibition, oxidative stress, and interference with Notch1 signaling.

    PubMed

    De Ford, C; Heidersdorf, B; Haun, F; Murillo, R; Friedrich, T; Borner, C; Merfort, I

    2016-01-28

    T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that preferentially affects children and adolescents. Over 50% of human T-ALLs possess activating mutations of Notch1. The clerodane diterpene casearin J (CJ) is a natural product that inhibits the sarcoendoplasmatic reticulum calcium ATPase (SERCA) pump and induces cell death in leukemia cells, but the molecular mechanism of cytotoxicity remains poorly understood. Here we show that owing to SERCA pump inhibition, CJ induces depletion of the endoplasmic reticulum calcium pools, oxidative stress, and apoptosis via the intrinsic signaling pathway. Moreover, Notch1 signaling is reduced in T-ALL cells with auto-activating mutations in the HD-domain of Notch1, but not in cells that do not depend on Notch1 signaling. CJ also provoked a slight activation of NF-κB, and consistent with this notion a combined treatment of CJ and the NF-κB inhibitor parthenolide (Pt) led to a remarkable synergistic cell death in T-ALL cells. Altogether, our data support the concept that inhibition of the SERCA pump may be a novel strategy for the treatment of T-ALL with HD-domain-mutant Notch1 receptors and that additional treatment with the NF-κB inhibitor parthenolide may have further therapeutic benefits.

  6. The clerodane diterpene casearin J induces apoptosis of T-ALL cells through SERCA inhibition, oxidative stress, and interference with Notch1 signaling

    PubMed Central

    De Ford, C; Heidersdorf, B; Haun, F; Murillo, R; Friedrich, T; Borner, C; Merfort, I

    2016-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that preferentially affects children and adolescents. Over 50% of human T-ALLs possess activating mutations of Notch1. The clerodane diterpene casearin J (CJ) is a natural product that inhibits the sarcoendoplasmatic reticulum calcium ATPase (SERCA) pump and induces cell death in leukemia cells, but the molecular mechanism of cytotoxicity remains poorly understood. Here we show that owing to SERCA pump inhibition, CJ induces depletion of the endoplasmic reticulum calcium pools, oxidative stress, and apoptosis via the intrinsic signaling pathway. Moreover, Notch1 signaling is reduced in T-ALL cells with auto-activating mutations in the HD-domain of Notch1, but not in cells that do not depend on Notch1 signaling. CJ also provoked a slight activation of NF-κB, and consistent with this notion a combined treatment of CJ and the NF-κB inhibitor parthenolide (Pt) led to a remarkable synergistic cell death in T-ALL cells. Altogether, our data support the concept that inhibition of the SERCA pump may be a novel strategy for the treatment of T-ALL with HD-domain-mutant Notch1 receptors and that additional treatment with the NF-κB inhibitor parthenolide may have further therapeutic benefits. PMID:26821066

  7. Crucial role of the Rap G protein signal in Notch activation and leukemogenicity of T-cell acute lymphoblastic leukemia.

    PubMed

    Doi, Keiko; Imai, Takahiko; Kressler, Christopher; Yagita, Hideo; Agata, Yasutoshi; Vooijs, Marc; Hamazaki, Yoko; Inoue, Joe; Minato, Nagahiro

    2015-01-23

    The Rap G protein signal regulates Notch activation in early thymic progenitor cells, and deregulated Rap activation (Rap(high)) results in the development of Notch-dependent T-cell acute lymphoblastic leukemia (T-ALL). We demonstrate that the Rap signal is required for the proliferation and leukemogenesis of established Notch-dependent T-ALL cell lines. Attenuation of the Rap signal by the expression of a dominant-negative Rap1A17 or Rap1GAP, Sipa1, in a T-ALL cell line resulted in the reduced Notch processing at site 2 due to impaired maturation of Adam10. Inhibition of the Rap1 prenylation with a geranylgeranyl transferase inhibitor abrogated its membrane-anchoring to Golgi-network and caused reduced proprotein convertase activity required for Adam10 maturation. Exogenous expression of a mature form of Adam10 overcame the Sipa1-induced inhibition of T-ALL cell proliferation. T-ALL cell lines expressed Notch ligands in a Notch-signal dependent manner, which contributed to the cell-autonomous Notch activation. Although the initial thymic blast cells barely expressed Notch ligands during the T-ALL development from Rap(high) hematopoietic progenitors in vivo, the ligands were clearly expressed in the T-ALL cells invading extrathymic vital organs. These results reveal a crucial role of the Rap signal in the Notch-dependent T-ALL development and the progression.

  8. Notch/Rbpjκ signaling regulates progenitor maintenance and differentiation of hypothalamic arcuate neurons

    PubMed Central

    Aujla, Paven K.; Naratadam, George T.; Xu, Liwen; Raetzman, Lori T.

    2013-01-01

    The hypothalamic arcuate nucleus (Arc), containing pro-opoiomelanocortin (POMC), neuropeptide Y (NPY) and growth hormone releasing hormone (GHRH) neurons, regulates feeding, energy balance and body size. Dysregulation of this homeostatic mediator underlies diseases ranging from growth failure to obesity. Despite considerable investigation regarding the function of Arc neurons, mechanisms governing their development remain unclear. Notch signaling factors such as Hes1 and Mash1 are present in hypothalamic progenitors that give rise to Arc neurons. However, how Notch signaling controls these progenitor populations is unknown. To elucidate the role of Notch signaling in Arc development, we analyzed conditional loss-of-function mice lacking a necessary Notch co-factor, Rbpjκ, in Nkx2.1-cre-expressing cells (Rbpjκ cKO), as well as mice with expression of the constitutively active Notch1 intracellular domain (NICD) in Nkx2.1-cre-expressing cells (NICD Tg). We found that loss of Rbpjκ results in absence of Hes1 but not of Hes5 within the primordial Arc at E13.5. Additionally, Mash1 expression is increased, coincident with increased proliferation and accumulation of Arc neurons at E13.5. At E18.5, Rbpjκ cKO mice have few progenitors and show increased numbers of differentiated Pomc, NPY and Ghrh neurons. By contrast, NICD Tg mice have increased hypothalamic progenitors, show an absence of differentiated Arc neurons and aberrant glial differentiation at E18.5. Subsequently, both Rbpjκ cKO and NICD Tg mice have changes in growth and body size during postnatal development. Taken together, our results demonstrate that Notch/Rbpjκ signaling regulates the generation and differentiation of Arc neurons, which contribute to homeostatic regulation of body size. PMID:23884446

  9. Proteolytic Cleavage of Notch: “HIT and RUN”

    PubMed Central

    van Tetering, G.; Vooijs, M.

    2014-01-01

    The Notch pathway is a highly conserved signaling pathway in multicellular eukaryotes essential in controlling spatial patterning, morphogenesis and homeostasis in embryonic and adult tissues. Notch proteins coordinate cell-cell communication through receptor-ligand interactions between adjacent cells. Notch signaling is frequently deregulated by oncogenic mutation or overexpression in many cancer types. Notch activity is controlled by three sequential cleavage steps leading to ectodomain shedding and transcriptional activation. Here we review the key regulatory steps in the activation of Notch, from receptor maturation to receptor activation (HIT) via a rate-limiting proteolytic cascade (RUN) in the context of species-specific differences. PMID:21506924

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

    PubMed Central

    Laky, Karen; Evans, Sharron; Perez-Diez, Ainhoa

    2015-01-01

    SUMMARY 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. PMID:25607460

  11. Human papillomavirus 16E6 and NFX1-123 potentiate notch signaling and differentiation without activating cellular arrest

    SciTech Connect

    Vliet-Gregg, Portia A.; Hamilton, Jennifer R.; Katzenellenbogen, Rachel A.

    2015-04-15

    High-risk human papillomavirus (HR HPV) oncoproteins bind host cell proteins to dysregulate and uncouple apoptosis, senescence, differentiation, and growth. These pathways are important for both the viral life cycle and cancer development. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and they collaboratively increase the growth and differentiation master regulator, Notch1. In 16E6 expressing keratinocytes (16E6 HFKs), the Notch canonical pathway genes Hes1 and Hes5 were increased with overexpression of NFX1-123, and their expression was directly linked to the activation or blockade of the Notch1 receptor. Keratinocyte differentiation genes Keratin 1 and Keratin 10 were also increased, but in contrast their upregulation was only indirectly associated with Notch1 receptor stimulation and was fully unlinked to growth arrest, increased p21{sup Waf1/CIP1}, or decreased proliferative factor Ki67. This leads to a model of 16E6, NFX1-123, and Notch1 differently regulating canonical and differentiation pathways and entirely uncoupling cellular arrest from increased differentiation. - Highlights: • 16E6 and NFX1-123 increased the Notch canonical pathway through Notch1. • 16E6 and NFX1-123 increased the differentiation pathway indirectly through Notch1. • 16E6 and NFX1-123 increased differentiation gene expression without growth arrest. • Increased NFX1-123 with 16E6 may create an ideal cellular phenotype for HPV.

  12. Expression of notch receptors and ligands in the adult gut.

    PubMed

    Sander, Guy R; Powell, Barry C

    2004-04-01

    The Notch signaling pathway has become recognized as a vitally important pathway in regulating proliferative/differentiative decisions and cell fate. To explore the involvement of the Notch pathway in adult gut, we investigated the expression of Notch receptors and their ligands by Northern blotting and in situ hybridization. Notch receptors and ligands were expressed in both proliferative and post-mitotic cells throughout adult rat gut, variously in epithelial, immune, and endothelial cells. Expression of Notch1, Jagged1, and Jagged2 frequently overlapped, whereas Notch2 expression was restricted to specific crypt cells, the lamina propria of the large intestine, and Peyer's patch lymphocytes. We propose that the expression of multiple Notch receptors and ligands in a range of different intestinal cell types indicates that this signaling pathway underpins many of the processes involved in the maintenance and function of the adult gut.

  13. The Notch meeting: an odyssey from structure to function.

    PubMed

    Chitnis, Ajay; Balle-Cuif, Laure

    2016-02-15

    The Notch signaling pathway plays fundamental roles in diverse developmental processes. Studies of the basic biology of Notch function have provided insights into how its dysfunction contributes to multi-systemic diseases and cancer. In addition, our understanding of Notch signaling in maintaining stem/progenitor cell populations is revealing new avenues for rekindling regeneration. The Notch IX meeting, which was held in Athens, Greece in October 2015, brought together scientists working on different model systems and studying Notch signaling in various contexts. Here, we provide a summary of the key points that were presented at the meeting. Although we focus on the molecular mechanisms that determine Notch signaling and its role in development, we also cover talks describing roles for Notch in adulthood. Together, the talks revealed how interactions between adjacent cells mediated by Notch regulate development and physiology at multiple levels. PMID:26884393

  14. Endogenous Gradients of Resting Potential Instructively Pattern Embryonic Neural Tissue via Notch Signaling and Regulation of Proliferation

    PubMed Central

    Pai, Vaibhav P.; Lemire, Joan M.; Paré, Jean-François; Lin, Gufa; Chen, Ying

    2015-01-01

    Biophysical forces play important roles throughout embryogenesis, but the roles of spatial differences in cellular resting potentials during large-scale brain morphogenesis remain unknown. Here, we implicate endogenous bioelectricity as an instructive factor during brain patterning in Xenopus laevis. Early frog embryos exhibit a characteristic hyperpolarization of cells lining the neural tube; disruption of this spatial gradient of the transmembrane potential (Vmem) diminishes or eliminates the expression of early brain markers, and causes anatomical mispatterning of the brain, including absent or malformed regions. This effect is mediated by voltage-gated calcium signaling and gap-junctional communication. In addition to cell-autonomous effects, we show that hyperpolarization of transmembrane potential (Vmem) in ventral cells outside the brain induces upregulation of neural cell proliferation at long range. Misexpression of the constitutively active form of Notch, a suppressor of neural induction, impairs the normal hyperpolarization pattern and neural patterning; forced hyperpolarization by misexpression of specific ion channels rescues brain defects induced by activated Notch signaling. Strikingly, hyperpolarizing posterior or ventral cells induces the production of ectopic neural tissue considerably outside the neural field. The hyperpolarization signal also synergizes with canonical reprogramming factors (POU and HB4), directing undifferentiated cells toward neural fate in vivo. These data identify a new functional role for bioelectric signaling in brain patterning, reveal interactions between Vmem and key biochemical pathways (Notch and Ca2+ signaling) as the molecular mechanism by which spatial differences of Vmem regulate organogenesis of the vertebrate brain, and suggest voltage modulation as a tractable strategy for intervention in certain classes of birth defects. PMID:25762681

  15. LIN-12/Notch signaling instructs postsynaptic muscle arm development by regulating UNC-40/DCC and MADD-2 in Caenorhabditis elegans.

    PubMed

    Li, Pengpeng; Collins, Kevin M; Koelle, Michael R; Shen, Kang

    2013-03-19

    The diverse cell types and the precise synaptic connectivity between them are the cardinal features of the nervous system. Little is known about how cell fate diversification is linked to synaptic target choices. Here we investigate how presynaptic neurons select one type of muscles, vm2, as a synaptic target and form synapses on its dendritic spine-like muscle arms. We found that the Notch-Delta pathway was required to distinguish target from non-target muscles. APX-1/Delta acts in surrounding cells including the non-target vm1 to activate LIN-12/Notch in the target vm2. LIN-12 functions cell-autonomously to up-regulate the expression of UNC-40/DCC and MADD-2 in vm2, which in turn function together to promote muscle arm formation and guidance. Ectopic expression of UNC-40/DCC in non-target vm1 muscle is sufficient to induce muscle arm extension from these cells. Therefore, the LIN-12/Notch signaling specifies target selection by selectively up-regulating guidance molecules and forming muscle arms in target cells. DOI:http://dx.doi.org/10.7554/eLife.00378.001.

  16. Structure and Function of the Su(H)-Hairless Repressor Complex, the Major Antagonist of Notch Signaling in Drosophila melanogaster

    PubMed Central

    Torella, Rubben; Preiss, Anette; Maier, Dieter; Kovall, Rhett A.

    2016-01-01

    Notch is a conserved signaling pathway that specifies cell fates in metazoans. Receptor-ligand interactions induce changes in gene expression, which is regulated by the transcription factor CBF1/Su(H)/Lag-1 (CSL). CSL interacts with coregulators to repress and activate transcription from Notch target genes. While the molecular details of the activator complex are relatively well understood, the structure-function of CSL-mediated repressor complexes is poorly defined. In Drosophila, the antagonist Hairless directly binds Su(H) (the fly CSL ortholog) to repress transcription from Notch targets. Here, we determine the X-ray structure of the Su(H)-Hairless complex bound to DNA. Hairless binding produces a large conformational change in Su(H) by interacting with residues in the hydrophobic core of Su(H), illustrating the structural plasticity of CSL molecules to interact with different binding partners. Based on the structure, we designed mutants in Hairless and Su(H) that affect binding, but do not affect formation of the activator complex. These mutants were validated in vitro by isothermal titration calorimetry and yeast two- and three-hybrid assays. Moreover, these mutants allowed us to solely characterize the repressor function of Su(H) in vivo. PMID:27404588

  17. Blood flow suppresses vascular Notch signalling via dll4 and is required for angiogenesis in response to hypoxic signalling

    PubMed Central

    Watson, Oliver; Novodvorsky, Peter; Gray, Caroline; Rothman, Alexander M.K.; Lawrie, Allan; Crossman, David C.; Haase, Andrea; McMahon, Kathryn; Gering, Martin; Van Eeden, Fredericus J.M.; Chico, Timothy J.A.

    2013-01-01

    Aims The contribution of blood flow to angiogenesis is incompletely understood. We examined the effect of blood flow on Notch signalling in the vasculature of zebrafish embryos, and whether blood flow regulates angiogenesis in zebrafish with constitutively up-regulated hypoxic signalling. Methods and results Developing zebrafish (Danio rerio) embryos survive via diffusion in the absence of circulation induced by knockdown of cardiac troponin T2 or chemical cardiac cessation. The absence of blood flow increased vascular Notch signalling in 48 h post-fertilization old embryos via up-regulation of the Notch ligand dll4. Despite this, patterning of the intersegmental vessels is not affected by absent blood flow. We therefore examined homozygous vhl mutant zebrafish that have constitutively up-regulated hypoxic signalling. These display excessive and aberrant angiogenesis from 72 h post-fertilization, with significantly increased endothelial number, vessel diameter, and length. The absence of blood flow abolished these effects, though normal vessel patterning was preserved. Conclusion We show that blood flow suppresses vascular Notch signalling via down-regulation of dll4. We have also shown that blood flow is required for angiogenesis in response to hypoxic signalling but is not required for normal vessel patterning. These data indicate important differences in hypoxia-driven vs. developmental angiogenesis. PMID:23812297

  18. Differential effects of targeting Notch receptors in a mouse model of liver cancer

    PubMed Central

    Huntzicker, Erik G.; Hötzel, Kathy; Choy, Lisa; Che, Li; Ross, Jed; Pau, Gregoire; Sharma, Neeraj; Siebel, Christian W.; Chen, Xin; French, Dorothy M.

