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Sample records for regulates notch receptor

  1. 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.

  2. The Role of Notch Receptors in Transcriptional Regulation

    PubMed Central

    WANG, HONGFANG; ZANG, CHONGZHI; LIU, X. SHIRLEY; ASTER, JON C.

    2015-01-01

    Notch signaling has pleiotropic context-specific functions that have essential roles in many processes, including embryonic development and maintenance and homeostasis of adult tissues. Aberrant Notch signaling (both hyper- and hypoactive) is implicated in a number of human developmental disorders and many cancers. Notch receptor signaling is mediated by tightly regulated proteolytic cleavages that lead to the assembly of a nuclear Notch transcription complex, which drives the expression of downstream target genes and thereby executes Notch’s functions. Thus, understanding regulation of gene expression by Notch is central to deciphering how Notch carries out its many activities. Here, we summarize the recent findings pertaining to the complex interplay between the Notch transcriptional complex and interacting factors involved in transcriptional regulation, including co-activators, cooperating transcription factors, and chromatin regulators, and discuss emerging data pertaining to the role of Notch-regulated noncoding RNAs in transcription. PMID:25418913

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. Delta/Notch-Like EGF-Related Receptor (DNER) Is Not a Notch Ligand.

    PubMed

    Greene, Maxwell; Lai, Yongjie; Pajcini, Kostandin; Bailis, Will; Pear, Warren S; Lancaster, Eric

    2016-01-01

    Delta/Notch-like EGF-related receptor (DNER) has been reported to act as a Notch ligand, despite lacking a Delta/Serrate/Lag (DSL) binding domain common to all other known ligands. The established Notch ligand Delta-like 1 (DLL1), but not DNER, activated Notch1 in a luciferase assay, prevented the differentiation of myoblasts through Notch signaling, and bound Notch-fc in a cell-based assay. DNER is not a Notch ligand and its true function remains unknown. PMID:27622512

  11. Delta/Notch-Like EGF-Related Receptor (DNER) Is Not a Notch Ligand.

    PubMed

    Greene, Maxwell; Lai, Yongjie; Pajcini, Kostandin; Bailis, Will; Pear, Warren S; Lancaster, Eric

    2016-01-01

    Delta/Notch-like EGF-related receptor (DNER) has been reported to act as a Notch ligand, despite lacking a Delta/Serrate/Lag (DSL) binding domain common to all other known ligands. The established Notch ligand Delta-like 1 (DLL1), but not DNER, activated Notch1 in a luciferase assay, prevented the differentiation of myoblasts through Notch signaling, and bound Notch-fc in a cell-based assay. DNER is not a Notch ligand and its true function remains unknown.

  12. Delta/Notch-Like EGF-Related Receptor (DNER) Is Not a Notch Ligand

    PubMed Central

    Lai, Yongjie; Pajcini, Kostandin; Bailis, Will; Pear, Warren S.; Lancaster, Eric

    2016-01-01

    Delta/Notch-like EGF-related receptor (DNER) has been reported to act as a Notch ligand, despite lacking a Delta/Serrate/Lag (DSL) binding domain common to all other known ligands. The established Notch ligand Delta-like 1 (DLL1), but not DNER, activated Notch1 in a luciferase assay, prevented the differentiation of myoblasts through Notch signaling, and bound Notch-fc in a cell-based assay. DNER is not a Notch ligand and its true function remains unknown. PMID:27622512

  13. 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

  14. 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.

  15. 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

  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. 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

  18. Notch1 Is Regulated by Chorionic Gonadotropin and Progesterone in Endometrial Stromal Cells and Modulates Decidualization in Primates

    PubMed Central

    Afshar, Yalda; Miele, Lucio

    2012-01-01

    No other tissue in the body undergoes such a vast and extensive growth and remodeling in a relatively short period of time as the primate endometrium. Endometrial integrity is coordinated by ovarian hormones, namely, estrogens, progesterone, and the embryonic hormone chorionic gonadotropin (CG). These regulated events modulate the menstrual cycle and decidualization. The Notch family of transmembrane receptors regulate cellular proliferation, differentiation, and apoptosis, cellular processes required to maintain endometrial integrity. In two primate models, the human and the simulated pregnant baboon model, we demonstrated that Notch1 is increased during the window of uterine receptivity, concomitant with CG. Furthermore, CG combined with estrogens and progesterone up-regulate the level of Notch1, whereas progesterone increases the intracellular transcriptionally competent Notch1, which binds in a complex with progesterone receptor. Inhibition of Notch1 prevented decidualization, and alternatively, when decidualization is biochemically recapitulated in vitro, Notch1 is down-regulated. A focused microarray demonstrated that the Notch inhibitor, Numb, dramatically increased when Notch1 decreased during decidualization. We propose that in the endometrium, Notch has a dual role during the window of uterine receptivity. Initially, Notch1 mediates a survival signal in the uterine endometrium in response to CG from the implanting blastocyst and progesterone, so that menstrual sloughing is averted. Subsequently, Notch1 down-regulation may be critical for the transition of stromal fibroblast to decidual cells, which is essential for the establishment of a successful pregnancy. PMID:22535768

  19. Angiopoietin-like proteins stimulate HSPC development through interaction with notch receptor signaling

    PubMed Central

    Lin, Michelle I; Price, Emily N; Boatman, Sonja; Hagedorn, Elliott J; Trompouki, Eirini; Satishchandran, Sruthi; Carspecken, Charles W; Uong, Audrey; DiBiase, Anthony; Yang, Song; Canver, Matthew C; Dahlberg, Ann; Lu, Zhigang; Zhang, Cheng Cheng; Orkin, Stuart H; Bernstein, Irwin D; Aster, Jon C; White, Richard M; Zon, Leonard I

    2015-01-01

    Angiopoietin-like proteins (angptls) are capable of ex vivo expansion of mouse and human hematopoietic stem and progenitor cells (HSPCs). Despite this intriguing ability, their mechanism is unknown. In this study, we show that angptl2 overexpression is sufficient to expand definitive HSPCs in zebrafish embryos. Angptl1/2 are required for definitive hematopoiesis and vascular specification of the hemogenic endothelium. The loss-of-function phenotype is reminiscent of the notch mutant mindbomb (mib), and a strong genetic interaction occurs between angptls and notch. Overexpressing angptl2 rescues mib while overexpressing notch rescues angptl1/2 morphants. Gene expression studies in ANGPTL2-stimulated CD34+ cells showed a strong MYC activation signature and myc overexpression in angptl1/2 morphants or mib restored HSPCs formation. ANGPTL2 can increase NOTCH activation in cultured cells and ANGPTL receptor interacted with NOTCH to regulate NOTCH cleavage. Together our data provide insight to the angptl-mediated notch activation through receptor interaction and subsequent activation of myc targets. DOI: http://dx.doi.org/10.7554/eLife.05544.001 PMID:25714926

  20. 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

  1. G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition

    PubMed Central

    Zhang, Panpan; He, Qiuping; Chen, Dongbo; Liu, Weixiao; Wang, Lu; Zhang, Chunxia; Ma, Dongyuan; Li, Wei; Liu, Bing; Liu, Feng