    2015-01-01

    Primary liver cancer encompasses both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). The Notch signaling pathway, known to be important for the proper development of liver architecture, is also a potential driver of primary liver cancer. However, with four known Notch receptors and several Notch ligands, it is not clear which Notch pathway members play the predominant role in liver cancer. To address this question we utilized antibodies to specifically target Notch1, Notch2, Notch3 or Jag1 in a mouse model of primary liver cancer driven by AKT and NRas. We show that inhibition of Notch2 reduces tumor burden by eliminating highly malignant hepatocellular carcinoma- and cholangiocarcinoma-like tumors. Inhibition of the Notch ligand Jag 1 had a similar effect, consistent with Jag1 acting in cooperation with Notch2. This effect was specific to Notch2, as Notch3 inhibition did not decrease tumor burden. Unexpectedly, Notch1 inhibition altered the relative proportion of tumor types, reducing HCC-like tumors but dramatically increasing CC-like tumors. Finally, we show that Notch2 and Jag1 are expressed in, and Notch2 signaling is activated in, a subset of human HCC samples. Conclusions: These findings underscore the distinct roles of different Notch receptors in the liver and suggest that inhibition of Notch2 signaling represents a novel therapeutic option in the treatment of liver cancer. PMID:25311838

  19. Fine-Tuning of the Kaposi’s Sarcoma-Associated Herpesvirus Life Cycle in Neighboring Cells through the RTA-JAG1-Notch Pathway

    PubMed Central

    He, Zhiheng; Liang, Deguang; Sun, Rui; Lan, Ke

    2016-01-01

    Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is an oncogenic pathogen that displays latent and lytic life cycles. In KS lesions, infiltrated immune cells, secreted viral and/or cellular cytokines, and hypoxia orchestrate a chronic pro-lytic microenvironment that can promote KSHV reactivation. However, only a small subset of viruses spontaneously undergoes lytic replication in this pro-lytic microenvironment while the majority remains in latency. Here, we show that the expression of the Notch ligand JAG1 is induced by KSHV-encoded replication and transcription activator (RTA) during reactivation. JAG1 up-regulation activates Notch signaling in neighboring cells and prevents viral lytic replication. The suppression of JAG1 and Notch1 with inhibitors or small interfering RNA promotes lytic replication in the presence of RTA induction or under conditions of hypoxia. The underlying mechanism involves the Notch downstream effector hairy and enhancer of split 1 (Hes1), which directly binds lytic gene promoters and attenuates viral lytic gene expression. RTA interacts with lymphoid enhancer-binding factor 1 (LEF1), disrupts LEF1/Groucho/TLE suppressive complexes and releases LEF1 to activate JAG1 expression. Taken together, our results suggest that cells with viral lytic replication can inhibit KSHV reactivation in neighboring cells through an RTA-JAG1-Notch pathway. These data provide insight into the mechanism by which the virus maintains the balance between lytic and latent infection in the pro-lytic tumor microenvironment. PMID:27760204

  20. Regulation of monocyte cell fate by blood vessels mediated by Notch signalling.

    PubMed

    Gamrekelashvili, Jaba; Giagnorio, Roberto; Jussofie, Jasmin; Soehnlein, Oliver; Duchene, Johan; Briseño, Carlos G; Ramasamy, Saravana K; Krishnasamy, Kashyap; Limbourg, Anne; Kapanadze, Tamar; Ishifune, Chieko; Hinkel, Rabea; Radtke, Freddy; Strobl, Lothar J; Zimber-Strobl, Ursula; Napp, L Christian; Bauersachs, Johann; Haller, Hermann; Yasutomo, Koji; Kupatt, Christian; Murphy, Kenneth M; Adams, Ralf H; Weber, Christian; Limbourg, Florian P

    2016-08-31

    A population of monocytes, known as Ly6C(lo) monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6C(hi) monocytes into Ly6C(lo) monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation.

  1. Regulation of monocyte cell fate by blood vessels mediated by Notch signalling

    PubMed Central

    Gamrekelashvili, Jaba; Giagnorio, Roberto; Jussofie, Jasmin; Soehnlein, Oliver; Duchene, Johan; Briseño, Carlos G.; Ramasamy, Saravana K.; Krishnasamy, Kashyap; Limbourg, Anne; Kapanadze, Tamar; Ishifune, Chieko; Hinkel, Rabea; Radtke, Freddy; Strobl, Lothar J.; Zimber-Strobl, Ursula; Napp, L. Christian; Bauersachs, Johann; Haller, Hermann; Yasutomo, Koji; Kupatt, Christian; Murphy, Kenneth M.; Adams, Ralf H.; Weber, Christian; Limbourg, Florian P.

    2016-01-01

    A population of monocytes, known as Ly6Clo monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6Chi monocytes into Ly6Clo monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation. PMID:27576369

  2. Regulation of monocyte cell fate by blood vessels mediated by Notch signalling.

    PubMed

    Gamrekelashvili, Jaba; Giagnorio, Roberto; Jussofie, Jasmin; Soehnlein, Oliver; Duchene, Johan; Briseño, Carlos G; Ramasamy, Saravana K; Krishnasamy, Kashyap; Limbourg, Anne; Kapanadze, Tamar; Ishifune, Chieko; Hinkel, Rabea; Radtke, Freddy; Strobl, Lothar J; Zimber-Strobl, Ursula; Napp, L Christian; Bauersachs, Johann; Haller, Hermann; Yasutomo, Koji; Kupatt, Christian; Murphy, Kenneth M; Adams, Ralf H; Weber, Christian; Limbourg, Florian P

    2016-01-01

    A population of monocytes, known as Ly6C(lo) monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6C(hi) monocytes into Ly6C(lo) monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation. PMID:27576369

  3. Evolution of the chordate regeneration blastema: Differential gene expression and conserved role of notch signaling during siphon regeneration in the ascidian Ciona.

    PubMed

    Hamada, Mayuko; Goricki, Spela; Byerly, Mardi S; Satoh, Noriyuki; Jeffery, William R

    2015-09-15

    The regeneration of the oral siphon (OS) and other distal structures in the ascidian Ciona intestinalis occurs by epimorphosis involving the formation of a blastema of proliferating cells. Despite the longstanding use of Ciona as a model in molecular developmental biology, regeneration in this system has not been previously explored by molecular analysis. Here we have employed microarray analysis and quantitative real time RT-PCR to identify genes with differential expression profiles during OS regeneration. The majority of differentially expressed genes were downregulated during OS regeneration, suggesting roles in normal growth and homeostasis. However, a subset of differentially expressed genes was upregulated in the regenerating OS, suggesting functional roles during regeneration. Among the upregulated genes were key members of the Notch signaling pathway, including those encoding the delta and jagged ligands, two fringe modulators, and to a lesser extent the notch receptor. In situ hybridization showed a complementary pattern of delta1 and notch gene expression in the blastema of the regenerating OS. Chemical inhibition of the Notch signaling pathway reduced the levels of cell proliferation in the branchial sac, a stem cell niche that contributes progenitor cells to the regenerating OS, and in the OS regeneration blastema, where siphon muscle fibers eventually re-differentiate. Chemical inhibition also prevented the replacement of oral siphon pigment organs, sensory receptors rimming the entrance of the OS, and siphon muscle fibers, but had no effects on the formation of the wound epidermis. Since Notch signaling is involved in the maintenance of proliferative activity in both the Ciona and vertebrate regeneration blastema, the results suggest a conserved evolutionary role of this signaling pathway in chordate regeneration. The genes identified in this investigation provide the foundation for future molecular analysis of OS regeneration.

  4. Evolution of the chordate regeneration blastema: Differential gene expression and conserved role of notch signaling during siphon regeneration in the ascidian Ciona.

    PubMed

    Hamada, Mayuko; Goricki, Spela; Byerly, Mardi S; Satoh, Noriyuki; Jeffery, William R

    2015-09-15

    The regeneration of the oral siphon (OS) and other distal structures in the ascidian Ciona intestinalis occurs by epimorphosis involving the formation of a blastema of proliferating cells. Despite the longstanding use of Ciona as a model in molecular developmental biology, regeneration in this system has not been previously explored by molecular analysis. Here we have employed microarray analysis and quantitative real time RT-PCR to identify genes with differential expression profiles during OS regeneration. The majority of differentially expressed genes were downregulated during OS regeneration, suggesting roles in normal growth and homeostasis. However, a subset of differentially expressed genes was upregulated in the regenerating OS, suggesting functional roles during regeneration. Among the upregulated genes were key members of the Notch signaling pathway, including those encoding the delta and jagged ligands, two fringe modulators, and to a lesser extent the notch receptor. In situ hybridization showed a complementary pattern of delta1 and notch gene expression in the blastema of the regenerating OS. Chemical inhibition of the Notch signaling pathway reduced the levels of cell proliferation in the branchial sac, a stem cell niche that contributes progenitor cells to the regenerating OS, and in the OS regeneration blastema, where siphon muscle fibers eventually re-differentiate. Chemical inhibition also prevented the replacement of oral siphon pigment organs, sensory receptors rimming the entrance of the OS, and siphon muscle fibers, but had no effects on the formation of the wound epidermis. Since Notch signaling is involved in the maintenance of proliferative activity in both the Ciona and vertebrate regeneration blastema, the results suggest a conserved evolutionary role of this signaling pathway in chordate regeneration. The genes identified in this investigation provide the foundation for future molecular analysis of OS regeneration. PMID:26206613

  5. Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway.

    PubMed

    Mah, In Kyoung; Soloff, Rachel; Hedrick, Stephen M; Mariani, Francesca V

    2015-11-10

    The number of stem/progenitor cells available can profoundly impact tissue homeostasis and the response to injury or disease. Here, we propose that an atypical PKC, Prkci, is a key player in regulating the switch from an expansion to a differentiation/maintenance phase via regulation of Notch, thus linking the polarity pathway with the control of stem cell self-renewal. Prkci is known to influence symmetric cell division in invertebrates; however a definitive role in mammals has not yet emerged. Using a genetic approach, we find that loss of Prkci results in a marked increase in the number of various stem/progenitor cells. The mechanism used likely involves inactivation and symmetric localization of NUMB, leading to the activation of NOTCH1 and its downstream effectors. Inhibition of atypical PKCs may be useful for boosting the production of pluripotent stem cells, multipotent stem cells, or possibly even primordial germ cells by promoting the stem cell/progenitor fate.

  6. Signaling Pathways in Thyroid Cancer and Their Therapeutic Implications

    PubMed Central

    Jin, Shan; Borkhuu, Oyungerel; Bao, Wuyuntu; Yang, Yun-Tian

    2016-01-01

    Thyroid cancer is a common malignancy of endocrine system, and has now become the fastest increasing cancer among all the malignancies. The development, progression, invasion, and metastasis are closely associated with multiple signaling pathways and the functions of related molecules, such as Src, Janus kinase (JAK)-signal transducers and activators of transcription (STAT), mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/Akt, NF-κB, thyroid stimulating hormone receptor (TSHR), Wnt-β-catenin and Notch signaling pathways. Each of the signaling pathways could exert its function singly or through network with other pathways. These pathways could cooperate, promote, antagonize, or interact with each other to form a complex network for the regulation. Dysfunction of this network could increase the development, progression, invasion, and metastasis of thyroid cancer. Inoperable thyroid cancer still has a poor prognosis. However, signaling pathway-related targeted therapies offer the hope of longer quality of meaningful life for this small group of patients. Signaling pathway-related targets provide unprecedented opportunities for further research and clinical development of novel treatment strategies for this cancer. In the present work, the advances in these signaling pathways and targeted treatments of thyroid cancer were reviewed. PMID:26985248

  7. O-fucose monosaccharide of Drosophila Notch has a temperature-sensitive function and cooperates with O-glucose glycan in Notch transport and Notch signaling activation.

    PubMed

    Ishio, Akira; Sasamura, Takeshi; Ayukawa, Tomonori; Kuroda, Junpei; Ishikawa, Hiroyuki O; Aoyama, Naoki; Matsumoto, Kenjiroo; Gushiken, Takuma; Okajima, Tetsuya; Yamakawa, Tomoko; Matsuno, Kenji

    2015-01-01

    Notch (N) is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell fate decisions. N has many epidermal growth factor-like repeats that are O-fucosylated by the protein O-fucosyltransferase 1 (O-Fut1), and the O-fut1 gene is essential for N signaling. However, the role of the monosaccharide O-fucose on N is unclear, because O-Fut1 also appears to have O-fucosyltransferase activity-independent functions, including as an N-specific chaperon. Such an enzymatic activity-independent function could account for the essential role of O-fut1 in N signaling. To evaluate the role of the monosaccharide O-fucose modification in N signaling, here we generated a knock-in mutant of O-fut1 (O-fut1(R245A knock-in)), which expresses a mutant protein that lacks O-fucosyltransferase activity but maintains the N-specific chaperon activity. Using O-fut1(R245A knock-in) and other gene mutations that abolish the O-fucosylation of N, we found that the monosaccharide O-fucose modification of N has a temperature-sensitive function that is essential for N signaling. The O-fucose monosaccharide and O-glucose glycan modification, catalyzed by Rumi, function redundantly in the activation of N signaling. We also showed that the redundant function of these two modifications is responsible for the presence of N at the cell surface. Our findings elucidate how different forms of glycosylation on a protein can influence the protein's functions.

  8. Gamma-glutamylcyclotransferase promotes the growth of human glioma cells by activating Notch-Akt signaling.

    PubMed

    Shen, Shang-Hang; Yu, Ning; Liu, Xi-Yao; Tan, Guo-Wei; Wang, Zhan-Xiang

    2016-03-18

    Glioma as an aggressive type tumor is rapidly growing and has become one of the leading cause of cancer-related death worldwide. γ-Glutamylcyclotransferase (GGCT) has been shown as a diagnostic marker in various cancers. To reveal whether there is a correlation between GGCT and human glioma, GGCT expression in human glioma tissues and cell lines was first determined. We found that GGCT expression was up-regulated in human glioma tissues and cell lines. Further, we demonstrate that GGCT knockdown inhibits glioma cell T98G and U251 proliferation and colony formation, whereas GGCT overexpression leads to oppose effects. GGCT overexpression promotes the expression of Notch receptors and activates Akt signaling in glioma cells, and Notch-Akt signaling is activated in glioma tissues with high expression of GGCT. Finally, we show that inhibition of Notch-Akt signaling with Notch inhibitor MK-0752 blocks the effects of GGCT on glioma proliferation and colony formation. In conclusion, GGCT plays a critical role in glioma cell proliferation and may be a potential cancer therapeutic target. PMID:26828272

  9. The Secret Role of microRNAs in Cancer Stem Cell Development and Potential Therapy: A Notch-Pathway Approach.

    PubMed

    Prokopi, Marianna; Kousparou, Christina A; Epenetos, Agamemnon A

    2014-01-01

    MicroRNAs (miRNAs) have been implicated in the development of some if not all cancer types and have been identified as attractive targets for prognosis, diagnosis, and therapy of the disease. miRNAs are a class of small non-coding RNAs (20-22 nt in length) that bind imperfectly to the 3'-untranslated region of target mRNA regulating gene expression. Aberrantly expressed miRNAs in cancer, sometimes known as oncomiRNAs, have been shown to play a major role in oncogenesis, metastasis, and drug resistance. Amplification of oncomiRNAs during cancer development correlates with the silencing of tumor suppressor genes; on the other hand, down-regulation of miRNAs has also been observed in cancer and cancer stem cells (CSCs). In both cases, miRNA regulation is inversely correlated with cancer progression. Growing evidence indicates that miRNAs are also involved in the metastatic process by either suppressing or promoting metastasis-related genes leading to the reduction or activation of cancer cell migration and invasion processes. In particular, circulating miRNAs (vesicle-encapsulated or non-encapsulated) have significant effects on tumorigenesis: membrane-particles, apoptotic bodies, and exosomes have been described as providers of a cell-to-cell communication system transporting oncogenic miRNAs from tumors to neighboring cells and distant metastatic sites. It is hypothesized that miRNAs control cancer development in a traditional manner, by regulating signaling pathways and factors. In addition, recent developments indicate a non-conventional mechanism of cancer regulation by stem cell reprograming via a regulatory network consisting of miRNAs and Wnt/β-catenin, Notch, and Hedgehog signaling pathways, all of which are involved in controlling stem cell functions of CSCs. In this review, we focus on the role of miRNAs in the Notch-pathway and how they regulate CSC self-renewal, differentiation and tumorigenesis by direct/indirect targeting of the Notch-pathway. PMID

  10. The Secret Role of microRNAs in Cancer Stem Cell Development and Potential Therapy: A Notch-Pathway Approach

    PubMed Central

    Prokopi, Marianna; Kousparou, Christina A.; Epenetos, Agamemnon A.

    2015-01-01

    MicroRNAs (miRNAs) have been implicated in the development of some if not all cancer types and have been identified as attractive targets for prognosis, diagnosis, and therapy of the disease. miRNAs are a class of small non-coding RNAs (20–22 nt in length) that bind imperfectly to the 3′-untranslated region of target mRNA regulating gene expression. Aberrantly expressed miRNAs in cancer, sometimes known as oncomiRNAs, have been shown to play a major role in oncogenesis, metastasis, and drug resistance. Amplification of oncomiRNAs during cancer development correlates with the silencing of tumor suppressor genes; on the other hand, down-regulation of miRNAs has also been observed in cancer and cancer stem cells (CSCs). In both cases, miRNA regulation is inversely correlated with cancer progression. Growing evidence indicates that miRNAs are also involved in the metastatic process by either suppressing or promoting metastasis-related genes leading to the reduction or activation of cancer cell migration and invasion processes. In particular, circulating miRNAs (vesicle-encapsulated or non-encapsulated) have significant effects on tumorigenesis: membrane-particles, apoptotic bodies, and exosomes have been described as providers of a cell-to-cell communication system transporting oncogenic miRNAs from tumors to neighboring cells and distant metastatic sites. It is hypothesized that miRNAs control cancer development in a traditional manner, by regulating signaling pathways and factors. In addition, recent developments indicate a non-conventional mechanism of cancer regulation by stem cell reprograming via a regulatory network consisting of miRNAs and Wnt/β-catenin, Notch, and Hedgehog signaling pathways, all of which are involved in controlling stem cell functions of CSCs. In this review, we focus on the role of miRNAs in the Notch-pathway and how they regulate CSC self-renewal, differentiation and tumorigenesis by direct/indirect targeting of the Notch-pathway

  11. Myoblast cytonemes mediate Wg signaling from the wing imaginal disc and Delta-Notch signaling to the air sac primordium

    PubMed Central

    Huang, Hai; Kornberg, Thomas B

    2015-01-01

    The flight muscles, dorsal air sacs, wing blades, and thoracic cuticle of the Drosophila adult function in concert, and their progenitor cells develop together in the wing imaginal disc. The wing disc orchestrates dorsal air sac development by producing decapentaplegic and fibroblast growth factor that travel via specific cytonemes in order to signal to the air sac primordium (ASP). Here, we report that cytonemes also link flight muscle progenitors (myoblasts) to disc cells and to the ASP, enabling myoblasts to relay signaling between the disc and the ASP. Frizzled (Fz)-containing myoblast cytonemes take up Wingless (Wg) from the disc, and Delta (Dl)-containing myoblast cytonemes contribute to Notch activation in the ASP. Wg signaling negatively regulates Dl expression in the myoblasts. These results reveal an essential role for cytonemes in Wg and Notch signaling and for a signal relay system in the myoblasts. DOI: http://dx.doi.org/10.7554/eLife.06114.001 PMID:25951303

  12. Retroactive Signaling in Short Signaling Pathways

    PubMed Central

    Sepulchre, Jacques-Alexandre; Merajver, Sofía D.; Ventura, Alejandra C.

    2012-01-01

    In biochemical signaling pathways without explicit feedback connections, the core signal transduction is usually described as a one-way communication, going from upstream to downstream in a feedforward chain or network of covalent modification cycles. In this paper we explore the possibility of a new type of signaling called retroactive signaling, offered by the recently demonstrated property of retroactivity in signaling cascades. The possibility of retroactive signaling is analysed in the simplest case of the stationary states of a bicyclic cascade of signaling cycles. In this case, we work out the conditions for which variables of the upstream cycle are affected by a change of the total amount of protein in the downstream cycle, or by a variation of the phosphatase deactivating the same protein. Particularly, we predict the characteristic ranges of the downstream protein, or of the downstream phosphatase, for which a retroactive effect can be observed on the upstream cycle variables. Next, we extend the possibility of retroactive signaling in short but nonlinear signaling pathways involving a few covalent modification cycles. PMID:22848403

  13. Signaling on the endocytic pathway.

    PubMed

    McPherson, P S; Kay, B K; Hussain, N K

    2001-06-01

    Ligand binding to receptor tyrosine kinases and G-protein-coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrin-coated pits and vesicles. While receptor-mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand-activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal-regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways.

  14. Signaling on the endocytic pathway.

    PubMed

    McPherson, P S; Kay, B K; Hussain, N K

    2001-06-01

    Ligand binding to receptor tyrosine kinases and G-protein-coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrin-coated pits and vesicles. While receptor-mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand-activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal-regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways. PMID:11389765

  15. Inhibition of Notch signaling ameliorates insulin resistance in a FoxO1–dependent manner

    PubMed Central

    Pajvani, Utpal B.; Shawber, Carrie J.; Samuel, Varman T.; Birkenfeld, Andreas L.; Shulman, Gerald I.; Kitajewski, Jan; Accili, Domenico

    2012-01-01

    Summary Transcription factor FoxO1 promotes hepatic glucose production. Genetic inhibition of FoxO1 function prevents diabetes in experimental animal models, providing impetus to identify pharmacological approaches to modulate its function. Altered Notch signaling is seen in tumorigenesis, and Notch antagonists are in clinical testing for cancer application. Here, we report that FoxO1 and Notch coordinately regulate hepatic glucose metabolism. Combined haploinsufficiency of FoxO1 and Notch1 markedly improves insulin sensitivity in diet-induced insulin resistance, as does liver-specific knockout of the Notch transcriptional effector, Rbp-Jk. Conversely, Notch1 gain-of-function promotes insulin resistance in a FoxO1-dependent manner and induces Glucose-6-phosphatase expression. Pharmacological blockade of Notch signaling with γ-secretase inhibitors improves insulin sensitivity following in vivo administration in lean and in obese, insulin-resistant mice. The data identify a heretofore unknown metabolic function of Notch, and suggest that Notch inhibition is beneficial to diabetes treatment, in part by helping to offset excessive FoxO1–driven hepatic glucose production. PMID:21804540

  16. The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling.

    PubMed

    Chintala, Hemabindu; Krupska, Izabela; Yan, Lulu; Lau, Lester; Grant, Maria; Chaqour, Brahim

    2015-07-01

    Physiological angiogenesis depends on the highly coordinated actions of multiple angiogenic regulators. CCN1 is a secreted cysteine-rich and integrin-binding matricellular protein required for proper cardiovascular development. However, our understanding of the cellular origins and activities of this molecule is incomplete. Here, we show that CCN1 is predominantly expressed in angiogenic endothelial cells (ECs) at the leading front of actively growing vessels in the mouse retina. Endothelial deletion of CCN1 in mice using a Cre-Lox system is associated with EC hyperplasia, loss of pericyte coverage and formation of dense retinal vascular networks lacking the normal hierarchical arrangement of arterioles, capillaries and venules. CCN1 is a product of an immediate-early gene that is transcriptionally induced in ECs in response to stimulation by vascular endothelial growth factor (VEGF). We found that CCN1 activity is integrated with VEGF receptor 2 (VEGF-R2) activation and downstream signaling pathways required for tubular network formation. CCN1-integrin binding increased the expression of and association between Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) and VEGF-R2, which leads to rapid dephosphorylation of VEGF-R2 tyrosine, thus preventing EC hyperproliferation. Predictably, CCN1 further brings receptors/signaling molecules into proximity that are otherwise spatially separated. Furthermore, CCN1 induces integrin-dependent Notch activation in cultured ECs, and its targeted gene inactivation in vivo alters Notch-dependent vascular specification and remodeling, suggesting that functional levels of Notch signaling requires CCN1 activity. These data highlight novel functions of CCN1 as a naturally optimized molecule, fine-controlling key processes in physiological angiogenesis and safeguarding against aberrant angiogenic responses.

  17. Endothelio-mesenchymal interaction controls runx1 expression and modulates the notch pathway to initiate aortic hematopoiesis

    PubMed Central

    Richard, Charlotte; Drevon, Cécile; Canto, Pierre-Yves; Villain, Gaelle; Bollérot, Karine; Lempereur, Aveline; Teillet, Marie-Aimée; Vincent, Christine; Castillo, Catalina Rosselló; Torres, Miguel; Piwarzyk, Eileen; Speck, Nancy A.; Souyri, Michèle; Jaffredo, Thierry

    2014-01-01

    SUMMARY Hematopoietic stem cells (HSCs) are produced by a small cohort of hemogenic endothelial cells (ECs) during development through the formation of intra-aortic hematopoietic cell (HC) clusters (HCs). The Runx1 transcription factor plays a key role in the EC to HC and HSC transition. We show that Runx1 expression in hemogenic ECs and the subsequent initiation of HC formation are tightly controlled by the sub-aortic mesenchyme, although the mesenchyme is not a source of HCs. Runx1 and Notch signaling are involved in this process, with Notch signaling decreasing with time in HCs. Inhibiting Notch signaling readily increases HC production in mouse and chicken embryos. In the mouse however, this increase is transient. Collectively, we show complementary roles of hemogenic ECs and mesenchymal compartments in triggering aortic hematopoiesis. The sub-aortic mesenchyme induces Runx1 expression in hemogenic-primed endothelial cells and collaborates with Notch dynamics to control aortic hematopoiesis. PMID:23537631

  18. Signaling Pathways in Melanogenesis

    PubMed Central

    D’Mello, Stacey A. N.; Finlay, Graeme J.; Baguley, Bruce C.; Askarian-Amiri, Marjan E.

    2016-01-01

    Melanocytes are melanin-producing cells found in skin, hair follicles, eyes, inner ear, bones, heart and brain of humans. They arise from pluripotent neural crest cells and differentiate in response to a complex network of interacting regulatory pathways. Melanins are pigment molecules that are endogenously synthesized by melanocytes. The light absorption of melanin in skin and hair leads to photoreceptor shielding, thermoregulation, photoprotection, camouflage and display coloring. Melanins are also powerful cation chelators and may act as free radical sinks. Melanin formation is a product of complex biochemical events that starts from amino acid tyrosine and its metabolite, dopa. The types and amounts of melanin produced by melanocytes are determined genetically and are influenced by a variety of extrinsic and intrinsic factors such as hormonal changes, inflammation, age and exposure to UV light. These stimuli affect the different pathways in melanogenesis. In this review we will discuss the regulatory mechanisms involved in melanogenesis and explain how intrinsic and extrinsic factors regulate melanin production. We will also explain the regulatory roles of different proteins involved in melanogenesis. PMID:27428965

  19. Signaling Pathways in Melanogenesis.

    PubMed

    D'Mello, Stacey A N; Finlay, Graeme J; Baguley, Bruce C; Askarian-Amiri, Marjan E

    2016-01-01

    Melanocytes are melanin-producing cells found in skin, hair follicles, eyes, inner ear, bones, heart and brain of humans. They arise from pluripotent neural crest cells and differentiate in response to a complex network of interacting regulatory pathways. Melanins are pigment molecules that are endogenously synthesized by melanocytes. The light absorption of melanin in skin and hair leads to photoreceptor shielding, thermoregulation, photoprotection, camouflage and display coloring. Melanins are also powerful cation chelators and may act as free radical sinks. Melanin formation is a product of complex biochemical events that starts from amino acid tyrosine and its metabolite, dopa. The types and amounts of melanin produced by melanocytes are determined genetically and are influenced by a variety of extrinsic and intrinsic factors such as hormonal changes, inflammation, age and exposure to UV light. These stimuli affect the different pathways in melanogenesis. In this review we will discuss the regulatory mechanisms involved in melanogenesis and explain how intrinsic and extrinsic factors regulate melanin production. We will also explain the regulatory roles of different proteins involved in melanogenesis. PMID:27428965

  20. NOTCHing the bone: Insights into multi-functionality

    PubMed Central

    Engin, Feyza; Lee, Brendan

    2010-01-01

    Evolutionarily conserved Notch signaling plays a critical role during embryonic and postnatal life. The importance of Notch signaling in the determination of cell fate, and the spatio-temporal regulation of proliferation, differentiation and apoptosis has been demonstrated in various different organ systems. However, how Notch signaling affects the bone development was unknown until now. The in vivo effects of Notch signaling in lineage commitment, bone formation and bone resorption were demonstrated in recent studies. In addition to regulation of osteoblastogenesis, osteoblast directed osteoclastogenesis by Notch signaling revealed a dimorphic effect for this signaling pathway providing another example of such in bone development. Moreover, identification of the cross-talk between the hematopoietic stem cell niche and osteoblasts through Notch signaling also suggested another important role for Notch signaling, i.e., the coupling of cellular components of the bone microenvironment. The association between the gain and loss of function of Notch activity in bone pathology highlights Notch as a potentially novel therapeutic target for the treatment of metabolic bone disease and bone cancer. In this review, we will focus primarily on the regulation of bone cells, i.e., osteoblasts and osteoclasts by Notch signaling. We will also review the importance of Notch in specifying bone-hematopoietic stem cell niche interactions within the bone microenvironment. Finally, we will discuss potential clinical implications and future directions for this field. PMID:19520195

  1. Stochastic Regulation of her1/7 Gene Expression Is the Source of Noise in the Zebrafish Somite Clock Counteracted by Notch Signalling

    PubMed Central

    Jenkins, Robert P.; Hanisch, Anja; Soza-Ried, Cristian; Sahai, Erik

    2015-01-01

    The somite segmentation clock is a robust oscillator used to generate regularly-sized segments during early vertebrate embryogenesis. It has been proposed that the clocks of neighbouring cells are synchronised via inter-cellular Notch signalling, in order to overcome the effects of noisy gene expression. When Notch-dependent communication between cells fails, the clocks of individual cells operate erratically and lose synchrony over a period of about 5 to 8 segmentation clock cycles (2–3 hours in the zebrafish). Here, we quantitatively investigate the effects of stochasticity on cell synchrony, using mathematical modelling, to investigate the likely source of such noise. We find that variations in the transcription, translation and degradation rate of key Notch signalling regulators do not explain the in vivo kinetics of desynchronisation. Rather, the analysis predicts that clock desynchronisation, in the absence of Notch signalling, is due to the stochastic dissociation of Her1/7 repressor proteins from the oscillating her1/7 autorepressed target genes. Using in situ hybridisation to visualise sites of active her1 transcription, we measure an average delay of approximately three minutes between the times of activation of the two her1 alleles in a cell. Our model shows that such a delay is sufficient to explain the in vivo rate of clock desynchronisation in Notch pathway mutant embryos and also that Notch-mediated synchronisation is sufficient to overcome this stochastic variation. This suggests that the stochastic nature of repressor/DNA dissociation is the major source of noise in the segmentation clock. PMID:26588097

  2. Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4.

    PubMed

    Vega, Maria E; Giroux, Véronique; Natsuizaka, Mitsuteru; Liu, Mingen; Klein-Szanto, Andres J; Stairs, Douglas B; Nakagawa, Hiroshi; Wang, Kenneth K; Wang, Timothy C; Lynch, John P; Rustgi, Anil K

    2014-01-01

    Barrett's esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.

  3. Inhibitory Role of Notch1 in Calcific Aortic Valve Disease

    PubMed Central

    Koenig, Sara N.; Nichols, Haley A.; Galindo, Cristi L.; Garner, Harold R.; Merrill, Walter H.; Hinton, Robert B.; Garg, Vidu

    2011-01-01

    Aortic valve calcification is the most common form of valvular heart disease, but the mechanisms of calcific aortic valve disease (CAVD) are unknown. NOTCH1 mutations are associated with aortic valve malformations and adult-onset calcification in families with inherited disease. The Notch signaling pathway is critical for multiple cell differentiation processes, but its role in the development of CAVD is not well understood. The aim of this study was to investigate the molecular changes that occur with inhibition of Notch signaling in the aortic valve. Notch signaling pathway members are expressed in adult aortic valve cusps, and examination of diseased human aortic valves revealed decreased expression of NOTCH1 in areas of calcium deposition. To identify downstream mediators of Notch1, we examined gene expression changes that occur with chemical inhibition of Notch signaling in rat aortic valve interstitial cells (AVICs). We found significant downregulation of Sox9 along with several cartilage-specific genes that were direct targets of the transcription factor, Sox9. Loss of Sox9 expression has been published to be associated with aortic valve calcification. Utilizing an in vitro porcine aortic valve calcification model system, inhibition of Notch activity resulted in accelerated calcification while stimulation of Notch signaling attenuated the calcific process. Finally, the addition of Sox9 was able to prevent the calcification of porcine AVICs that occurs with Notch inhibition. In conclusion, loss of Notch signaling contributes to aortic valve calcification via a Sox9-dependent mechanism. PMID:22110751

  4. BEND6 is a nuclear antagonist of Notch signaling during self-renewal of neural stem cells

    PubMed Central

    Dai, Qi; Andreu-Agullo, Celia; Insolera, Ryan; Wong, Li Chin; Shi, Song-Hai; Lai, Eric C.