    2015-01-01

    In vertebrates, embryonic hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of endothelial cells, the hemogenic endothelium (HE), through the endothelial-to-hematopoietic transition (EHT). Notch signaling is essential for HSPC development during embryogenesis across vertebrates. However, whether and how it regulates EHT remains unclear. Here, we show that G protein-coupled receptor 183 (Gpr183) signaling serves as an indispensable switch for HSPC emergence by repressing Notch signaling before the onset of EHT. Inhibition of Gpr183 significantly upregulates Notch signaling and abolishes HSPC emergence. Upon activation by its ligand 7α-25-OHC, Gpr183 recruits β-arrestin1 and the E3 ligase Nedd4 to degrade Notch1 in specified HE cells and then facilitates the subsequent EHT. Importantly, 7α-25-OHC stimulation promotes HSPC emergence in vivo and in vitro, providing an attractive strategy for enhancing the in vitro generation of functional HSPCs. PMID:26358189

  2. 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.

  3. Stress-induced switch in Numb isoforms enhances Notch-dependent expression of subtype-specific transient receptor potential channel.

    PubMed

    Kyriazis, George A; Belal, Cherine; Madan, Meenu; Taylor, David G; Wang, Jang; Wei, Zelan; Pattisapu, Jogi V; Chan, Sic L

    2010-02-26

    The Notch signaling pathway plays an essential role in the regulation of cell specification by controlling differentiation, proliferation, and apoptosis. Numb is an intrinsic regulator of the Notch pathway and exists in four alternative splice variants that differ in the length of their phosphotyrosine-binding domain (PTB) and proline-rich region domains. The physiological relevance of the existence of the Numb splice variants and their exact regulation are still poorly understood. We previously reported that Numb switches from isoforms containing the insertion in PTB to isoforms lacking this insertion in neuronal cells subjected to trophic factor withdrawal (TFW). The functional relevance of the TFW-induced switch in Numb isoforms is not known. Here we provide evidence that the TFW-induced switch in Numb isoforms regulates Notch signaling strength and Notch target gene expression. PC12 cells stably overexpressing Numb isoforms lacking the PTB insertion exhibited higher basal Notch activity and Notch-dependent transcription of the transient receptor potential channel 6 (TRPC6) when compared with those overexpressing Numb isoforms with the PTB insertion. The differential regulation of TRPC6 expression is correlated with perturbed calcium signaling and increased neuronal vulnerability to TFW-induced death. Pharmacological inhibition of the Notch pathway or knockdown of TRPC6 function ameliorates the adverse effects caused by the TFW-induced switch in Numb isoforms. Taken together, our results indicate that Notch and Numb interaction may influence the sensitivity of neuronal cells to injurious stimuli by modulating calcium-dependent apoptotic signaling cascades.

  4. 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

  5. Notch in the intestine: regulation of homeostasis and pathogenesis.

    PubMed

    Noah, Taeko K; Shroyer, Noah F

    2013-01-01

    The small and large intestines are tubular organs composed of several tissue types. The columnar epithelium that lines the inner surface of the intestines distinguishes the digestive physiology of each region of the intestine and consists of several distinct cell types that are rapidly and continually renewed by intestinal stem cells that reside near the base of the crypts of Lieberkühn. Notch signaling controls the fate of intestinal stem cells by regulating the expression of Hes genes and by repressing Atoh1. Alternate models of Notch pathway control of cell fate determination are presented. Roles for Notch signaling in development of the intestine, including mesenchymal and neural cells, are discussed. The oncogenic activities of Notch in colorectal cancer, as well as the tumor suppressive activities of Atoh1, are reviewed. Therapeutic targeting of the Notch pathway in colorectal cancers is discussed, along with potential caveats.

  6. Sequential Notch activation regulates ventricular chamber development

    PubMed Central

    D'Amato, Gaetano; Luxán, Guillermo; del Monte-Nieto, Gonzalo; Martínez-Poveda, Beatriz; Torroja, Carlos; Walter, Wencke; Bochter, Matthew S.; Benedito, Rui; Cole, Susan; Martinez, Fernando; Hadjantonakis, Anna-Katerina; Uemura, Akiyoshi; Jiménez-Borreguero, Luis J.; de la Pompa, José Luis

    2016-01-01

    Ventricular chambers are essential for the rhythmic contraction and relaxation occurring in every heartbeat throughout life. Congenital abnormalities in ventricular chamber formation cause severe human heart defects. How the early trabecular meshwork of myocardial fibres forms and subsequently develops into mature chambers is poorly understood. We show that Notch signalling first connects chamber endocardium and myocardium to sustain trabeculation, and later coordinates ventricular patterning and compaction with coronary vessel development to generate the mature chamber, through a temporal sequence of ligand signalling determined by the glycosyltransferase manic fringe (MFng). Early endocardial expression of MFng promotes Dll4–Notch1 signalling, which induces trabeculation in the developing ventricle. Ventricular maturation and compaction require MFng and Dll4 downregulation in the endocardium, which allows myocardial Jag1 and Jag2 signalling to Notch1 in this tissue. Perturbation of this signalling equilibrium severely disrupts heart chamber formation. Our results open a new research avenue into the pathogenesis of cardiomyopathies. PMID:26641715

  7. 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

  8. 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.

  9. Notch and the Skeleton▿

    PubMed Central

    Zanotti, Stefano; Canalis, Ernesto

    2010-01-01

    Notch receptors are transmembrane receptors that regulate cell fate decisions. There are four Notch receptors in mammals. Upon binding to members of the Delta and Jagged family of transmembrane proteins, Notch is cleaved and the Notch intracellular domain (NICD) is released. NICD then translocates to the nucleus, where it associates with the CBF-1, Suppressor of Hairless, and Lag-2 (CSL) and Mastermind-Like (MAML) proteins. This complex activates the transcription of Notch target genes, such as Hairy Enhancer of Split (Hes) and Hes-related with YRPF motif (Hey). Notch signaling is critical for the regulation of mesenchymal stem cell differentiation. Misexpression of Notch in skeletal tissue indicates a role as an inhibitor of skeletal development and postnatal bone formation. Overexpression of Notch inhibits endochondral bone formation and osteoblastic differentiation, causing severe osteopenia. Conditional inactivation of Notch in the skeleton causes an increase in cancellous bone volume and enhanced osteoblastic differentiation. Notch ligands are expressed in the hematopoietic stem cell niche and are critical for the regulation of hematopoietic stem cell self-renewal. Dysregulation of Notch signaling is the underlying cause of diseases affecting the skeletal tissue, including Alagille syndrome, spondylocostal dysostosis, and possibly, osteosarcoma. PMID:19995916

  10. 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.