    2013-01-01

    The activity of the Notch pathway revolves around a CSL-class transcription factor, which recruits distinct complexes that activate or repress target gene expression. The co-activator complex is deeply conserved and includes the cleaved Notch intracellular domain (NICD) and Mastermind. By contrast, numerous CSL co-repressor proteins have been identified, and these are mostly different between invertebrate and vertebrate systems. In this study, we demonstrate that mammalian BEND6 is a neural BEN-solo factor that shares many functional attributes with Drosophila Insensitive, a co-repressor for the Drosophila CSL factor. BEND6 binds the mammalian CSL protein CBF1 and antagonizes Notch-dependent target activation. In addition, its association with Notch- and CBF1-regulated enhancers is promoted by CBF1 and antagonized by activated Notch. In utero electroporation experiments showed that ectopic BEND6 inhibited Notch-mediated self-renewal of neocortical neural stem cells and promoted neurogenesis. Conversely, knockdown of BEND6 increased NSC self-renewal in wild-type neocortex, and exhibited genetic interactions with gain and loss of Notch pathway activity. We recapitulated all of these findings in cultured neurospheres, in which overexpression and depletion of BEND6 caused reciprocal effects on neural stem cell renewal and neurogenesis. These data reveal a novel mammalian CSL co-repressor in the nervous system, and show that the Notch-inhibitory activity of certain BEN-solo proteins is conserved between flies and mammals. PMID:23571214

  5. Fatty Acid-binding Protein 4, a Point of Convergence for Angiogenic and Metabolic Signaling Pathways in Endothelial Cells*

    PubMed Central

    Harjes, Ulrike; Bridges, Esther; McIntyre, Alan; Fielding, Barbara A.; Harris, Adrian L.

    2014-01-01

    Fatty acid-binding protein 4 (FABP4) is an adipogenic protein and is implicated in atherosclerosis, insulin resistance, and cancer. In endothelial cells, FABP4 is induced by VEGFA, and inhibition of FABP4 blocks most of the VEGFA effects. We investigated the DLL4-NOTCH-dependent regulation of FABP4 in human umbilical vein endothelial cells by gene/protein expression and interaction analyses following inhibitor treatment and RNA interference. We found that FABP4 is directly induced by NOTCH. Stimulation of NOTCH signaling with human recombinant DLL4 led to FABP4 induction, independently of VEGFA. FABP4 induction by VEGFA was reduced by blockade of DLL4 binding to NOTCH or inhibition of NOTCH signal transduction. Chromatin immunoprecipitation of the NOTCH intracellular domain showed increased binding to two specific regions in the FABP4 promoter. The induction of FABP4 gene expression was dependent on the transcription factor FOXO1, which was essential for basal expression of FABP4, and FABP4 up-regulation following stimulation of the VEGFA and/or the NOTCH pathway. Thus, we show that the DLL4-NOTCH pathway mediates endothelial FABP4 expression. This indicates that induction of the angiogenesis-restricting DLL4-NOTCH can have pro-angiogenic effects via this pathway. It also provides a link between DLL4-NOTCH and FOXO1-mediated regulation of endothelial gene transcription, and it shows that DLL4-NOTCH is a nodal point in the integration of pro-angiogenic and metabolic signaling in endothelial cells. This may be crucial for angiogenesis in the tumor environment. PMID:24939870

  6. Notch Signaling Contributes to Liver Inflammation by Regulation of Interleukin-22-Producing Cells in Hepatitis B Virus Infection

    PubMed Central

    Wei, Xin; Wang, Jiu-Ping; Hao, Chun-Qiu; Yang, Xiao-Fei; Wang, Lin-Xu; Huang, Chang-Xing; Bai, Xue-Fan; Lian, Jian-Qi; Zhang, Ye

    2016-01-01

    The mechanism of hepatitis B virus (HBV) induced liver inflammation is not fully elucidated. Notch signaling augmented interleukin (IL)-22 secretion in CD4+ T cells, and Notch-IL-22 axis fine-tuned inflammatory response. We previously demonstrated a proinflammatory role of IL-22 in HBV infection. Thus, in this study, we analyzed the role of Notch in development of IL-22-producing cells in HBV infection by inhibition of Notch signaling using γ-secretase inhibitor DAPT in both hydrodynamic induced HBV-infected mouse model and in peripheral blood cells isolated from patients with HBV infection. mRNA expressions of Notch1 and Notch2 were significantly increased in livers and CD4+ T cells upon HBV infection. Inhibition of Notch signaling in vivo leaded to the reduction in NKp46+ innate lymphoid cells 22 (ILC22) and lymphoid tissue inducer 4 (LTi4) cells in the liver. This process was accompanied by downregulating the expressions of IL-22 and related proinflammatory cytokines and chemokines in the liver, as well as blocking the recruitment of antigen-non-specific inflammatory cells into the liver and subsequent liver injury, but did not affect HBV antigens production and IL-22 secretion in the serum. Furthermore, IL-22 production in HBV non-specific cultured CD4+ T cells, but not HBV-specific CD4+ T cells, was reduced in response to in vitro inhibition of Notch signaling. In conclusion, Notch siganling appears to be an important mediator of the liver inflammation by modulating hepatic ILC22. The potential proinflammatory effect of Notch-mediated ILC22 may be significant for the development of new therapeutic approaches for treatment of hepatitis B. PMID:27800305

  7. Mesoporous silica nanoparticle-based substrates for cell directed delivery of Notch signalling modulators to control myoblast differentiation

    NASA Astrophysics Data System (ADS)

    Böcking, Dominique; Wiltschka, Oliver; Niinimäki, Jenni; Shokry, Hussein; Brenner, Rolf; Lindén, Mika; Sahlgren, Cecilia

    2014-01-01

    Biochemical cues are critical to control stem cell function and can be utilized to develop smart biomaterials for stem cell engineering. The challenge is to deliver these cues in a restricted manner with spatial and temporal control. Here we have developed bilayer films of mesoporous silica nanoparticles for delayed cellular delivery of Notch modulators to promote muscle stem cell differentiation. We demonstrate that drug-loaded particles are internalized from the particle-covered surface, which allows for direct delivery of the drug into the cell and a delayed and confined drug release. Substrates of particles loaded with γ-secretase-inhibitors, which block the Notch signalling pathway, promoted efficient differentiation of myoblasts. The particle substrates were fully biocompatible and did not interfere with the inherent differentiation process. We further demonstrate that impregnating commercially available, biocompatible polymer scaffolds with MSNs allows for a free standing substrate for cell directed drug delivery.Biochemical cues are critical to control stem cell function and can be utilized to develop smart biomaterials for stem cell engineering. The challenge is to deliver these cues in a restricted manner with spatial and temporal control. Here we have developed bilayer films of mesoporous silica nanoparticles for delayed cellular delivery of Notch modulators to promote muscle stem cell differentiation. We demonstrate that drug-loaded particles are internalized from the particle-covered surface, which allows for direct delivery of the drug into the cell and a delayed and confined drug release. Substrates of particles loaded with γ-secretase-inhibitors, which block the Notch signalling pathway, promoted efficient differentiation of myoblasts. The particle substrates were fully biocompatible and did not interfere with the inherent differentiation process. We further demonstrate that impregnating commercially available, biocompatible polymer scaffolds with

  8. Tetrandrine induces autophagy and differentiation by activating ROS and Notch1 signaling in leukemia cells.

    PubMed

    Liu, Ting; Men, Qiuxu; Wu, Guixian; Yu, Chunrong; Huang, Zan; Liu, Xin; Li, Wenhua

    2015-04-10

    All-trans retinoic acid (ATRA) is a differentiating agent for the treatment of acute promyelocytic leukemia (APL). However, the therapeutic efficacy of ATRA has limitations. Tetrandrine is a traditional Chinese medicinal herb extract with antitumor effects. In this study, we investigated the effects of tetrandrine on human PML-RARα-positive acute promyelocytic leukemia cells. Tetrandrine inhibited tumors in vivo. It induced autophagy and differentiation by triggering ROS generation and activating Notch1 signaling. Tetrandrine induced autophagy and differentiation in M5 type patient primary leukemia cells. The in vivo results indicated that low concentrations of tetrandrine inhibited leukemia cells proliferation and induced autophagy and then facilitated their differentiation, by activating ROS and Notch1 signaling. We suggest that tetrandrine is a potential agent for the treatment of APL by inducing differentiation of leukemia cells. PMID:25797266

  9. Stromal cell-mediated glycolytic switch in CLL cells involves Notch-c-Myc signaling.

    PubMed

    Jitschin, Regina; Braun, Martina; Qorraj, Mirjeta; Saul, Domenica; Le Blanc, Katarina; Zenz, Thorsten; Mougiakakos, Dimitrios

    2015-05-28

    It is well established that the stromal niche exerts a protective effect on chronic lymphocytic leukemia (CLL) cells, thereby also affecting their drug sensitivity. One hallmark of malignant cells is metabolic reprogramming, which is mostly represented by a glycolytic shift known as the Warburg effect. Because treatment resistance can be linked to metabolic alterations, we investigated whether bone marrow stromal cells impact the bioenergetics of primary CLL cells. In fact, stromal contact led to an increase of aerobic glycolysis and the cells' overall glycolytic capacity accompanied by an increased glucose uptake, expression of glucose transporter, and glycolytic enzymes. Activation of Notch signaling and of its direct transcriptional target c-Myc contributed to this metabolic switch. Based on these observations, CLL cells' acquired increased glucose dependency as well as Notch-c-Myc signaling could be therapeutically exploited in an effort to overcome stroma-mediated drug resistance.

  10. The Histone Deacetylase Inhibitor Vaproic Acid Induces Cell Growth Arrest in Hepatocellular Carcinoma Cells via Suppressing Notch Signaling

    PubMed Central

    Sun, Guangchun; Mackey, Lily V.; Coy, David H.; Yu, Cui-Yun; Sun, Lichun

    2015-01-01

    Hepatocellular carcinoma (HCC) is a type of malignant cancer. Notch signaling is aberrantly expressed in HCC tissues with more evidence showing that this signaling plays a critical role in HCCs. In the present study, we investigate the effects of the anti-convulsant drug valproic acid (VPA) in HCC cells and its involvement in modulating Notch signaling. We found that VPA, acting as a histone deacetylase (HDAC) inhibitor, induced a decrease in HDAC4 and an increase in acetylated histone 4 (AcH4) and suppressed HCC cell growth. VPA also induced down-regulation of Notch signaling via suppressing the expression of Notch1 and its target gene HES1, with an increase of tumor suppressor p21 and p63. Furthermore, Notch1 activation via overexpressing Notch1 active form ICN1 induced HCC cell proliferation and anti-apoptosis, indicating Notch signaling played an oncogenic role in HCC cells. Meanwhile, VPA could reverse Notch1-induced increase of cell proliferation. Interestingly, VPA was also observed to stimulate the expression of G protein-coupled somatostatin receptor type 2 (SSTR2) that has been used in receptor-targeting therapies. This discovery supports a combination therapy of VPA with the SSTR2-targeting agents. Our in vitro assay did show that the combination of VPA and the peptide-drug conjugate camptothecin-somatostatin (CPT-SST) displayed more potent anti-proliferative effects on HCC cells than did each alone. VPA may be a potential drug candidate in the development of anti-HCC drugs via targeting Notch signaling, especially in combination with receptor-targeting cytotoxic agents. PMID:26366213

  11. Homemade notch filter to suppress strong FM or DAB - T/DVB - T signals

    NASA Astrophysics Data System (ADS)

    Monstein, Christian

    2016-04-01

    Many of the current 116 solar radio spectrometer instruments in the e-Callisto network are suffering from strong interference from FM-radio, DAB-T or DVB-T broadcast stations. With simple surface mount device (SMD) components a cheap notch (trap)filter can be produced to suppress these strong signals that otherwise may saturate the low noise amplifier and/or the receiver.

  12. The Sex-determining region Y-box 4 and homeobox C6 transcriptional networks in prostate cancer progression: crosstalk with the Wnt, Notch, and PI3K pathways.

    PubMed

    Moreno, Carlos S

    2010-02-01

    The transforming growth factor beta, Hedgehog, Notch, and Wnt signaling pathways all play critical roles in the development and progression of prostate cancer. It is becoming increasingly apparent that these pathways may intersect with developmentally important transcription factors such as the sex-determining region Y-box 4 (SOX4), homeobox C6, enhancer of zeste 2, and ETS-related gene, which are up-regulated in prostate cancers. For example, identification of the downstream targets of SOX4 and homeobox C6 suggests that these factors may cooperate to activate the Notch pathway and the PI3K/AKT pathway, possibly in response to Wnt signals. PI3K/AKT activation likely occurs indirectly via up-regulation of growth factor receptors, while Notch activation is secondary to up-regulation of Notch pathway components. In addition, SOX4 may affect terminal differentiation via regulation of other transcription factors such as NKX3.1 and MLL, and regulation of components of the microRNA pathway such as Dicer and Argonaute 1. The evidence supporting activation of these pathways in prostate cancer progression suggests that combinations of compounds targeting them may be of benefit to patients with aggressive, metastatic disease.

  13. Targeting Notch degradation system provides promise for breast cancer therapeutics.

    PubMed

    Liu, Jing; Shen, Jia-Xin; Wen, Xiao-Fen; Guo, Yu-Xian; Zhang, Guo-Jun

    2016-08-01

    Notch receptor signaling pathways play an important role, not only in normal breast development but also in breast cancer development and progression. As a group of ligand-induced proteins, different subtypes of mammalian Notch (Notch1-4) are sensitive to subtle changes in protein levels. Thus, a clear understanding of mechanisms of Notch protein turnover is essential for understanding normal and pathological mechanisms of Notch functions. It has been suggested that there is a close relationship between the carcinogenesis and the dysregulation of Notch degradation. However, this relationship remains mostly undefined in the context of breast cancer, as protein degradation is mediated by numerous signaling pathways as well as certain molecule modulators (activators/inhibitors). In this review, we summarize the published data regarding the regulation of Notch family member degradation in breast cancer, while emphasizing areas that are likely to provide new therapeutic modalities for mechanism-based anti-cancer drugs.

  14. Notch signal reception is required in vascular smooth muscle cells for ductus arteriosus closure.

    PubMed

    Krebs, Luke T; Norton, Christine R; Gridley, Thomas

    2016-02-01

    The ductus arteriosus is an arterial vessel that shunts blood flow away from the lungs during fetal life, but normally occludes after birth to establish the adult circulation pattern. Failure of the ductus arteriosus to close after birth is termed patent ductus arteriosus, and is one of the most common congenital heart defects. Our previous work demonstrated that vascular smooth muscle cell expression of the Jag1 gene, which encodes a ligand for Notch family receptors, is essential for postnatal closure of the ductus arteriosus in mice. However, it was not known what cell population was responsible for receiving the Jag1-mediated signal. Here we show, using smooth muscle cell-specific deletion of the Rbpj gene, which encodes a transcription factor that mediates all canonical Notch signaling, that Notch signal reception in the vascular smooth muscle cell compartment is required for ductus arteriosus closure. These data indicate that homotypic vascular smooth muscle cell interactions are required for proper contractile smooth muscle cell differentiation and postnatal closure of the ductus arteriosus in mice.

  15. Aberrant activation of canonical Notch1 signaling in the mouse uterus decreases progesterone receptor by hypermethylation and leads to infertility.

    PubMed

    Su, Ren-Wei; Strug, Michael R; Jeong, Jae-Wook; Miele, Lucio; Fazleabas, Asgerally T

    2016-02-23

    In mammalian reproduction, implantation is one of the most critical events. Failure of implantation and the subsequent decidualization contribute to more than 75% of pregnancy losses in women. Our laboratory has previously reported that inhibition of Notch signaling results in impaired decidualization in both women and a transgenic mouse model. In this study, we generated a Notch gain-of-function transgenic mouse by conditionally overexpressing the Notch1 intracellular domain (N1ICD) in the reproductive tract driven by a progesterone receptor (Pgr) -Cre. We show that the overexpression of N1ICD in the uterus results in complete infertility as a consequence of multiple developmental and physiological defects, including the absence of uterine glands and dysregulation of progesterone and estrogen signaling by a Recombination Signal Binding Protein Jκ-dependent signaling mechanism. We further show that the inhibition of progesterone signaling is caused by hypermethylation of its receptor Pgr by Notch1 overexpression through the transcription factor PU.1 and DNA methyltransferase 3b (Dnmt3b). We have generated a mouse model to study the consequence of increased Notch signaling in female reproduction and provide the first evidence, to our knowledge, that Notch signaling can regulate epigenetic modification of the Pgr.