  11. Notch receptor expression in human brain arteriovenous malformations.

    PubMed

    Hill-Felberg, Sandra; Wu, Hope Hueizhi; Toms, Steven A; Dehdashti, Amir R

    2015-08-01

    The roles of the Notch pathway proteins in normal adult vascular physiology and the pathogenesis of brain arteriovenous malformations are not well-understood. Notch 1 and 4 have been detected in human and mutant mice vascular malformations respectively. Although mutations in the human Notch 3 gene caused a genetic form of vascular stroke and dementia, its role in arteriovenous malformations development has been unknown. In this study, we performed immunohistochemistry screening on tissue microarrays containing eight surgically resected human brain arteriovenous malformations and 10 control surgical epilepsy samples. The tissue microarrays were evaluated for Notch 1-4 expression. We have found that compared to normal brain vascular tissue Notch-3 was dramatically increased in brain arteriovenous malformations. Similarly, Notch 4 labelling was also increased in vascular malformations and was confirmed by western blot analysis. Notch 2 was not detectable in any of the human vessels analysed. Using both immunohistochemistry on microarrays and western blot analysis, we have found that Notch-1 expression was detectable in control vessels, and discovered a significant decrease of Notch 1 expression in vascular malformations. We have demonstrated that Notch 3 and 4, and not Notch 1, were highly increased in human arteriovenous malformations. Our findings suggested that Notch 4, and more importantly, Notch 3, may play a role in the development and pathobiology of human arteriovenous malformations.

  12. 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.

  13. 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

  14. 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

  15. Prognostic values of four Notch receptor mRNA expression in gastric cancer

    PubMed Central

    Wu, Xiaoyu; Liu, Wentao; Tang, Ding; Xiao, Haijuan; Wu, Zhenfeng; Chen, Che; Yao, Xuequan; Liu, Fukun; Li, Gang

    2016-01-01

    Notch ligands and receptors are frequently deregulated in several human malignancies including gastric cancer. The activation of Notch signaling has been reported to contribute to gastric carcinogenesis and progression. However, the prognostic roles of individual Notch receptors in gastric cancer patients remain elusive. In the current study, we accessed the prognostic roles of four Notch receptors, Notch 1–4, in gastric cancer patients through “The Kaplan-Meier plotter” (KM plotter) database, in which updated gene expression data and survival information include a total of 876 gastric cancer patients. All four Notch receptors’ high mRNA expression was found to be correlated to worsen overall survival (OS) for all gastric cancer patients followed for 20 years. We further accessed the prognostic roles of individual Notch receptors in different clinicopathological features using Lauren classification, pathological grades, clinical grades, HER2 status and different choices of treatments of gastric cancer patients. These results indicate that there are critical prognostic values of the four Notch receptors in gastric cancer. This information will be useful for better understanding of the heterogeneity and complexity in the molecular biology of gastric cancer and to develop tools to more accurately predict their prognosis. PMID:27363496

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. Notch-1 promotes breast cancer cells proliferation by regulating LncRNA GAS5

    PubMed Central

    Pei, Jing; Wang, Benzhong

    2015-01-01

    Background: Notch signaling is indicated as novel therapeutic targets to prevent recurrence of breast cancer. LncRNAs were identified as downstream target of Notch pathway. However, the exact mechanisms involved in Notch signaling, lncRNAs and breast cancer remain to be explained. Objective: This original research aimed to determine the prognostic implications of Notch-1 for breast cancer, and explain mechanisms involved in regulation of lnRNA GAS5 by Notch-1, and identify the function of this mechanism on breast cancer. Method: Thirty breast cancer patients were included from The First Affiliated Hospital of Anhui Medical University (China) since January 2006 in this study. The mRNA level by RT-PCR and protein level of Notch-1 by western blot in tumor tissues and adjacent normal tissues were evaluated and 5-year survival analysis was applied to examine the significance of Notch-1. The levels of ten reported lncRNAs were determined by RT-PCR, and subsequently linear analysis was applied to analyze the relationship between these four unique lncRNAs and protein level of Notch-1, which identified the most relevant lncRNA GAS5 with Notch-1 in breast cancer. Subsequently, Notch1-siRNA was applied to influence the expression of Notch-1 in T47D, then the level of RSA5 was measured by RT-PCR, and CCK-8 assay was applied to measure the proliferation of T47D cells. Results: High level of Notch-1 provided a poor prognosis in breast cancer. Interference of Notch-1 significantly suppressed proliferation of T47D cell (P < 0.05), and significantly increased the level of GAS5. Conclusion: Notch-1 promotes breast cancer cells proliferation by regulating LncRNA GAS5. PMID:26550436

  4. Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression

    PubMed Central

    Franciosa, G; Diluvio, G; Gaudio, F Del; Giuli, M V; Palermo, R; Grazioli, P; Campese, A F; Talora, C; Bellavia, D; D'Amati, G; Besharat, Z M; Nicoletti, C; Siebel, C W; Choy, L; Rustighi, A; Sal, G Del; Screpanti, I; Checquolo, S

    2016-01-01

    Deregulated Notch signaling is associated with T-cell Acute Lymphoblastic Leukemia (T-ALL) development and progression. Increasing evidence reveals that Notch pathway has an important role in the invasion ability of tumor cells, including leukemia, although the underlying molecular mechanisms remain mostly unclear. Here, we show that Notch3 is a novel target protein of the prolyl-isomerase Pin1, which is able to regulate Notch3 protein processing and to stabilize the cleaved product, leading to the increased expression of the intracellular domain (N3IC), finally enhancing Notch3-dependent invasiveness properties. We demonstrate that the combined inhibition of Notch3 and Pin1 in the Notch3-overexpressing human leukemic TALL-1 cells reduces their high invasive potential, by decreasing the expression of the matrix metalloprotease MMP9. Consistently, Pin1 depletion in a mouse model of Notch3-induced T-ALL, by reducing N3IC expression and signaling, impairs the expansion/invasiveness of CD4+CD8+ DP cells in peripheral lymphoid and non-lymphoid organs. Notably, in in silico gene expression analysis of human T-ALL samples we observed a significant correlation between Pin1 and Notch3 expression levels, which may further suggest a key role of the newly identified Notch3-Pin1 axis in T-ALL aggressiveness and progression. Thus, combined suppression of Pin1 and Notch3 proteins may be exploited as an additional target therapy for T-ALL. PMID:26876201

  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. NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

    PubMed Central

    White, Mark P.; Theodoris, Christina V.; Liu, Lei; Collins, William J.; Blue, Kathleen W.; Lee, Joon Ho; Meng, Xianzhong; Robbins, Robert C.; Ivey, Kathryn N.; Srivastava, Deepak

    2015-01-01

    Valvular and vascular calcification are common causes of cardiovascular morbidity and mortality. Developing effective treatments requires understanding the molecular underpinnings of these processes. Shear stress is thought to play a role in inhibiting calcification. Furthermore, NOTCH1 regulates vascular and valvular endothelium, and human mutations in NOTCH1 can cause calcific aortic valve disease. Here, we determined the genome-wide impact of altering shear stress and NOTCH signaling on aortic valve endothelium. mRNA-sequencing of human aortic valve endothelial cells (HAVECs) with or without knockdown of NOTCH1, in the presence or absence of shear stress, revealed NOTCH1-dependency of the atherosclerosis-related gene connexin 40 (GJA5), and numerous repressors of endochondral ossification. Among these, Matrix GLA Protein (MGP) is highly expressed in aortic valve and vasculature, and inhibits soft tissue calcification by sequestering bone morphogenetic proteins (BMPs). Altering NOTCH1 levels affected MGP mRNA and protein in HAVECs. Furthermore, shear stress activated NOTCH signaling and MGP in a NOTCH1-dependent manner. NOTCH1 positively regulated endothelial MGP in vivo through specific binding motifs upstream of MGP. Our studies suggest that shear stress activates NOTCH1 in primary human aortic valve endothelial cells leading to downregulation of osteoblast-like gene networks that play a role in tissue calcification. PMID:25871831

  7. Notch-1 regulates proliferation and differentiation of human bladder cancer cell lines by inhibiting expression of Krüppel-like factor 4.

    PubMed

    Ai, Xing; Jia, Zhuomin; Liu, Shuanglin; Wang, Jiajun; Zhang, Xu

    2014-10-01

    Inhibition of Notch signaling pathways, consisting of 4 highly conserved receptors (Notch 1-4), induces expression of Krüppel-like transcription factors (KLFs) linked to bladder cancer tumorigenesis and metastasis. Effects of Notch-1 knockdown on cell proliferation, differentiation and KLF4 levels in bladder cancer cell lines were investigated. PsiRNA1‑mediated Notch-1 and KLF4 knockdown models and control model without the psiRNA1 vector were constructed using bladder cancer cell lines T24 and BIU87. Cell proliferation, apoptosis and expression of Notch-1 and KLF4 were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry assay with Annexin V-FITC/PI staining, and reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis, respectively. Proliferation was assessed in Notch-1 and/or KLF4 knockdown. The results showed that Notch-1 mRNA and protein expression levels were significantly lower following psiRNA1 vector transfection in both cell lines (P<0.05). Growth and proliferation of both cell lines were significantly inhibited by Notch-1 knockdown (P<0.05), and more G0/G1 arrest and apoptosis were observed compared to those in the control groups (P<0.05). The effects were time-dependent, peaking between 24-48 h and declining by 72 h. KLF4 expression was significantly higher in the Notch-1 knockdown group than in control cells (P<0.05). Notch-1 knockdown cell proliferation was significantly lower than that of Notch-1 and KLF4 knockdown (P<0.05). In conclusion, Notch-1 may act as an oncogene, regulating the proliferation and differentiation of bladder cancer cells by inhibiting KLF4. Pending further exploration of pathway variations and crosstalk, these pathways may be useful targets for bladder cancer therapy.

  8. Distinct prognostic values of four-Notch-receptor mRNA expression in ovarian cancer.

    PubMed

    Zhou, Xinling; Teng, Lingling; Wang, Min

    2016-05-01

    Notch signaling pathway includes ligands and Notch receptors, which are frequently deregulated in several human malignancies including ovarian cancer. Aberrant activation of Notch signaling has been linked to ovarian carcinogenesis and progression. In the current study, we used the "Kaplan-Meier plotter" (KM plotter) database, in which updated gene expression data and survival information from a total of 1306 ovarian cancer patients were used to access the prognostic value of four Notch receptors in ovarian cancer patients. Hazard ratio (HR), 95 % confidence intervals, and log-rank P were calculated. Notch1 messenger RNA (mRNA) high expression was not found to be correlated to overall survival (OS) for all ovarian cancer, as well as in serous and endometrioid cancer patients followed for 20 years. However, Notch1 mRNA high expression is significantly associated with worsen OS in TP53 wild-type ovarian cancer patients, while it is significantly associated with better OS in TP53 mutation-type ovarian cancer patients. Notch2 mRNA high expression was found to be significantly correlated to worsen OS for all ovarian cancer patients, as well as in grade II ovarian cancer patients. Notch3 mRNA high expression was found to be significantly correlated to better OS for all ovarian cancer patients, but not in serous cancer patients and endometrioid cancer patients. Notch4 mRNA high expression was not found to be significantly correlated to OS for all ovarian cancer patients, serous cancer patients, and endometrioid cancer patients. These results indicate that there are distinct prognostic values of four Notch receptors in ovarian cancer. This information will be useful for better understanding of the heterogeneity and complexity in the molecular biology of ovarian cancer and for developing tools to more accurately predict their prognosis. Based on our results, Notch1 could be a potential drug target of TP53 wild-type ovarian cancer and Notch2 could be a potential drug

  9. 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

  10. 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

  11. 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

  12. Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells

    PubMed Central

    Marcel, Nimi; Sarin, Apurva

    2016-01-01

    Cell survival is one of several processes regulated by the Notch pathway in mammalian cells. Here we report functional outcomes of non-nuclear Notch signaling to activate autophagy, a conserved cellular response to nutrient stress, regulating survival in murine natural T-regulatory cells (Tregs), an immune subset controlling tolerance and inflammation. Induction of autophagy required ligand-dependent, Notch intracellular domain (NIC) activity, which controlled mitochondrial organization and survival of activated Tregs. Consistently, NIC immune-precipitated Beclin and Atg14, constituents of the autophagy initiation complex. Further, ectopic expression of an effector of autophagy (Atg3) or recombinant NIC tagged to a nuclear export signal (NIC-NES), restored autophagy and suppressor function in Notch1-/- Tregs. Furthermore, Notch1 deficiency in the Treg lineage resulted in immune hyperactivity, implicating Notch activity in Treg homeostasis. Notch1 integration with autophagy, revealed in these experiments, holds implications for Notch regulated cell-fate decisions governing differentiation. DOI: http://dx.doi.org/10.7554/eLife.14023.001 PMID:27267497

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. Transcriptional regulation of Notch and Delta: requirement for neuroblast segregation in Drosophila.

    PubMed

    Seugnet, L; Simpson, P; Haenlin, M

    1997-05-01

    Segregation of a single neural precursor from each proneural cluster in Drosophila relies on Notch-mediated lateral signalling. Studies concerning the spacing of precursors for the microchaetes of the peripheral nervous system suggested the existence of a regulatory loop between Notch and its ligand Delta within each cell that is under transcriptional control. Activation of Notch leads to repression of the achaete-scute genes which themselves regulate transcription of Delta, perhaps directly. Here we have tested a requirement for transcriptional regulation of Notch and/or Delta during neuroblast segregation in embryos, by providing Notch and Delta ubiquitously at uniform levels. Neuroblast segregation occurs normally under conditions of uniform Notch expression. Under conditions of uniform Delta expression, a single neuroblast segregates from each proneural group in 80% of the cases, more than one in the remaining 20%. Thus transcriptional regulation of Delta is largely dispensable. We discuss the possibility that segregation of single precursors in the central nervous system may rely on a heterogeneous distribution of neural potential between different cells of the proneural group. Notch signalling would enable all cells to mutually repress each other and only a cell with an elevated neural potential could overcome this repression. PMID:9169848

  20. 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

  1. 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

  2. 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.