  16. Insulin signals control the competence of the Drosophila female germline stem cell niche to respond to Notch ligands.

    PubMed

    Hsu, Hwei-Jan; Drummond-Barbosa, Daniela

    2011-02-15

    Adult stem cells reside in specialized microenvironments, or niches, that are essential for their function in vivo. Stem cells are physically attached to the niche, which provides secreted factors that promote their self-renewal and proliferation. Despite intense research on the role of the niche in regulating stem cell function, much less is known about how the niche itself is controlled. We previously showed that insulin signals directly stimulate germline stem cell (GSC) division and indirectly promote GSC maintenance via the niche in Drosophila. Insulin-like peptides are required for maintenance of cap cells (a major component of the niche) via modulation of Notch signaling, and they also control attachment of GSCs to cap cells and E-cadherin levels at the cap cell-GSC junction. Here, we further dissect the molecular and cellular mechanisms underlying these processes. We show that insulin and Notch ligands directly stimulate cap cells to maintain their numbers and indirectly promote GSC maintenance. We also report that insulin signaling, via phosphoinositide 3-kinase and FOXO, intrinsically controls the competence of cap cells to respond to Notch ligands and thereby be maintained. Contrary to a previous report, we also find that Notch ligands originated in GSCs are not required either for Notch activation in the GSC niche, or for cap cell or GSC maintenance. Instead, the niche itself produces ligands that activate Notch signaling within cap cells, promoting stability of the GSC niche. Finally, insulin signals control cap cell-GSC attachment independently of their role in Notch signaling. These results are potentially relevant to many systems in which Notch signaling modulates stem cells and demonstrate that complex interactions between local and systemic signals are required for proper stem cell niche function.

  17. The Ser/Thr Phosphatase PP2A Regulatory Subunit Widerborst Inhibits Notch Signaling

    PubMed Central

    Bose, Anasua; Majot, Adam T.; Bidwai, Ashok P.

    2014-01-01

    Drosophila Enhancer of split M8, an effector of Notch signaling, is regulated by protein kinase CK2. The phosphatase PP2A is thought to play an opposing (inhibitory) role, but the identity of the regulatory subunit was unknown. The studies described here reveal a role for the PP2A regulatory subunit widerborst (wdb) in three developmental contexts; the bristle, wing and the R8 photoreceptors of the eye. wdb overexpression elicits bristle and wing defects akin to reduced Notch signaling, whereas hypomorphic mutations in this PP2A subunit elicit opposite effects. We have also evaluated wdb functions using mutations in Notch and E(spl) that affect the eye. We find that the eye and R8 defects of the well-known Nspl mutation are enhanced by a hypomorphic allele of wdb, whereas they are strongly rescued by wdb overexpression. Similarly, ectopic wdb rescues the eye and R8 defects of the E(spl)D mutation, which affects the m8 gene. In addition, wdb overexpression also rescues the bristle defects of ectopically expressed M8, or the eye and R8 defects of its CK2 phosphomimetic variant M8-S159D. The latter finding suggests that PP2A may target M8 at highly conserved residues in the vicinity of the CK2 site, whose phosphorylation controls repression of Atonal and the R8 fate. Together, the studies identify PP2A-Wdb as a participant in Notch signaling, and suggest that M8 activity is controlled by phosphorylation and dephosphorylation. The conservation of the phosphorylation sites between Drosophila E(spl) and the HES/HER proteins from mammals, reptiles, amphibians, birds and fish raises the prospect that this mode of regulation is widespread. PMID:25006677

  18. YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway

    SciTech Connect

    Lin, Yi-Ting; Ding, Jing-Ya; Li, Ming-Yang; Yeh, Tien-Shun; Wang, Tsu-Wei; Yu, Jenn-Yah

    2012-09-10

    Tight regulation of cell numbers by controlling cell proliferation and apoptosis is important during development. Recently, the Hippo pathway has been shown to regulate tissue growth and organ size in Drosophila. In mammalian cells, it also affects cell proliferation and differentiation in various tissues, including the nervous system. Interplay of several signaling cascades, such as Notch, Wnt, and Sonic Hedgehog (Shh) pathways, control cell proliferation during neuronal differentiation. However, it remains unclear whether the Hippo pathway coordinates with other signaling cascades in regulating neuronal differentiation. Here, we used P19 cells, a mouse embryonic carcinoma cell line, as a model to study roles of YAP, a core component of the Hippo pathway, in neuronal differentiation. P19 cells can be induced to differentiate into neurons by expressing a neural bHLH transcription factor gene Ascl1. Our results showed that YAP promoted cell proliferation and inhibited neuronal differentiation. Expression of Yap activated Shh but not Wnt or Notch signaling activity during neuronal differentiation. Furthermore, expression of Yap increased the expression of Patched homolog 1 (Ptch1), a downstream target of the Shh signaling. Knockdown of Gli2, a transcription factor of the Shh pathway, promoted neuronal differentiation even when Yap was over-expressed. We further demonstrated that over-expression of Yap inhibited neuronal differentiation in primary mouse cortical progenitors and Gli2 knockdown rescued the differentiation defect in Yap over-expressing cells. In conclusion, our study reveals that Shh signaling acts downstream of YAP in regulating neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer YAP promotes cell proliferation and inhibits neuronal differentiation in P19 cells. Black-Right-Pointing-Pointer YAP promotes Sonic hedgehog signaling activity during neuronal differentiation. Black-Right-Pointing-Pointer Knockdown of Gli2 rescues the Yap

  19. Targeting Notch to overcome radiation resistance

    PubMed Central

    Yahyanejad, Sanaz; Theys, Jan; Vooijs, Marc

    2016-01-01

    Radiotherapy represents an important therapeutic strategy in the treatment of cancer cells. However, it often fails to eliminate all tumor cells because of the intrinsic or acquired treatment resistance, which is the most common cause of tumor recurrence. Emerging evidences suggest that the Notch signaling pathway is an important pathway mediating radiation resistance in tumor cells. Successful targeting of Notch signaling requires a thorough understanding of Notch regulation and the context-dependent interactions between Notch and other therapeutically relevant pathways. Understanding these interactions will increase our ability to design rational combination regimens that are more likely to be safe and effective. Here we summarize the role of Notch in mediating resistance to radiotherapy, the different strategies to block Notch in cancer cells and how treatment scheduling can improve tumor response. Finally, we discuss a need for reliable Notch related biomarkers in specific tumors to measure pathway activity and to allow identification of a subset of patients who are likely to benefit from Notch targeted therapies. PMID:26713603

  20. Notch-1 expression levels in 3T3-L1 cells influence ras signaling and transformation by oncogenic ras.

    PubMed

    Ruiz-Hidalgo, M J; Garcés, C; Laborda, J

    1999-04-01

    Notch proteins participate in interactions between several cell types involved on the specification of numerous cell fates during development. We previously showed that enforced downregulation of Notch-1 expression prevented adipogenesis of 3T3-L1 cells. Since adipogenesis of 3T3-L1 cells can be induced by oncogenic ras, we studied whether this was also the case in 3T3-L1 cells with decreased levels of Notch-1 expression. We found that oncogenic ras induces transformation and not differentiation of 3T3-L1 cells with diminished levels of Notch-1. This result suggests that Notch-1 is implicated in the interpretation of signals leading to activation of p21 Ras.

  1. The Wnt3a/β-catenin target gene Mesogenin1 controls the segmentation clock by activating a Notch signaling program

    PubMed Central

    Chalamalasetty, Ravindra B.; Dunty, William C.; Biris, Kristin K.; Ajima, Rieko; Iacovino, Michelina; Beisaw, Arica; Feigenbaum, Lionel; Chapman, Deborah L.; Yoon, Jeong Kyo; Kyba, Michael; Yamaguchi, Terry P.

    2013-01-01

    Summary Segmentation is an organizing principle of body plans. The segmentation clock, a molecular oscillator best illustrated by the cyclic expression of Notch signaling genes, controls the periodic cleavage of somites from unsegmented presomitic mesoderm (PSM) during vertebrate segmentation. Wnt3a controls the spatiotemporal expression of cyclic Notch genes, however the underlying mechanisms remain obscure. Transcriptional profiling of Wnt3a−/− embryos led to the identification of the bHLH transcription factor, Mesogenin1 (Msgn1), as a direct target gene of Wnt3a. To identify Msgn1 targets, we performed genome-wide studies of Msgn1 activity in embryonic stem cells. Here we show that Msgn1 is a major transcriptional activator of a Notch signaling program, synergizing with Notch to trigger clock gene expression. Msgn1 also indirectly regulates cyclic genes in the Fgf and Wnt pathways. Thus, Msgn1 is a central component of a transcriptional cascade that translates a spatial Wnt3a gradient into a temporal pattern of clock gene expression. PMID:21750544

  2. Salinomycin Suppresses PDGFRβ, MYC, and Notch Signaling in Human Medulloblastoma

    PubMed Central

    Zhou, Shuang; Wang, Fengfei; Zhang, Ying; Johnson, Max R; Qian, Steven; Wu, Min; Wu, Erxi

    2014-01-01

    Medulloblastoma (MB) is the most common childhood brain tumor. Despite improved therapy and management, approximately 30% of patients die of the disease. To search for a more effective therapeutic strategy, the effects of salinomycin were tested on cell proliferation, cell death, and cell cycle progression in human MB cell lines. The results demonstrated that salinomycin inhibits cell proliferation, induces cell death , and disrupts cell cycle progression in MB cells. Salinomycin was also tested on the expression levels of key genes involved in proliferation and survival signaling and revealed that salinomycin down-regulates the expression of PDGFRβ, MYC, p21 and Bcl-2 as well as up-regulates the expression of cyclin A. In addition, the results reveal that salinomycin suppresses the expression of Hes1 and Hes5 in MB cells. Our data shed light on the potential of using salinomycin as a novel therapeutic agent for patients with MB. PMID:25478603

  3. Notch1-Dll4 signalling and mechanical force regulate leader cell formation during collective cell migration.

    PubMed

    Riahi, Reza; Sun, Jian; Wang, Shue; Long, Min; Zhang, Donna D; Wong, Pak Kin

    2015-03-13

    At the onset of collective cell migration, a subset of cells within an initially homogenous population acquires a distinct 'leader' phenotype with characteristic morphology and motility. However, the factors driving the leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined. Here we use single-cell gene expression analysis and computational modelling to show that the leader cell identity is dynamically regulated by Dll4 signalling through both Notch1 and cellular stress in a migrating epithelium. Time-lapse microscopy reveals that Dll4 is induced in leader cells after the creation of the cell-free region and leader cells are regulated via Notch1-Dll4 lateral inhibition. Furthermore, mechanical stress inhibits Dll4 expression and leader cell formation in the monolayer. Collectively, our findings suggest that a reduction of mechanical force near the boundary promotes Notch1-Dll4 signalling to dynamically regulate the density of leader cells during collective cell migration.

  4. The effects of notch filters on the correlation properties of a PN signal

    NASA Technical Reports Server (NTRS)

    Sussman, S. M.; Ferrari, E. J.

    1974-01-01

    With wideband pseudo-noise (PN) communications systems, it is sometimes desirable to supplement the inherent interference rejection capabilities by adding notch filters to attenuate relatively narrowband interference. This correspondence presents an investigation of the effects of notch filters on the performance of PN correlation receivers. A theoretical analysis of the correlation drop due to filter distortion has been conducted and confirmed by experimentation. Additional measurements and analysis have established the trade-off between correlation drop and interference suppression as a function of interference bandwidth. A typical result is that by incurring a penalty of a 1-dB drop in correlation peak, interfering signals having bandwidths of 2 to 3% of the PN chip rate can be attenuated by 25 dB.

  5. Metabolism and transportation pathways of GDP-fucose that are required for the O-fucosylation of Notch.

    PubMed

    Yamakawa, Tomoko; Ayukawa, Tomonori; Matsuno, Kenji

    2012-01-01

    Notch is a single-pass transmembrane receptor that mediates the local cell-cell interactions necessary for many cell-fate decisions. The extra cellular domain of Notch contains a tandem array of epidermal growth factor-like (EGF-like) repeats. Some of these EGF-like repeats are O-fucosylated by protein O-fucosyltransferase 1 (O-fut1), which is essential for Notch signaling in Drosophila and mouse. This O-fucose is further modified by Fringe, a GlcNAc transferase and other glycosyltransferases (O-fut1 in Drosophila and Pofut1 in mouse), to form an O-linked tetrasaccharide, which modulates Notch's selective binding to its ligands.

  6. Stilbenoids remodel the DNA methylation patterns in breast cancer cells and inhibit oncogenic NOTCH signaling through epigenetic regulation of MAML2 transcriptional activity

    PubMed Central

    Lubecka, Katarzyna; Kurzava, Lucinda; Flower, Kirsty; Buvala, Hannah; Zhang, Hao; Teegarden, Dorothy; Camarillo, Ignacio; Suderman, Matthew; Kuang, Shihuan; Andrisani, Ourania; Flanagan, James M.; Stefanska, Barbara

    2016-01-01

    DNA hypomethylation was previously implicated in cancer progression and metastasis. The purpose of this study was to examine whether stilbenoids, resveratrol and pterostilbene thought to exert anticancer effects, target genes with oncogenic function for de novo methylation and silencing, leading to inactivation of related signaling pathways. Following Illumina 450K, genome-wide DNA methylation analysis reveals that stilbenoids alter DNA methylation patterns in breast cancer cells. On average, 75% of differentially methylated genes have increased methylation, and these genes are enriched for oncogenic functions, including NOTCH signaling pathway. MAML2, a coactivator of NOTCH targets, is methylated at the enhancer region and transcriptionally silenced in response to stilbenoids, possibly explaining the downregulation of NOTCH target genes. The increased DNA methylation at MAML2 enhancer coincides with increased occupancy of repressive histone marks and decrease in activating marks. This condensed chromatin structure is associated with binding of DNMT3B and decreased occupancy of OCT1 transcription factor at MAML2 enhancer, suggesting a role of DNMT3B in increasing methylation of MAML2 after stilbenoid treatment. Our results deliver a novel insight into epigenetic regulation of oncogenic signals in cancer and provide support for epigenetic-targeting strategies as an effective anticancer approach. PMID:27207652

  7. Androgens Up-regulate Transcription of the Notch Inhibitor Numb in C2C12 Myoblasts via Wnt/β-Catenin Signaling to T Cell Factor Elements in the Numb Promoter*

    PubMed Central

    Liu, Xin-Hua; Wu, Yong; Yao, Shen; Levine, Alice C.; Kirschenbaum, Alexander; Collier, Lauren; Bauman, William A.; Cardozo, Christopher P.