  3. 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

  4. Notch1 is associated with the multidrug resistance of hypoxic osteosarcoma by regulating MRP1 gene expression.

    PubMed

    Li, C; Guo, D; Tang, B; Zhang, Y; Zhang, K; Nie, L

    2016-01-01

    Hypoxia and Notch signaling pathway are closely related and both participate in cell proliferation and drug resistance of tumors. However, the molecular mechanisms of hypoxia and Notch signaling pathway in cell proliferation and drug resistance of osteosarcoma (OS) remain unclear. In this study, to further evaluate the role of hypoxia and Notch1 on drug resistance of OS, we investigated the influence of inhibiting Notch1 pathway by Notch1 small interference RNA (siRNA) on human MG-63 OS cells in hypoxia. Our data showed that hypoxia promoted OS cell proliferation, induced the G0/G1-S-G2/M phase transition, and increased multidrug resistance of human OS cells. Western blot analysis suggested that hypoxia increased the expression of HIF-1α, Notch1, and multidrug resistance protein-1 (MRP1) in human OS cells. Notch1 siRNA inhibits proliferation and increases apoptosis of hypoxic OS cells. Finally, these hypoxic OS cells can be sensitized to multidrug treatment through inhibition of the Notch protein expression by siRNA. Repression of the Notch protein expression resulted in down-regulation of MRP1 protein. These data support the conclusion that Notch signaling is up-regulated in human OS cells under hypoxia and Notch1 may represent a viable target to overcome chemoresistant OS cells in a hypoxic niche by regulating MRP1 gene expression. PMID:27468877

  5. Notch-mediated lateral inhibition regulates proneural wave propagation when combined with EGF-mediated reaction diffusion.

    PubMed

    Sato, Makoto; Yasugi, Tetsuo; Minami, Yoshiaki; Miura, Takashi; Nagayama, Masaharu

    2016-08-30

    Notch-mediated lateral inhibition regulates binary cell fate choice, resulting in salt and pepper patterns during various developmental processes. However, how Notch signaling behaves in combination with other signaling systems remains elusive. The wave of differentiation in the Drosophila visual center or "proneural wave" accompanies Notch activity that is propagated without the formation of a salt and pepper pattern, implying that Notch does not form a feedback loop of lateral inhibition during this process. However, mathematical modeling and genetic analysis clearly showed that Notch-mediated lateral inhibition is implemented within the proneural wave. Because partial reduction in EGF signaling causes the formation of the salt and pepper pattern, it is most likely that EGF diffusion cancels salt and pepper pattern formation in silico and in vivo. Moreover, the combination of Notch-mediated lateral inhibition and EGF-mediated reaction diffusion enables a function of Notch signaling that regulates propagation of the wave of differentiation. PMID:27535937

  6. Notch-mediated lateral inhibition regulates proneural wave propagation when combined with EGF-mediated reaction diffusion

    PubMed Central

    Sato, Makoto; Yasugi, Tetsuo; Minami, Yoshiaki; Miura, Takashi; Nagayama, Masaharu

    2016-01-01

    Notch-mediated lateral inhibition regulates binary cell fate choice, resulting in salt and pepper patterns during various developmental processes. However, how Notch signaling behaves in combination with other signaling systems remains elusive. The wave of differentiation in the Drosophila visual center or “proneural wave” accompanies Notch activity that is propagated without the formation of a salt and pepper pattern, implying that Notch does not form a feedback loop of lateral inhibition during this process. However, mathematical modeling and genetic analysis clearly showed that Notch-mediated lateral inhibition is implemented within the proneural wave. Because partial reduction in EGF signaling causes the formation of the salt and pepper pattern, it is most likely that EGF diffusion cancels salt and pepper pattern formation in silico and in vivo. Moreover, the combination of Notch-mediated lateral inhibition and EGF-mediated reaction diffusion enables a function of Notch signaling that regulates propagation of the wave of differentiation. PMID:27535937

  7. 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.

  8. 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

  9. 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

  10. 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

  11. Epigenetic regulation of Delta-Like1 controls Notch1 activation in gastric cancer.

    PubMed

    Piazzi, Giulia; Fini, Lucia; Selgrad, Michael; Garcia, Melissa; Daoud, Yahya; Wex, Thomas; Malfertheiner, Peter; Gasbarrini, Antonio; Romano, Marco; Meyer, Richard L; Genta, Robert M; Fox, James G; Boland, C Richard; Bazzoli, Franco; Ricciardiello, Luigi

    2011-12-01

    The Notch signaling pathway drives proliferation, differentiation, apoptosis, cell fate, and maintenance of stem cells in several tissues. Aberrant activation of Notch signaling has been described in several tumours and in gastric cancer (GC), activated Notch1 has been associated with de-differentiation of lineage-committed stomach cells into stem progenitors and GC progression. However, the specific role of the Notch1 ligand DLL1 in GC has not yet been elucidated. To assess the role of DLL1 in GC cancer, the expression of Notch1 and its ligands DLL1 and Jagged1, was analyzed in 8 gastric cancer cell lines (KATOIII, SNU601, SNU719, AGS, SNU16, MKN1, MKN45, TMK1). DLL1 expression was absent in KATOIII, SNU601, SNU719 and AGS. The lack of DLL1 expression in these cells was associated with promoter hypermethylation and 5-aza-2'dC caused up-regulation of DLL1. The increase in DLL1 expression was associated with activation of Notch1 signalling, with an increase in cleaved Notch1 intracellular domain (NICD) and Hes1, and down-regulation in Hath1. Concordantly, Notch1 signalling was activated with the overexpression of DLL1. Moreover, Notch1 signalling together with DLL1 methylation were evaluated in samples from 52 GC patients and 21 healthy control as well as in INS-GAS mice infected with H. pylori and randomly treated with eradication therapy. In GC patients, we found a correlation between DLL1 and Hes1 expression, while DLL1 methylation and Hath1 expression were associated with the diffuse and mixed type of gastric cancer. Finally, none of the samples from INS-GAS mice infected with H. pylori, a model of intestinal-type gastric tumorigenesis, showed promoter methylation of DLL1. This study shows that Notch1 activity in gastric cancer is controlled by the epigenetic silencing of the ligand DLL1, and that Notch1 inhibition is associated with the diffuse type of gastric cancer. PMID:22249198

  12. 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

  13. 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

  14. Regulation of Nematostella neural progenitors by SoxB, Notch and bHLH genes.

    PubMed

    Richards, Gemma Sian; Rentzsch, Fabian

    2015-10-01

    Notch signalling, SoxB and Group A bHLH 'proneural' genes are conserved regulators of the neurogenic program in many bilaterians. However, the ancestry of their functions and interactions is not well understood. We address this question in the sea anemone Nematostella vectensis, a representative of the Cnidaria, the sister clade to the Bilateria. It has previously been found that the SoxB orthologue NvSoxB(2) is expressed in neural progenitor cells (NPCs) in Nematostella and promotes the development of both neurons and nematocytes, whereas Notch signalling has been implicated in the negative regulation of neurons and the positive regulation of nematocytes. Here, we clarify the role of Notch by reporting that inhibition of Notch signalling increases the numbers of both neurons and nematocytes, as well as increasing the number of NvSoxB(2)-expressing cells. This suggests that Notch restricts neurogenesis by limiting the generation of NPCs. We then characterise NvAth-like (Atonal/Neurogenin family) as a positive regulator of neurogenesis that is co-expressed with NvSoxB(2) in a subset of dividing NPCs, while we find that NvAshA (Achaete-scute family) and NvSoxB(2) are co-expressed in non-dividing cells only. Reciprocal knockdown experiments reveal a mutual requirement for NvSoxB(2) and NvAth-like in neural differentiation; however, the primary expression of each gene is independent of the other. Together, these data demonstrate that Notch signalling and NvSoxB(2) regulate Nematostella neural progenitors via parallel yet interacting mechanisms; with different aspects of these interactions being shared with Drosophila and/or vertebrate neurogenesis. PMID:26443634

  15. Regulation of Nematostella neural progenitors by SoxB, Notch and bHLH genes

    PubMed Central

    Richards, Gemma Sian; Rentzsch, Fabian

    2015-01-01

    Notch signalling, SoxB and Group A bHLH ‘proneural’ genes are conserved regulators of the neurogenic program in many bilaterians. However, the ancestry of their functions and interactions is not well understood. We address this question in the sea anemone Nematostella vectensis, a representative of the Cnidaria, the sister clade to the Bilateria. It has previously been found that the SoxB orthologue NvSoxB(2) is expressed in neural progenitor cells (NPCs) in Nematostella and promotes the development of both neurons and nematocytes, whereas Notch signalling has been implicated in the negative regulation of neurons and the positive regulation of nematocytes. Here, we clarify the role of Notch by reporting that inhibition of Notch signalling increases the numbers of both neurons and nematocytes, as well as increasing the number of NvSoxB(2)-expressing cells. This suggests that Notch restricts neurogenesis by limiting the generation of NPCs. We then characterise NvAth-like (Atonal/Neurogenin family) as a positive regulator of neurogenesis that is co-expressed with NvSoxB(2) in a subset of dividing NPCs, while we find that NvAshA (Achaete-scute family) and NvSoxB(2) are co-expressed in non-dividing cells only. Reciprocal knockdown experiments reveal a mutual requirement for NvSoxB(2) and NvAth-like in neural differentiation; however, the primary expression of each gene is independent of the other. Together, these data demonstrate that Notch signalling and NvSoxB(2) regulate Nematostella neural progenitors via parallel yet interacting mechanisms; with different aspects of these interactions being shared with Drosophila and/or vertebrate neurogenesis. PMID:26443634

  16. Haploinsufficiency of the NOTCH1 Receptor as a Cause of Adams-Oliver Syndrome with Variable Cardiac Anomalies

    PubMed Central

    Southgate, Laura; Sukalo, Maja; Karountzos, Anastasios S.V.; Taylor, Edward J.; Collinson, Claire S.; Ruddy, Deborah; Snape, Katie M.; Dallapiccola, Bruno; Joss, Shelagh; Brancati, Francesco; Digilio, M. Cristina; Graul-Neumann, Luitgard M.; Salviati, Leonardo; Coerdt, Wiltrud; Jacquemin, Emmanuel; Wuyts, Wim; Zenker, Martin

    2015-01-01

    Background Adams-Oliver syndrome (AOS) is a rare disorder characterized by congenital limb defects and scalp cutis aplasia. In a proportion of cases, notable cardiac involvement is also apparent. Despite recent advances in the understanding of the genetic basis of AOS, for the majority of affected subjects the underlying molecular defect remains unresolved. This study aimed to identify novel genetic determinants of AOS. Methods and Results Whole-exome sequencing was performed for 12 probands, each with a clinical diagnosis of AOS. Analyses led to the identification of novel heterozygous truncating NOTCH1 mutations (c.1649dupA and c.6049_6050delTC) in two kindreds in which AOS was segregating as an autosomal dominant trait. Screening a cohort of 52 unrelated AOS subjects, we detected 8 additional unique NOTCH1 mutations, including three de novo amino-acid substitutions, all within the ligand-binding domain. Congenital heart anomalies were noted in 47% (8/17) of NOTCH1-positive probands and affected family members. In leucocyte-derived RNA from subjects harboring NOTCH1 extracellular domain mutations, we observed significant reduction of NOTCH1 expression, suggesting instability and degradation of mutant mRNA transcripts by the cellular machinery. Transient transfection of mutagenized NOTCH1 missense constructs also revealed significant reduction in gene expression. Mutant NOTCH1 expression was associated with down-regulation of the Notch target genes HEY1 and HES1, indicating that NOTCH1-related AOS arises through dysregulation of the Notch signaling pathway. Conclusions These findings highlight a key role for NOTCH1 across a range of developmental anomalies that include cardiac defects, and implicate NOTCH1 haploinsufficiency as a likely molecular mechanism for this group of disorders. PMID:25963545

  17. 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

  18. Regulation of dendritic-cell differentiation by bone marrow stroma via different Notch ligands

    PubMed Central

    Cheng, Pingyan; Nefedova, Yulia; Corzo, Cesar A.; Gabrilovich, Dmitry I.

    2007-01-01

    Notch is a major factor mediating interaction between hematopoietic progenitor cells (HPCs) and bone marrow stroma (BMS). However its contribution to dendritic cell (DC) differentiation is controversial. We found that main Notch ligands Delta-1 and Jagged-1 had the opposite effect on DC differentiation. Delta-1 promoted generation of fully differentiated DCs, whereas Jagged-1 stimulated accumulation of DC precursors but prevented their transition to terminally differentiated DCs. BMS expressed a substantially higher level of Jagged-1 than Delta-1. Just the opposite expression pattern was observed in spleen stroma (SS). The BMS effect on DC differentiation was similar to that of Jagged-1, whereas the effect of SS was similar to the effect of Delta-1. Down-regulation of Jagged-1 in BMS substantially increased DC differentiation. Experiments in vivo with adoptive transfer of DC precursors further supported the different roles of BMS and SS in DC development. Jagged-1 and Delta-1 equally activated CBF-1/RBPJκ transcription factor, which is a major Notch target. However, they produced a different pattern of activation of Notch target gene Hes1. Overexpression of Hes1 resulted in increased DC differentiation from HPCs. Thus, this study not only revealed the different role of Notch ligands in DC differentiation but also may provide a new insight into regulation of DC differentiation by BMS. PMID:16973960

  19. 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

  20. 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

  1. 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.

  2. Constitutive expression of genes encoding notch receptors and ligands in developing lymphocytes, nTreg cells and dendritic cells in the human thymus.