    2013-01-01

    Androgen signaling via the androgen receptor is a key pathway that contributes to development, cell fate decisions, and differentiation, including that of myogenic progenitors. Androgens and synthetic steroids have well established anabolic actions on skeletal muscle. Wnt and Notch signaling pathways are also essential to myogenic cell fate decisions during development and tissue repair. However, the interactions among these pathways are largely unknown. Androgenic regulation of Wnt signaling has been reported. Nandrolone, an anabolic steroid, has been shown to inhibit Notch signaling and up-regulate Numb, a Notch inhibitor. To elucidate the mechanisms of interaction between nandrolone and Wnt/Notch signaling, we investigated the effects of nandrolone on Numb expression and Wnt signaling and determined the roles of Wnt signaling in nandrolone-induced Numb expression in C2C12 myoblasts. Nandrolone increased Numb mRNA and protein levels and T cell factor (Tcf) transcriptional activity via inhibition of glycogen synthase kinase 3β. Up-regulation of Numb expression by nandrolone was blocked by the Wnt inhibitors, sFRP1 and DKK1, whereas Wnt3a increased Numb mRNA and protein expression. In addition, we observed that the proximal promoter of the Numb gene had functional Tcf binding elements to which β-catenin was recruited in a manner enhanced by both nandrolone and Wnt3a. Moreover, site-directed mutagenesis indicated that the Tcf binding sites in the Numb promoter are required for the nandrolone-induced Numb transcriptional activation in this cell line. These results reveal a novel molecular mechanism underlying up-regulation of Numb transcription with a critical role for increased canonical Wnt signaling. In addition, the data identify Numb as a novel target gene of the Wnt signaling pathway by which Wnts would be able to inhibit Notch signaling. PMID:23649620

  8. Notch4 Signaling Induces a Mesenchymal-Epithelial-like Transition in Melanoma Cells to Suppress Malignant Behaviors.

    PubMed

    Bonyadi Rad, Ehsan; Hammerlindl, Heinz; Wels, Christian; Popper, Ulrich; Ravindran Menon, Dinoop; Breiteneder, Heimo; Kitzwoegerer, Melitta; Hafner, Christine; Herlyn, Meenhard; Bergler, Helmut; Schaider, Helmut

    2016-04-01

    The effects of Notch signaling are context-dependent and both oncogenic and tumor-suppressive functions have been described. Notch signaling in melanoma is considered oncogenic, but clinical trials testing Notch inhibition in this malignancy have not proved successful. Here, we report that expression of the constitutively active intracellular domain of Notch4 (N4ICD) in melanoma cells triggered a switch from a mesenchymal-like parental phenotype to an epithelial-like phenotype. The epithelial-like morphology was accompanied by strongly reduced invasive, migratory, and proliferative properties concomitant with the downregulation of epithelial-mesenchymal transition markers Snail2 (SNAI2), Twist1, vimentin (VIM), and MMP2 and the reexpression of E-cadherin (CDH1). The N4ICD-induced phenotypic switch also resulted in significantly reduced tumor growth in vivo Immunohistochemical analysis of primary human melanomas and cutaneous metastases revealed a significant correlation between Notch4 and E-cadherin expression. Mechanistically, we demonstrate that N4ICD induced the expression of the transcription factors Hey1 and Hey2, which bound directly to the promoter regions of Snail2 and Twist1 and repressed gene transcription, as determined by EMSA and luciferase assays. Taken together, our findings indicate a role for Notch4 as a tumor suppressor in melanoma, uncovering a potential explanation for the poor clinical efficacy of Notch inhibitors observed in this setting. Cancer Res; 76(7); 1690-7. ©2016 AACR. PMID:26801977

  9. Activation of nuclear PTEN by inhibition of Notch signaling induces G2/M cell cycle arrest in gastric cancer.

    PubMed

    Kim, S-J; Lee, H-W; Baek, J-H; Cho, Y-H; Kang, H G; Jeong, J S; Song, J; Park, H-S; Chun, K-H

    2016-01-14

    Mutation in PTEN has not yet been detected, but its function as a tumor suppressor is inactivated in many cancers. In this study we determined that, activated Notch signaling disables PTEN by phosphorylation and thereby contributes to gastric tumorigenesis. Notch inhibition by small interfering RNA or γ-secretase inhibitor (GSI) induced mitotic arrest and apoptosis in gastric cancer cells. Notch inhibition induced dephosphorylation in the C-terminal domain of PTEN, which led to PTEN nuclear localization. Overexpression of activated Notch1-induced phosphorylation of PTEN and reversed GSI-induced mitotic arrest. Dephosphorylated nuclear PTEN caused prometaphase arrest by interaction with the cyclin B1-CDK1 complex, resulting in their accumulation in the nucleus and subsequent apoptosis. We found a correlation between high expression levels of Notch1 and low survival rates and, similarly, between reduced nuclear PTEN expression and increasing the TNM classification of malignant tumours stages in malignant tissues from gastric cancer patients. The growth of Notch1-depleted gastric tumors was significantly retarded in xenografted mice, and in addition, PTEN deletion restored growth similar to control tumors. We also demonstrated that combination treatment with GSI and chemotherapeutic agents significantly reduced the orthotopically transplanted gastric tumors in mice without noticeable toxicity. Overall, our findings suggest that inhibition of Notch signaling can be employed as a PTEN activator, making it a potential target for gastric cancer therapy.

  10. Effect of Tongxinluo on Nephrin Expression via Inhibition of Notch1/Snail Pathway in Diabetic Rats

    PubMed Central

    Cui, Fangqiang; Zou, Dawei; Gao, Yanbin; Zhang, Na; Wang, Jinyang; Xu, Liping; Geng, Jianguo; Li, Jiaoyang; Zhou, Shengnan; Wang, Xinyao

    2015-01-01

    Podocyte injury is an important mechanism of diabetic nephropathy (DN). Accumulating evidence suggests that nephrin expression is decreased in podocyte in DN. Moreover, it has been demonstrated that tongxinluo (TXL) can ameliorate renal structure disruption and dysfunction in DN. However, the effect of TXL on podocyte injury in DN and its molecular mechanism is unclear. In order to explore the effect of TXL on podocyte injury and its molecular mechanism in DN, our in vivo and in vitro studies were performed. Our results showed that TXL increased nephrin expression in diabetic rats and in high glucose cultured podocyte. Meanwhile, TXL decreased ICN1 (the intracellular domain of notch), HES1, and snail expression in podocyte in vivo and in vitro. More importantly, we found that TXL protected podocyte from injury in DN. The results demonstrated that TXL inhibited the activation of notch1/snail pathway and increased nephrin expression, which may be a mechanism of protecting effect on podocyte injury in DN. PMID:26417374

  11. Two pathways for importing GDP-fucose into the endoplasmic reticulum lumen function redundantly in the O-fucosylation of Notch in Drosophila.

    PubMed

    Ishikawa, Hiroyuki O; Ayukawa, Tomonori; Nakayama, Minoru; Higashi, Shunsuke; Kamiyama, Shin; Nishihara, Shoko; Aoki, Kazuhisa; Ishida, Nobuhiro; Sanai, Yutaka; Matsuno, Kenji

    2010-02-01

    Notch is a transmembrane receptor that shares homology with proteins containing epidermal growth factor-like repeats and mediates the cell-cell interactions necessary for many cell fate decisions. In Drosophila, O-fucosyltransferase 1 catalyzes the O-fucosylation of these epidermal growth factor-like repeats. This O-fucose elongates, resulting in an O-linked tetrasaccharide that regulates the signaling activities of Notch. Fucosyltransferases utilize GDP-fucose, which is synthesized in the cytosol, but fucosylation occurs in the lumen of the endoplasmic reticulum (ER) and Golgi. Therefore, GDP-fucose uptake into the ER and Golgi is essential for fucosylation. However, although GDP-fucose biosynthesis is well understood, the mechanisms and intracellular routes of GDP-fucose transportation remain unclear. Our previous study on the Drosophila Golgi GDP-fucose transporter (Gfr), which specifically localizes to the Golgi, suggested that another GDP-fucose transporter(s) exists in Drosophila. Here, we identified Efr (ER GDP-fucose transporter), a GDP-fucose transporter that localizes specifically to the ER. Efr is a multifunctional nucleotide sugar transporter involved in the biosynthesis of heparan sulfate-glycosaminoglycan chains and the O-fucosylation of Notch. Comparison of the fucosylation defects in the N-glycans in Gfr and Efr mutants revealed that Gfr and Efr made distinct contributions to this modification; Gfr but not Efr was crucial for the fucosylation of N-glycans. We also found that Gfr and Efr function redundantly in the O-fucosylation of Notch, although they had different localizations and nucleotide sugar transportation specificities. These results indicate that two pathways for the nucleotide sugar supply, involving two nucleotide sugar transporters with distinct characteristics and distributions, contribute to the O-fucosylation of Notch. PMID:19948734

  12. Endothelial cells downregulate apolipoprotein D expression in mural cells through paracrine secretion and Notch signaling

    PubMed Central

    Pajaniappan, Mohanasundari; Glober, Nancy K.; Kennard, Simone; Liu, Hua; Zhao, Ning

    2011-01-01

    Endothelial and mural cell interactions are vitally important for proper formation and function of blood vessels. These two cell types communicate to regulate multiple aspects of vessel function. In studying genes regulated by this interaction, we identified apolipoprotein D (APOD) as one gene that is downregulated in mural cells by coculture with endothelial cells. APOD is a secreted glycoprotein that has been implicated in governing stress response, lipid metabolism, and aging. Moreover, APOD is known to regulate smooth muscle cells and is found in abundance within atherosclerotic lesions. Our data show that the regulation of APOD in mural cells is bimodal. Paracrine secretion by endothelial cells causes partial downregulation of APOD expression. Additionally, cell contact-dependent Notch signaling plays a role. NOTCH3 on mural cells promotes the downregulation of APOD, possibly through interaction with the JAGGED-1 ligand on endothelial cells. Our results show that NOTCH3 contributes to the downregulation of APOD and by itself is sufficient to attenuate APOD transcript expression. In examining the consequence of decreased APOD expression in mural cells, we show that APOD negatively regulates cell adhesion. APOD attenuates adhesion by reducing focal contacts; however, it has no effect on stress fiber formation. These data reveal a novel mechanism in which endothelial cells control neighboring mural cells through the downregulation of APOD, which, in turn, influences mural cell function by modulating adhesion. PMID:21705670

  13. Endothelial cells downregulate apolipoprotein D expression in mural cells through paracrine secretion and Notch signaling.

    PubMed

    Pajaniappan, Mohanasundari; Glober, Nancy K; Kennard, Simone; Liu, Hua; Zhao, Ning; Lilly, Brenda

    2011-09-01

    Endothelial and mural cell interactions are vitally important for proper formation and function of blood vessels. These two cell types communicate to regulate multiple aspects of vessel function. In studying genes regulated by this interaction, we identified apolipoprotein D (APOD) as one gene that is downregulated in mural cells by coculture with endothelial cells. APOD is a secreted glycoprotein that has been implicated in governing stress response, lipid metabolism, and aging. Moreover, APOD is known to regulate smooth muscle cells and is found in abundance within atherosclerotic lesions. Our data show that the regulation of APOD in mural cells is bimodal. Paracrine secretion by endothelial cells causes partial downregulation of APOD expression. Additionally, cell contact-dependent Notch signaling plays a role. NOTCH3 on mural cells promotes the downregulation of APOD, possibly through interaction with the JAGGED-1 ligand on endothelial cells. Our results show that NOTCH3 contributes to the downregulation of APOD and by itself is sufficient to attenuate APOD transcript expression. In examining the consequence of decreased APOD expression in mural cells, we show that APOD negatively regulates cell adhesion. APOD attenuates adhesion by reducing focal contacts; however, it has no effect on stress fiber formation. These data reveal a novel mechanism in which endothelial cells control neighboring mural cells through the downregulation of APOD, which, in turn, influences mural cell function by modulating adhesion.

  14. Pharmacology of intracellular signalling pathways

    PubMed Central

    Nahorski, Stefan R

    2006-01-01

    This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca2+ is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future. PMID:16402119

  15. Notch 2 signaling contributes to cell growth, anti-apoptosis and metastasis in laryngeal squamous cell carcinoma

    PubMed Central

    Zou, You; Fang, Fang; Ding, Yong-Jun; Dai, Meng-Yuan; Yi, Xing; Chen, Chen; Tao, Ze-Zhang; Chen, Shi-Ming

    2016-01-01

    Notch signaling is important during the development of a variety of human tumors. Depending on the context, Notch signaling can be either oncogenic or anti-proliferative, and therefore, its effects in cancer are unpredictable. The aim of the present study was to identify the importance of Notch 2 in the cell growth and metastasis of laryngeal squamous cell carcinoma (LSCC). The current study performed quantum dots-based immunofluorescence histochemistry to determine expression of Notch 2 in 72 LSCC samples without lymph node metastasis, 23 LSCC samples with lymph node metastasis and 31 samples from vocal cord polyps. It was observed that Notch 2 was upregulated in LSCC tissue compared with normal vocal cord polyps. This upregulation was further enhanced in LSCC tissues with lymph node metastasis compared with LSCC tissues without lymph node metastasis. Following knockdown of NOTCH2 expression in LSCC cells, the in vitro tumorigenicity of Hep-2 cells was inhibited, with growth, migration, invasion and proliferation reduced, and apoptosis induced. Additionally, following downregulation of Notch 2 protein expression, the protein expression levels of phosphor-mitogen-activated protein kinase 1 (p-ERK), v-myc avian myelocytomatosis viral oncogene homolog and B-cell CLL/lymphoma 2 (Bcl2) were also downregulated, whereas, Bcl2-associated X protein expression was upregulated. There were no changes detected in the protein expression levels of total-ERK, phospho-v-akt murine thymoma viral oncogene homolog 1 (p-Akt) and total-Akt. The results of the present study suggest that Notch 2 is important for the cell growth, anti-apoptosis and metastasis of LSCC. Therefore, Notch 2 inhibitors may have therapeutic potential for the treatment of patients with LSCC via the inhibition of cancer cell growth and metastasis. PMID:27572051

  16. Signaling Pathways in Cartilage Repair

    PubMed Central

    Mariani, Erminia; Pulsatelli, Lia; Facchini, Andrea

    2014-01-01

    In adult healthy cartilage, chondrocytes are in a quiescent phase characterized by a fine balance between anabolic and catabolic activities. In ageing, degenerative joint diseases and traumatic injuries of cartilage, a loss of homeostatic conditions and an up-regulation of catabolic pathways occur. Since cartilage differentiation and maintenance of homeostasis are finely tuned by a complex network of signaling molecules and biophysical factors, shedding light on these mechanisms appears to be extremely relevant for both the identification of pathogenic key factors, as specific therapeutic targets, and the development of biological approaches for cartilage regeneration. This review will focus on the main signaling pathways that can activate cellular and molecular processes, regulating the functional behavior of cartilage in both physiological and pathological conditions. These networks may be relevant in the crosstalk among joint compartments and increased knowledge in this field may lead to the development of more effective strategies for inducing cartilage repair. PMID:24837833

  17. PlexinD1 Is a Novel Transcriptional Target and Effector of Notch Signaling in Cancer Cells

    PubMed Central

    Rehman, Michael; Capparuccia, Lorena

    2016-01-01

    The secreted semaphorin Sema3E controls cell migration and invasiveness in cancer cells. Sema3E-receptor, PlexinD1, is frequently upregulated in melanoma, breast, colon, ovarian and prostate cancers; however, the mechanisms underlying PlexinD1 upregulation and the downstream events elicited in tumor cells are still unclear. Here we show that the canonical RBPjk-dependent Notch signaling cascade controls PlexinD1 expression in primary endothelial and cancer cells. Transcriptional activation was studied by quantitative PCR and promoter activity reporter assays. We found that Notch ligands and constitutively activated intracellular forms of Notch receptors upregulated PlexinD1 expression; conversely RNAi-based knock-down, or pharmacological inhibition of Notch signaling by gamma-secretase inhibitors, downregulated PlexinD1 levels. Notably, both Notch1 and Notch3 expression positively correlates with PlexinD1 levels in prostate cancer, as well as in other tumor types. In prostate cancer cells, Sema3E-PlexinD1 axis was previously reported to regulate migration; however, implicated mechanisms were not elucidated. Here we show that in these cells PlexinD1 activity induces the expression of the transcription factor Slug, downregulates E-cadherin levels and enhances cell migration. Moreover, our mechanistic data identify PlexinD1 as a pivotal mediator of this signaling axis downstream of Notch in prostate cancer cells. In fact, on one hand, PlexinD1 is required to mediate cell migration and E-cadherin regulation elicited by Notch. On the other hand, PlexinD1 upregulation is sufficient to induce prostate cancer cell migration and metastatic potential in mice, leading to functional rescue in the absence of Notch. In sum, our work identifies PlexinD1 as a novel transcriptional target induced by Notch signaling, and reveals its role promoting prostate cancer cell migration and downregulating E-cadherin levels in Slug-dependent manner. Collectively, these findings suggest that

  18. Quantum dot nanoprobe-based high-content monitoring of notch pathway inhibition of breast cancer stem cell by capsaicin.

    PubMed

    Shim, Yumi; Song, Joon Myong

    2015-12-01

    Breast cancer is the major cause of cancer death for women worldwide. Breast cancer patients are treated with chemotherapy and radiotherapy. Although chemotherapy and radiotherapy are applied, some cancer cells still survive. These cells, called cancer stem cell (CSC), exhibit special capabilities, such as drug and radio resistance. The remaining CSC can trigger cancer recurrence. Thus, it is critical to find an effective way to target CSC. Capsaicin has been reported to affect anticancer activity in many cancers. It also has been shown that capsaicin induces apoptosis in the MCF-7 breast cancer cell line. In this study, we demonstrate that capsaicin causes dose-dependent growth disruption in breast CSC and inhibits translocation of notch intracellular membrane domain (NICD) into the nucleus. MCF-7 cells were treated with capsaicin at various concentrations (5 μM, 10 μM, and 20 μM) for 24 h. After capsaicin treatment, it was found that the number of breast CSC (%) decreased as the treatment concentration of capsaicin increased. This result was also confirmed with FACS. NICD translocation to the nucleus and apoptotic cell death of breast CSC were concurrently observed at the single breast CSC level using highly sensitive quantum dot (Qdot)-antibody nanoprobes. The control breast CSCs without the capsaicin treatment were able to translocate NICD into the nucleus. On the other hand, translocation of NICD into the nucleus was not observed in capsaicin-treated cells. In addition, apoptotic cell death was caused when the breast CSC were treated with capsaicin at more than 10 μM. Although many studies have shown that capsaicin produces anticancer activity in cancer cell lines, the present result is the first report to demonstrate that capsaicin is capable of causing breast CSC apoptotic cell death via inhibiting its notch signaling pathway.

  19. Myeloid-Specific Blockade of Notch Signaling Attenuates Choroidal Neovascularization through Compromised Macrophage Infiltration and Polarization in Mice

    PubMed Central

    Dou, Guo-Rui; Li, Na; Chang, Tian-Fang; Zhang, Ping; Gao, Xiang; Yan, Xian-Chun; Liang, Liang; Han, Hua; Wang, Yu-Sheng

    2016-01-01

    Macrophages have been recognized as an important inflammatory component in choroidal neovascularization (CNV). However, it is unclear how these cells are activated and polarized, how they affect angiogenesis and what the underlining mechanisms are during CNV. Notch signaling has been implicated in macrophage activation. Previously we have shown that inducible disruption of RBP-J, the critical transcription factor of Notch signaling, in adult mice results in enhanced CNV, but it is unclear what is the role of macrophage-specific Notch signaling in the development of CNV. In the current study, by using the myeloid specific RBP-J knockout mouse model combined with the laser-induced CNV model, we show that disruption of Notch signaling in macrophages displayed attenuated CNV growth, reduced macrophage infiltration and activation, and alleviated angiogenic response after laser induction. The inhibition of CNV occurred with reduced expression of VEGF and TNF-α in infiltrating inflammatory macrophages in myeloid specific RBP-J knockout mice. These changes might result in direct inhibition of EC lumen formation, as shown in an in vitro study. Therefore, clinical intervention of Notch signaling in CNV needs to pinpoint myeloid lineage to avoid the counteractive effects of global inhibition. PMID:27339903

  20. Notch1–STAT3–ETBR signaling axis controls reactive astrocyte proliferation after brain injury

    PubMed Central

    LeComte, Matthew D.; Shimada, Issei S.; Sherwin, Casey; Spees, Jeffrey L.

    2015-01-01

    Defining the signaling network that controls reactive astrogliosis may provide novel treatment targets for patients with diverse CNS injuries and pathologies. We report that the radial glial cell antigen RC2 identifies the majority of proliferating glial fibrillary acidic protein-positive (GFAP+) reactive astrocytes after stroke. These cells highly expressed endothelin receptor type B (ETBR) and Jagged1, a Notch1 receptor ligand. To study signaling in adult reactive astrocytes, we developed a model based on reactive astrocyte-derived neural stem cells isolated from GFAP-CreER-Notch1 conditional knockout (cKO) mice. By loss- and gain-of-function studies and promoter activity assays, we found that Jagged1/Notch1 signaling increased ETBR expression indirectly by raising the level of phosphorylated signal transducer and activator of transcription 3 (STAT3), a previously unidentified EDNRB transcriptional activator. Similar to inducible transgenic GFAP-CreER-Notch1-cKO mice, GFAP-CreER-ETBR-cKO mice exhibited a defect in reactive astrocyte proliferation after cerebral ischemia. Our results indicate that the Notch1–STAT3–ETBR axis connects a signaling network that promotes reactive astrocyte proliferation after brain injury. PMID:26124113

  1. BCAS2 Regulates Delta-Notch Signaling Activity through Delta Pre-mRNA Splicing in Drosophila Wing Development

    PubMed Central

    Huang, Chu-Wei; Chen, Po-Han; Chan, Shih-Peng; Tsao, Yeou-Ping; Lee, Hsiu-Hsiang; Wu, June-Tai; Chen, Show-Li

    2015-01-01

    Previously, we showed that BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing. In this study, we provide strong evidence that BCAS2 regulates the activity of Delta-Notch signaling via Delta pre-mRNA splicing. Depletion of dBCAS2 reduces Delta mRNA expression and leads to accumulation of Delta pre-mRNA, resulting in diminished transcriptions of Delta-Notch signaling target genes, such as cut and E(spl)m8. Furthermore, ectopic expression of human BCAS2 (hBCAS2) and Drosophila BCAS2 (dBCAS2) in a dBCAS2-deprived fly can rescue dBCAS2 depletion-induced wing damage to the normal phenotypes. These rescued phenotypes are correlated with the restoration of Delta pre-mRNA splicing, which affects Delta-Notch signaling activity. Additionally, overexpression of Delta can rescue the wing deformation by deprivation of dBCAS2; and the depletion of dBCAS2 can restore the aberrant eye associated with Delta-overexpressing retinas; providing supporting evidence for the regulation of Delta-Notch signaling by dBCAS2. Taken together, dBCAS2 participates in Delta pre-mRNA splicing that affects the regulation of Delta-Notch signaling in Drosophila wing development. PMID:26091239

  2. Preclinical profile of a potent gamma-secretase inhibitor targeting notch signaling with in vivo efficacy and pharmacodynamic properties.

    PubMed

    Luistro, Leopoldo; He, Wei; Smith, Melissa; Packman, Kathryn; Vilenchik, Maria; Carvajal, Daisy; Roberts, John; Cai, James; Berkofsky-Fessler, Windy; Hilton, Holly; Linn, Michael; Flohr, Alexander; Jakob-Røtne, Roland; Jacobsen, Helmut; Glenn, Kelli; Heimbrook, David; Boylan, John F

    2009-10-01

    Notch signaling is an area of great interest in oncology. RO4929097 is a potent and selective inhibitor of gamma-secretase, producing inhibitory activity of Notch signaling in tumor cells. The RO4929097 IC50 in cell-free and cellular assays is in the low nanomolar range with >100-fold selectivity with respect to 75 other proteins of various types (receptors, ion channels, and enzymes). RO4929097 inhibits Notch processing in tumor cells as measured by the reduction of intracellular Notch expression by Western blot. This leads to reduced expression of the Notch transcriptional target gene Hes1. RO4929097 does not block tumor cell proliferation or induce apoptosis but instead produces a less transformed, flattened, slower-growing phenotype. RO4929097 is active following oral dosing. Antitumor activity was shown in 7 of 8 xenografts tested on an intermittent or daily schedule in the absence of body weight loss or Notch-related toxicities. Importantly, efficacy is maintained after dosing is terminated. Angiogenesis reverse transcription-PCR array data show reduced expression of several key angiogenic genes. In addition, comparative microarray analysis suggests tumor cell differentiation as an additional mode of action. These preclinical results support evaluation of RO4929097 in clinical studies using an intermittent dosing schedule. A multicenter phase I dose escalation study in oncology is under way. PMID:19773430

  3. Preclinical profile of a potent gamma-secretase inhibitor targeting notch signaling with in vivo efficacy and pharmacodynamic properties.

    PubMed

    Luistro, Leopoldo; He, Wei; Smith, Melissa; Packman, Kathryn; Vilenchik, Maria; Carvajal, Daisy; Roberts, John; Cai, James; Berkofsky-Fessler, Windy; Hilton, Holly; Linn, Michael; Flohr, Alexander; Jakob-Røtne, Roland; Jacobsen, Helmut; Glenn, Kelli; Heimbrook, David; Boylan, John F

    2009-10-01

    Notch signaling is an area of great interest in oncology. RO4929097 is a potent and selective inhibitor of gamma-secretase, producing inhibitory activity of Notch signaling in tumor cells. The RO4929097 IC50 in cell-free and cellular assays is in the low nanomolar range with >100-fold selectivity with respect to 75 other proteins of various types (receptors, ion channels, and enzymes). RO4929097 inhibits Notch processing in tumor cells as measured by the reduction of intracellular Notch expression by Western blot. This leads to reduced expression of the Notch transcriptional target gene Hes1. RO4929097 does not block tumor cell proliferation or induce apoptosis but instead produces a less transformed, flattened, slower-growing phenotype. RO4929097 is active following oral dosing. Antitumor activity was shown in 7 of 8 xenografts tested on an intermittent or daily schedule in the absence of body weight loss or Notch-related toxicities. Importantly, efficacy is maintained after dosing is terminated. Angiogenesis reverse transcription-PCR array data show reduced expression of several key angiogenic genes. In addition, comparative microarray analysis suggests tumor cell differentiation as an additional mode of action. These preclinical results support evaluation of RO4929097 in clinical studies using an intermittent dosing schedule. A multicenter phase I dose escalation study in oncology is under way.

  4. Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and β-catenin signaling

    PubMed Central

    Bannai, Yuka; Aminova, Leila R.; Faulkner, Melinda J.; Ho, Mengfei; Wilson, Brenda A.

    2012-01-01

    The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric Gq, Gi, and G12/13 proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of β-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and β-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G12/13 signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on Wnt/β-catenin and Notch1 signaling, and in particular the role of Gq and G12/13 in mediating PMT effects on Rho/ROCK and Notch1 signaling. PMID:22919671

  5. Association of Notch pathway down-regulation with Triple Negative/Basal-like breast carcinomas and high tumor-infiltrating FOXP3+ Tregs.

    PubMed

    Ortiz-Martínez, Fernando; Gutiérrez-Aviñó, Francisco José; Sanmartín, Elena; Pomares-Navarro, Eloy; Villalba-Riquelme, Cristina; García-Martínez, Araceli; Lerma, Enrique; Peiró, Gloria

    2016-06-01

    T regulatory cells (Tregs) are a lineage of lymphocytes involved in immune response suppression that are characterized by the expression of the forkhead box P3 (FOXP3) transcription factor. Notch pathway regulates FOXP3 transcription in Tregs, but its role in breast cancer is unknown. We aimed at studying whether Notch pathway regulates FOXP3 expression and Tregs content in breast cancer, and its association with luminal breast carcinomas. We analyzed by quantitative Real-Time PCR the mRNA levels of FOXP3, Notch pathway genes (Notch1, Notch2, Notch4 and Jagged1) and STAT3 in a series of 152 breast carcinomas including hormone receptor-positive and -negative phenotypes (luminal and Triple Negative/Basal-like). We also studied the protein expression of Notch1, STAT3 and FOXP3 by immunohistochemistry. High FOXP3 mRNA levels correlated with larger tumor size (p=0.010), histological grade 3 (p=0.008) and positive lymph-node status (p=0.031). Also, low levels of Notch pathway genes mRNA correlated with poor prognostic factors such as larger tumor size, positive lymph-node status, tumor phenotype and infiltrating tumor Tregs. A survival analysis for the patients showed that large tumor size, histological grade 3, vascular invasion, infiltrating Tregs and low Notch1 mRNA expression were significantly associated with a decreased patients' overall survival (p≤0.05). On a multivariate analysis, high Tregs content (HR=3.00, 95% CI 1.04-8.90, p=0.042) and low Notch1 mRNA levels (HR=3.33, 95% CI 1.02-10.86, p=0.046) were independent markers for overall survival. Our results support that the Notch pathway up-regulation promotes luminal breast carcinomas, whereas down-regulation correlates with the expression of FOXP3, favors tumor Tregs infiltration and associates with Triple Negative/Basal-like tumors. PMID:27118257

  6. Nox2 contributes to the arterial endothelial specification of mouse induced pluripotent stem cells by upregulating Notch signaling

    PubMed Central

    Kang, Xueling; Wei, Xiangxiang; Wang, Xinhong; Jiang, Li; Niu, Cong; Zhang, Jianyi; Chen, Sifeng; Meng, Dan

    2016-01-01

    Reactive oxygen species (ROS) have a crucial role in stem-cell differentiation; however, the mechanisms by which ROS regulate the differentiation of stem cells into endothelial cells (ECs) are unknown. Here, we determine the role of ROS produced by NADPH oxidase 2 (Nox2) in the endothelial-lineage specification of mouse induced-pluripotent stem cells (miPSCs). When wild-type (WT) and Nox2-knockout (Nox2−/−) miPSCs were differentiated into ECs (miPSC-ECs), the expression of endothelial markers, arterial endothelial markers, pro-angiogenic cytokines, and Notch pathway components was suppressed in the Nox2−/− cells but increased in both WT and Nox2−/− miPSCs when Nox2 expression was upregulated. Higher levels of Nox2 expression increased Notch signaling and arterial EC differentiation, and this increase was abolished by the inhibition of ROS generation or by the silencing of Notch1 expression. Nox2 deficiency was associated with declines in the survival and angiogenic potency of miPSC-ECs, and capillary and arterial density were lower in the ischemic limbs of mice after treatment with Nox2−/− miPSC-ECs than WT miPSC-EC treatment. Taken together, these observations indicate that Nox2-mediated ROS production promotes arterial EC specification in differentiating miPSCs by activating the Notch signaling pathway and contributes to the angiogenic potency of transplanted miPSC-derived ECs. PMID:27642005

  7. Cis-interactions between Notch and its ligands block ligand-independent Notch activity

    PubMed Central

    Palmer, William Hunt; Jia, Dongyu; Deng, Wu-Min

    2014-01-01

    The Notch pathway is integrated into numerous developmental processes and therefore is fine-tuned on many levels, including receptor production, endocytosis, and degradation. Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling. We show that cells without both cis- and trans-ligands can mediate Notch-dependent developmental events during Drosophila oogenesis, indicating ligand-independent Notch activity occurs when the receptor is free of cis- and trans-ligands. Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling. We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity. DOI: http://dx.doi.org/10.7554/eLife.04415.001 PMID:25486593

  8. RIPping notch apart: a new role for endocytosis in signal transduction?

    PubMed

    Krämer, H

    2000-04-25

    Notch proteins are receptors that are important in mediating several developmental processes. Notch receptors are activated upon binding transmembrane ligands, the DSL proteins. Notch is cleaved at several sites and activation of Notch leads to the cleavage of the intracellular domain, which then is translocated to the nucleus and regulates the transcription of target genes. Krämer discusses how binding of Notch to the DSL ligand, Delta, leads to cleavage and trans-endocytosis of the Notch extracellular domain into the Delta-expressing cell. This trans-endocytosis event contributes to the cleavage and release of the active Notch intracellular domain. The Perspective is accompanied by a movie illustrating the trans-endocytosis of Notch.

  9. Inhibition of Notch signaling by Dll4-Fc promotes reperfusion of acutely ischemic tissues

    SciTech Connect

    Liu, Ren; Trindade, Alexandre; Sun, Zhanfeng; Kumar, Ram; Weaver, Fred A.; Krasnoperov, Valery; Naga, Kranthi; Duarte, Antonio; Gill, Parkash S.

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer Low dose Dll4-Fc increases vascular proliferation and overall perfusion. Black-Right-Pointing-Pointer Low dose Dll4-Fc helps vascular injury recovery in hindlimb ischemia model. Black-Right-Pointing-Pointer Low dose Dll4-Fc helps vascular injury recovery in skin flap model. Black-Right-Pointing-Pointer Dll4 heterozygous deletion promotes vascular injury recovery. Black-Right-Pointing-Pointer Dll4 overexpression delays vascular injury recovery. -- Abstract: Notch pathway regulates vessel development and maturation. Dll4, a high-affinity ligand for Notch, is expressed predominantly in the arterial endothelium and is induced by hypoxia among other factors. Inhibition of Dll4 has paradoxical effects of reducing the maturation and perfusion in newly forming vessels while increasing the density of vessels. We hypothesized that partial and/or intermittent inhibition of Dll4 may lead to increased vascular response and still allow vascular maturation to occur. Thus tissue perfusion can be restored rapidly, allowing quicker recovery from ischemia or tissue injury. Our studies in two different models (hindlimb ischemia and skin flap) show that inhibition of Dll4 at low dose allows faster recovery from vascular and tissue injury. This opens a new possibility for Dll4 blockade's therapeutic application in promoting recovery from vascular injury and restoring blood supply to ischemic tissues.