    PubMed

    Bento-de-Souza, Luciana; Victor, Jefferson R; Bento-de-Souza, Luiz C; Arrais-Santos, Magaly; Rangel-Santos, Andréia C; Pereira-Costa, Érica; Raniero-Fernandes, Elaine; Seixas-Duarte, Maria I; Oliveira-Filho, João B; Silva Duarte, Alberto J

    2016-01-01

    The thymus is the site of T cell maturation. Notch receptors (Notch1-4) and ligands (DLL1-3 and Jagged1-2) constitute one of several pathways involved in this process. Our data revealed differential constitutive expression of Notch genes and ligands in T lymphocytes and thymic dendritic cells (tDCs), suggesting their participation in human thymocyte maturation. nTreg analyses indicated that the Notch components function in parallel to promote maturation in the thymus. PMID:27504259

  3. 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

  4. 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

  5. TspanC8 tetraspanins differentially regulate the cleavage of ADAM10 substrates, Notch activation and ADAM10 membrane compartmentalization.

    PubMed

    Jouannet, Stéphanie; Saint-Pol, Julien; Fernandez, Laurent; Nguyen, Viet; Charrin, Stéphanie; Boucheix, Claude; Brou, Christel; Milhiet, Pierre-Emmanuel; Rubinstein, Eric

    2016-05-01

    The metalloprotease ADAM10 mediates the shedding of the ectodomain of various cell membrane proteins, including APP, the precursor of the amyloid peptide Aβ, and Notch receptors following ligand binding. ADAM10 associates with the members of an evolutionary conserved subgroup of tetraspanins, referred to as TspanC8, which regulate its exit from the endoplasmic reticulum. Here we show that 4 of these TspanC8 (Tspan5, Tspan14, Tspan15 and Tspan33) which positively regulate ADAM10 surface expression levels differentially impact ADAM10-dependent Notch activation and the cleavage of several ADAM10 substrates, including APP, N-cadherin and CD44. Sucrose gradient fractionation, single molecule tracking and quantitative mass-spectrometry analysis of the repertoire of molecules co-immunoprecipitated with Tspan5, Tspan15 and ADAM10 show that these two tetraspanins differentially regulate ADAM10 membrane compartmentalization. These data represent a unique example where several tetraspanins differentially regulate the function of a common partner protein through a distinct membrane compartmentalization. PMID:26686862

  6. Dual roles of Notch in regulation of apically restricted mitosis and apicobasal polarity of neuroepithelial cells.

    PubMed

    Ohata, Shinya; Aoki, Ryo; Kinoshita, Shigeharu; Yamaguchi, Masahiro; Tsuruoka-Kinoshita, Sachiko; Tanaka, Hideomi; Wada, Hironori; Watabe, Shugo; Tsuboi, Takashi; Masai, Ichiro; Okamoto, Hitoshi

    2011-01-27

    How the mitosis of neuroepithelial stem cells is restricted to the apical ventricular area remains unclear. In zebrafish, the mosaic eyes(rw306) (moe/epb41l5(rw306)) mutation disrupts the interaction between the putative adaptor protein Moe and the apicobasal polarity regulator Crumbs (Crb), and impairs the maintenance of neuroepithelial apicobasal polarity. While Crb interacts directly with Notch and inhibits its activity, Moe reverses this inhibition. In the moe(rw306) hindbrain, Notch activity is significantly reduced, and the number of cells that proliferate basally away from the apical area is increased. Surprisingly, activation of Notch in the moe(rw306) mutant rescues not only the basally localized proliferation but also the aberrant neuroepithelial apicobasal polarity. We present evidence that the Crb⋅Moe complex and Notch play key roles in a positive feedback loop to maintain the apicobasal polarity and the apical-high basal-low gradient of Notch activity in neuroepithelial cells, both of which are essential for their apically restricted mitosis. PMID:21262462

  7. 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

  8. Combinatorial microenvironmental regulation of liver progenitor differentiation by Notch ligands, TGFβ, and extracellular matrix

    PubMed Central

    Kaylan, Kerim B.; Ermilova, Viktoriya; Yada, Ravi Chandra; Underhill, Gregory H.

    2016-01-01

    The bipotential differentiation of liver progenitor cells underlies liver development and bile duct formation as well as liver regeneration and disease. TGFβ and Notch signaling are known to play important roles in the liver progenitor specification process and tissue morphogenesis. However, the complexity of these signaling pathways and their currently undefined interactions with other microenvironmental factors, including extracellular matrix (ECM), remain barriers to complete mechanistic understanding. Utilizing a series of strategies, including co-cultures and cellular microarrays, we identified distinct contributions of different Notch ligands and ECM proteins in the fate decisions of bipotential mouse embryonic liver (BMEL) progenitor cells. In particular, we demonstrated a cooperative influence of Jagged-1 and TGFβ1 on cholangiocytic differentiation. We established ECM-specific effects using cellular microarrays consisting of 32 distinct combinations of collagen I, collagen III, collagen IV, fibronectin, and laminin. In addition, we demonstrated that exogenous Jagged-1, Delta-like 1, and Delta-like 4 within the cellular microarray format was sufficient for enhancing cholangiocytic differentiation. Further, by combining Notch ligand microarrays with shRNA-based knockdown of Notch ligands, we systematically examined the effects of both cell-extrinsic and cell-intrinsic ligand. Our results highlight the importance of divergent Notch ligand function and combinatorial microenvironmental regulation in liver progenitor fate specification. PMID:27025873

  9. 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

  10. Autophagy regulates Notch degradation and modulates stem cell development and neurogenesis

    PubMed Central

    Wu, Xiaoting; Fleming, Angeleen; Ricketts, Thomas; Pavel, Mariana; Virgin, Herbert; Menzies, Fiona M.; Rubinsztein, David C.

    2016-01-01

    Autophagy is a conserved, intracellular, lysosomal degradation pathway. While mechanistic aspects of this pathway are increasingly well defined, it remains unclear how autophagy modulation impacts normal physiology. It is, however, becoming clear that autophagy may play a key role in regulating developmental pathways. Here we describe for the first time how autophagy impacts stem cell differentiation by degrading Notch1. We define a novel route whereby this plasma membrane-resident receptor is degraded by autophagy, via uptake into ATG16L1-positive autophagosome-precursor vesicles. We extend our findings using a physiologically relevant mouse model with a hypomorphic mutation in Atg16L1, a crucial autophagy gene, which shows developmental retention of early-stage cells in various tissues where the differentiation of stem cells is retarded and thus reveal how modest changes in autophagy can impact stem cell fate. This may have relevance for diverse disease conditions, like Alzheimer's Disease or Crohn's Disease, associated with altered autophagy. PMID:26837467

  11. Autophagy regulates Notch degradation and modulates stem cell development and neurogenesis.

    PubMed

    Wu, Xiaoting; Fleming, Angeleen; Ricketts, Thomas; Pavel, Mariana; Virgin, Herbert; Menzies, Fiona M; Rubinsztein, David C

    2016-01-01

    Autophagy is a conserved, intracellular, lysosomal degradation pathway. While mechanistic aspects of this pathway are increasingly well defined, it remains unclear how autophagy modulation impacts normal physiology. It is, however, becoming clear that autophagy may play a key role in regulating developmental pathways. Here we describe for the first time how autophagy impacts stem cell differentiation by degrading Notch1. We define a novel route whereby this plasma membrane-resident receptor is degraded by autophagy, via uptake into ATG16L1-positive autophagosome-precursor vesicles. We extend our findings using a physiologically relevant mouse model with a hypomorphic mutation in Atg16L1, a crucial autophagy gene, which shows developmental retention of early-stage cells in various tissues where the differentiation of stem cells is retarded and thus reveal how modest changes in autophagy can impact stem cell fate. This may have relevance for diverse disease conditions, like Alzheimer's Disease or Crohn's Disease, associated with altered autophagy. PMID:26837467

  12. Notch stimulates growth by direct regulation of genes involved in the control of glycolysis and the tricarboxylic acid cycle

    PubMed Central

    Slaninova, Vera; Krafcikova, Michaela; Perez-Gomez, Raquel; Steffal, Pavel; Trantirek, Lukas; Bray, Sarah J.