  10. Integration of TGF-β/Smad and Jagged1/Notch signalling in epithelial-to-mesenchymal transition

    PubMed Central

    Zavadil, Jiri; Cermak, Lukas; Soto-Nieves, Noemi; Böttinger, Erwin P

    2004-01-01

    Epithelial-to-mesenchymal transitions (EMTs) underlie cell plasticity required in embryonic development and frequently observed in advanced carcinogenesis. Transforming growth factor-β (TGF-β) induces EMT phenotypes in epithelial cells in vitro and has been associated with EMT in vivo. Here we report that expression of the hairy/enhancer-of-split-related transcriptional repressor Hey1, and the Notch-ligand Jagged1 (Jag1), was induced by TGF-β at the onset of EMT in epithelial cells from mammary gland, kidney tubules, and epidermis. The HEY1 expression profile was biphasic, consisting of immediate-early Smad3-dependent, Jagged1/Notch-independent activation, followed by delayed, indirect Jagged1/Notch-dependent activation. TGF-β-induced EMT was blocked by RNA silencing of HEY1 or JAG1, and by chemical inactivation of Notch. The EMT phenotype, biphasic activation of Hey1, and delayed expression of Jag1 were induced by TGF-β in wild-type, but not in Smad3-deficient, primary mouse kidney tubular epithelial cells. Our findings identify a new mechanism for functional integration of Jagged1/Notch signalling and coordinated activation of the Hey1 transcriptional repressor controlled by TGF-β/Smad3, and demonstrate functional roles for Smad3, Hey1, and Jagged1/Notch in mediating TGF-β-induced EMT. PMID:14976548

  11. Mastermind-like transcriptional co-activator-mediated Notch signaling is indispensable for maintaining conjunctival epithelial identity

    PubMed Central

    Zhang, Yujin; Lam, Oliver; Nguyen, Minh-Thanh T.; Ng, Gracia; Pear, Warren S.; Ai, Walden; Wang, I-Jong; Kao, Winston W.-Y.; Liu, Chia-Yang

    2013-01-01

    Conjunctival goblet cells primarily synthesize mucins to lubricate the ocular surface, which is essential for normal vision. Notch signaling has been known to associate with goblet cell differentiation in intestinal and respiratory tracts, but its function in ocular surface has yet to be fully characterized. Herein, we demonstrate that conditional inhibition of canonical Notch signaling by expressing dominant negative mastermind-like 1 (dnMaml1) in ocular surface epithelia resulted in complete suppression of goblet cell differentiation during and subsequent to development. When compared with the ocular surface of wild-type mice (OSWt), expression of dnMaml1 at the ocular surface (OSdnMaml1) caused conjunctival epithelial hyperplasia, aberrant desquamation, failure of Mucin 5ac (Muc5ac) synthesis, subconjunctival inflammation and epidermal metaplasia in cornea. In addition, conditional deletion of Notch1 from the ocular surface epithelia partially recapitulated OSdnMaml1 phenotypes. We have demonstrated that N1-ICD (Notch1 intracellular domain) transactivated the mouse Krüppel-like factor 4 (Klf) promoter and that Klf4 directly bound to and significantly potentiated the Muc5ac promoter. By contrast, OSdnMaml1 dampened Klf4 and Klf5 expression, and diminished Muc5ac synthesis. Collectively, these findings indicated that Maml-mediated Notch signaling plays a pivotal role in the initiation and maintenance of goblet cell differentiation for normal ocular surface morphogenesis and homeostasis through regulation of Klf4 and Klf5. PMID:23293291

  12. Current perspectives of the signaling pathways directing neural crest induction.

    PubMed

    Stuhlmiller, Timothy J; García-Castro, Martín I

    2012-11-01

    The neural crest is a migratory population of embryonic cells with a tremendous potential to differentiate and contribute to nearly every organ system in the adult body. Over the past two decades, an incredible amount of research has given us a reasonable understanding of how these cells are generated. Neural crest induction involves the combinatorial input of multiple signaling pathways and transcription factors, and is thought to occur in two phases from gastrulation to neurulation. In the first phase, FGF and Wnt signaling induce NC progenitors at the border of the neural plate, activating the expression of members of the Msx, Pax, and Zic families, among others. In the second phase, BMP, Wnt, and Notch signaling maintain these progenitors and bring about the expression of definitive NC markers including Snail2, FoxD3, and Sox9/10. In recent years, additional signaling molecules and modulators of these pathways have been uncovered, creating an increasingly complex regulatory network. In this work, we provide a comprehensive review of the major signaling pathways that participate in neural crest induction, with a focus on recent developments and current perspectives. We provide a simplified model of early neural crest development and stress similarities and differences between four major model organisms: Xenopus, chick, zebrafish, and mouse. PMID:22547091

  13. Current perspectives of the signaling pathways directing neural crest induction.

    PubMed

    Stuhlmiller, Timothy J; García-Castro, Martín I

    2012-11-01

    The neural crest is a migratory population of embryonic cells with a tremendous potential to differentiate and contribute to nearly every organ system in the adult body. Over the past two decades, an incredible amount of research has given us a reasonable understanding of how these cells are generated. Neural crest induction involves the combinatorial input of multiple signaling pathways and transcription factors, and is thought to occur in two phases from gastrulation to neurulation. In the first phase, FGF and Wnt signaling induce NC progenitors at the border of the neural plate, activating the expression of members of the Msx, Pax, and Zic families, among others. In the second phase, BMP, Wnt, and Notch signaling maintain these progenitors and bring about the expression of definitive NC markers including Snail2, FoxD3, and Sox9/10. In recent years, additional signaling molecules and modulators of these pathways have been uncovered, creating an increasingly complex regulatory network. In this work, we provide a comprehensive review of the major signaling pathways that participate in neural crest induction, with a focus on recent developments and current perspectives. We provide a simplified model of early neural crest development and stress similarities and differences between four major model organisms: Xenopus, chick, zebrafish, and mouse.

  14. Pattern formation: a focus on notch in butterfly eyespots.

    PubMed

    French, Vernon; Brakefield, Paul M

    2004-08-24

    New observations of early and dynamic expression of Notch in developing lepidopteran wings suggests that this signalling pathway may function in defining the central focus that will specify the butterfly eyespot colour pattern. PMID:15324685

  15. Pattern formation: a focus on notch in butterfly eyespots.

    PubMed

    French, Vernon; Brakefield, Paul M

    2004-08-24

    New observations of early and dynamic expression of Notch in developing lepidopteran wings suggests that this signalling pathway may function in defining the central focus that will specify the butterfly eyespot colour pattern.

  16. A self-organizing miR-132/Ctbp2 circuit regulates bimodal notch signals and glial progenitor fate choice during spinal cord maturation.

    PubMed

    Salta, Evgenia; Lau, Pierre; Sala Frigerio, Carlo; Coolen, Marion; Bally-Cuif, Laure; De Strooper, Bart

    2014-08-25

    Radial glial progenitors play pivotal roles in the development and patterning of the spinal cord, and their fate is controlled by Notch signaling. How Notch is shaped to regulate their crucial transition from expansion toward differentiation remains, however, unknown. miR-132 in the developing zebrafish dampens Notch signaling via a cascade involving the transcriptional corepressor Ctbp2 and the Notch suppressor Sirt1. At early embryonic stages, high Ctbp2 levels sustain Notch signaling and radial glial expansion and concomitantly induce miR-132 expression via a double-negative feedback loop involving Rest inhibition. The changing balance in miR-132 and Ctbp2 interaction gradually drives the switch in Notch output and radial glial progenitor fate as part of the larger developmental program involved in the transition from embryonic to larval spinal cord.

  17. Regulation of a Notch3-Hes1 pathway and protective effect by a tocopherol-omega alkanol chain derivative in muscle atrophy.

    PubMed

    von Grabowiecki, Yannick; Licona, Cynthia; Palamiuc, Lavinia; Abreu, Paula; Vidimar, Vania; Coowar, Djalil; Mellitzer, Georg; Gaiddon, Christian

    2015-01-01

    Muscular atrophy, a physiopathologic process associated with severe human diseases such as amyotrophic lateral sclerosis (ALS) or cancer, has been linked to reactive oxygen species (ROS) production. The Notch pathway plays a role in muscle development and in muscle regeneration upon physical injury. In this study, we explored the possibility that the Notch pathway participates in the ROS-related muscular atrophy occurring in cancer-associated cachexia and ALS. We also tested whether hybrid compounds of tocopherol, harboring antioxidant activity, and the omega-alkanol chain, presenting cytoprotective activity, might reduce muscle atrophy and impact the Notch pathway. We identified one tocopherol-omega alkanol chain derivative, AGT251, protecting myoblastic cells against known cytotoxic agents. We showed that this compound presenting antioxidant activity counteracts the induction of the Notch pathway by cytotoxic stress, leading to a decrease of Notch1 and Notch3 expression. At the functional level, these regulations correlated with a repression of the Notch target gene Hes1 and the atrophy/remodeling gene MuRF1. Importantly, we also observed an induction of Notch3 and Hes1 expression in two murine models of muscle atrophy: a doxorubicin-induced cachexia model and an ALS murine model expressing mutated superoxide dismutase 1. In both models, the induction of Notch3 and Hes1 were partially opposed by AGT251, which correlated with ameliorations in body and muscle weight, reduction of muscular atrophy markers, and improved survival. Altogether, we identified a compound of the tocopherol family that protects against muscle atrophy in various models, possibly through the regulation of the Notch pathway.

  18. Regulation of a Notch3-Hes1 pathway and protective effect by a tocopherol-omega alkanol chain derivative in muscle atrophy.

    PubMed

    von Grabowiecki, Yannick; Licona, Cynthia; Palamiuc, Lavinia; Abreu, Paula; Vidimar, Vania; Coowar, Djalil; Mellitzer, Georg; Gaiddon, Christian

    2015-01-01

    Muscular atrophy, a physiopathologic process associated with severe human diseases such as amyotrophic lateral sclerosis (ALS) or cancer, has been linked to reactive oxygen species (ROS) production. The Notch pathway plays a role in muscle development and in muscle regeneration upon physical injury. In this study, we explored the possibility that the Notch pathway participates in the ROS-related muscular atrophy occurring in cancer-associated cachexia and ALS. We also tested whether hybrid compounds of tocopherol, harboring antioxidant activity, and the omega-alkanol chain, presenting cytoprotective activity, might reduce muscle atrophy and impact the Notch pathway. We identified one tocopherol-omega alkanol chain derivative, AGT251, protecting myoblastic cells against known cytotoxic agents. We showed that this compound presenting antioxidant activity counteracts the induction of the Notch pathway by cytotoxic stress, leading to a decrease of Notch1 and Notch3 expression. At the functional level, these regulations correlated with a repression of the Notch target gene Hes1 and the atrophy/remodeling gene MuRF1. Importantly, we also observed an induction of Notch3 and Hes1 expression in two murine models of muscle atrophy: a doxorubicin-induced cachexia model and an ALS murine model expressing mutated superoxide dismutase 1. In both models, the induction of Notch3 and Hes1 were partially opposed by AGT251, which correlated with ameliorations in body and muscle weight, reduction of muscular atrophy markers, and improved survival. Altogether, we identified a compound of the tocopherol family that protects against muscle atrophy in various models, possibly through the regulation of the Notch pathway. PMID:25326132

  19. Notch Signaling Coordinates Progenitor Cell-Mediated Biliary Regeneration Following Partial Hepatectomy

    PubMed Central

    Lu, Jie; Zhou, Yingqun; Hu, Tianyuan; Zhang, Hui; Shen, Miao; Cheng, Ping; Dai, Weiqi; Wang, Fan; Chen, Kan; Zhang, Yan; Wang, Chengfeng; Li, Jingjing; Zheng, Yuanyuan; Yang, Jing; Zhu, Rong; Wang, Jianrong; Lu, Wenxia; Zhang, Huawei; Wang, Junshan; Xia, Yujing; De Assuncao, Thiago M.; Jalan-Sakrikar, Nidhi; Huebert, Robert C.; Bin Zhou; Guo, Chuanyong

    2016-01-01

    Aberrant transcriptional regulation contributes to the pathogenesis of both congenital and adult forms of liver disease. Although the transcription factor RBPJ is essential for liver morphogenesis and biliary development, its specific function in the differentiation of hepatic progenitor cells (HPC) has not been investigated, and little is known about its role in adult liver regeneration. HPCs are bipotent liver stem cells that can self-replicate and differentiate into hepatocytes or cholangiocytes in vitro. HPCs are thought to play an important role in liver regeneration and repair responses. While the coordinated repopulation of both hepatocyte and cholangiocyte compartment is pivotal to the structure and function of the liver after regeneration, the mechanisms coordinating biliary regeneration remain vastly understudied. Here, we utilized complex genetic manipulations to drive liver-specific deletion of the Rbpj gene in conjunction with lineage tracing techniques to delineate the precise functions of RBPJ during biliary development and HPC-associated biliary regeneration after hepatectomy. Furthermore, we demonstrate that RBPJ promotes HPC differentiation toward cholangiocytes in vitro and blocks hepatocyte differentiation through mechanisms involving Hippo-Notch crosstalk. Overall, this study demonstrates that the Notch-RBPJ signaling axis critically regulates biliary regeneration by coordinating the fate decision of HPC and clarifies the molecular mechanisms involved. PMID:26951801

  20. Vaccarin attenuates the human EA.hy926 endothelial cell oxidative stress injury through inhibition of Notch signaling.

    PubMed

    Xie, Fengshan; Cai, Weiwei; Liu, Yanling; Li, Yue; Du, Bin; Feng, Lei; Qiu, Liying

    2015-01-01

    Endothelial cell injury is an essential component of atherosclerosis and hypertension. Atherosclerosis and other macrovascular diseases are the most common complications of diabetes. Vaccarin is a major flavonoid glycoside in Vaccariae semen, and is expected to be useful in the treatment of vascular diseases. The aim of the present study was to evaluate the possible effects of vaccarin in human umbilical vein endothelial cells (EA.hy926) induced by hydrogen peroxide (H2O2) and its underlying mechanism in the prevention and treatment of H2O2 injury. In this study, the EA.hy926 cells were exposed to 250, 500 and 1000 µM H2O2 for 2 and 4 h in the absence or presence of vaccarin, and the cell injury induced by H2O2 was examined via SRB. Cell migratory ability, lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA) levels and decreasing superoxide dismutase (SOD) activity were evaluated by the wound healing assay and corresponding assay kits. Cell apoptosis was detected by flow cytometry with Annexin V-fluorescein isothiocyanate/propidium iodide Apoptosis Detection kit and Hoechst staining. Furthermore, western blot detected the protein expressions of Notch1, Hes1 and caspase-3. Following treatment with H2O2, it was found that H2O2 stimulated cell injury in a dose-dependent manner, including reducing cell viability and cell migratory ability, increasing LDH leakage and MDA levels, and decreasing SOD activity. H2O2 further accelerated cell apoptosis via activation of Notch1 and the downstream molecule Hes1. Preincubation with vaccarin was found to protect EA.hy926 cells from H2O2-induced cell oxidative stress injury, which promoted cell viability and cell migratory ability, inhibited the level of LDH and MDA, but enhanced the activity of SOD. In particular, in addition to downregulation Notch signaling, vaccarin treatments also downregulated caspase-3, a cell apoptotic pathway-related protein. These findings indicated that vaccarin may be able to selectively protect

  1. MicroRNA-449a Overexpression, Reduced NOTCH1 Signals and Scarce Goblet Cells Characterize the Small Intestine of Celiac Patients

    PubMed Central

    Tinto, Nadia; Montanaro, Donatella; Capobianco, Valentina; Izzo, Valentina; Tucci, Francesca; Troncone, Giancarlo; Greco, Luigi; Sacchetti, Lucia

    2011-01-01

    MiRNAs play a relevant role in regulating gene expression in a variety of physiological and pathological conditions including autoimmune disorders. MiRNAs are also important in the differentiation and function of the mouse intestinal epithelium. Our study was aimed to look for miRNA-based modulation of gene expression in celiac small intestine, and particularly for genes involved in cell intestinal differentiation/proliferation mechanisms. A cohort of 40 children (20 with active CD, 9 on a gluten-free diet (GFD), and 11 controls), were recruited at the Paediatrics Department (University of Naples Federico II). The expression of 365 human miRNAs was quantified by TaqMan low-density arrays. We used bioinformatics to predict putative target genes of miRNAs and to select biological pathways. The presence of NOTCH1, HES1, KLF4, MUC-2, Ki67 and beta-catenin proteins in the small intestine of CD and control children was tested by immunohistochemistry. The expression of about 20% of the miRNAs tested differed between CD and control children. We found that high miR-449a levels targeted and reduced both NOTCH1 and KLF4 in HEK-293 cells. NOTCH1, KLF4 signals and the number of goblet cells were lower in small intestine of children with active CD and in those on a GFD than in controls, whereas more nuclear beta-catenin staining, as a sign of the WNT pathway activation, and more Ki67 staining, as sign of proliferation, were present in crypts from CD patients than in controls. In conclusion we first demonstrate a miRNA mediated gene regulation in small intestine of CD patients. We also highlighted a reduced NOTCH1 pathway in our patients, irrespective of whether the disease was active or not. We suggest that NOTCH pathway could be constitutively altered in the celiac small intestine and could drive the increased proliferation and the decreased differentiation of intestinal cells towards the secretory goblet cell lineage. PMID:22194996

  2. NOTCH activation interferes with cell fate specification in the gastrulating mouse