    2016-01-01

    Glycolytic shift is a characteristic feature of rapidly proliferating cells, such as cells during development and during immune response or cancer cells, as well as of stem cells. It results in increased glycolysis uncoupled from mitochondrial respiration, also known as the Warburg effect. Notch signalling is active in contexts where cells undergo glycolytic shift. We decided to test whether metabolic genes are direct transcriptional targets of Notch signalling and whether upregulation of metabolic genes can help Notch to induce tissue growth under physiological conditions and in conditions of Notch-induced hyperplasia. We show that genes mediating cellular metabolic changes towards the Warburg effect are direct transcriptional targets of Notch signalling. They include genes encoding proteins involved in glucose uptake, glycolysis, lactate to pyruvate conversion and repression of the tricarboxylic acid cycle. The direct transcriptional upregulation of metabolic genes is PI3K/Akt independent and occurs not only in cells with overactivated Notch but also in cells with endogenous levels of Notch signalling and in vivo. Even a short pulse of Notch activity is able to elicit long-lasting metabolic changes resembling the Warburg effect. Loss of Notch signalling in Drosophila wing discs as well as in human microvascular cells leads to downregulation of glycolytic genes. Notch-driven tissue overgrowth can be rescued by downregulation of genes for glucose metabolism. Notch activity is able to support growth of wing during nutrient-deprivation conditions, independent of the growth of the rest of the body. Notch is active in situations that involve metabolic reprogramming, and the direct regulation of metabolic genes may be a common mechanism that helps Notch to exert its effects in target tissues. PMID:26887408

  13. An Expanded Notch-Delta Model Exhibiting Long-Range Patterning and Incorporating MicroRNA Regulation

    PubMed Central

    Chen, Jerry S.; Gumbayan, Abygail M.; Zeller, Robert W.; Mahaffy, Joseph M.

    2014-01-01

    Notch-Delta signaling is a fundamental cell-cell communication mechanism that governs the differentiation of many cell types. Most existing mathematical models of Notch-Delta signaling are based on a feedback loop between Notch and Delta leading to lateral inhibition of neighboring cells. These models result in a checkerboard spatial pattern whereby adjacent cells express opposing levels of Notch and Delta, leading to alternate cell fates. However, a growing body of biological evidence suggests that Notch-Delta signaling produces other patterns that are not checkerboard, and therefore a new model is needed. Here, we present an expanded Notch-Delta model that builds upon previous models, adding a local Notch activity gradient, which affects long-range patterning, and the activity of a regulatory microRNA. This model is motivated by our experiments in the ascidian Ciona intestinalis showing that the peripheral sensory neurons, whose specification is in part regulated by the coordinate activity of Notch-Delta signaling and the microRNA miR-124, exhibit a sparse spatial pattern whereby consecutive neurons may be spaced over a dozen cells apart. We perform rigorous stability and bifurcation analyses, and demonstrate that our model is able to accurately explain and reproduce the neuronal pattern in Ciona. Using Monte Carlo simulations of our model along with miR-124 transgene over-expression assays, we demonstrate that the activity of miR-124 can be incorporated into the Notch decay rate parameter of our model. Finally, we motivate the general applicability of our model to Notch-Delta signaling in other animals by providing evidence that microRNAs regulate Notch-Delta signaling in analogous cell types in other organisms, and by discussing evidence in other organisms of sparse spatial patterns in tissues where Notch-Delta signaling is active. PMID:24945987

  14. 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

  15. 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

  16. 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.

  17. Notch signaling affects biliary fibrosis via transcriptional regulation of RBP-jκ in an animal model of chronic liver disease

    PubMed Central

    Lee, Sun-Jae; Kim, Kyung-Hyun; Pak, Sok Cheon; Kang, Yu-Na; Yoon, Ghil-Suk; Park, Kwan-Kyu

    2015-01-01

    Liver repair in patients with a chronic liver disease requires the orchestrated action of epithelial, mesenchymal, and inflammatory cells. Notch components are expressed in both the epithelial and mesenchymal compartments of the adult liver and are differentially regulated after injury. However, the functional role of Notch signaling in regulating epithelial/mesenchymal cross-talk during fibrogenic pathologic repair remains unknown. The aim of this study was to investigate how proliferation of the bile duct influences biliary fibrosis and to recognize the effect of inhibiting Notch signaling in biliary fibrotic tissue of the injured liver. We designed a synthetic decoy oligodeoxynucleotide (ODN) for recombination signal binding protein immunoglobulin kappa J (RBP-jκ), which is a common DNA-binding partner of Notch receptors. The effect of blocking RBP-jκ on fibrogenesis was assessed in the 3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet mouse model. We observed the reduced fibrosis and decreased expression of associated signaling molecules after the RBP-jκ decoy ODN treatment. These data demonstrate that Notch signaling may play an important role in progression of ductular reaction and fibrosis. Further studies are required to unveil how ductular cells interact with other liver cell types, such as hepatic stellate cells or Kupffer cells,in patients with cholestatic liver diseases based on Notch signaling. These results suggest that controlling the ductular reaction using a synthetic ring type decoy RBP-jκ ODN will help develop a novel therapeutic approach targeting biliary fibrosis in patients with chronic liver diseases. PMID:26722458

  18. Androgen receptor genomic regulation

    PubMed Central

    Jin, Hong-Jian; Kim, Jung

    2013-01-01

    The transcriptional activity of the androgen receptor (AR) is not only critical for the normal development and function of the prostate but also pivotal to the onset and progression of prostate cancer (PCa). The studies of AR transcriptional regulation were previously limited to a handful of AR-target genes. Owing to the development of various high-throughput genomic technologies, significant advances have been made in recent years. Here we discuss the discoveries of genome-wide androgen-regulated genes in PCa cell lines, animal models and tissues using expression microarray and sequencing, the mapping of genomic landscapes of AR using Combining Chromatin Immunoprecipitation (ChIP)-on-chip and ChIP-seq assays, the interplay of transcriptional cofactors in defining AR binding profiles, and the genomic regulation and AR reprogramming in advanced PCa. PMID:25237629

  19. 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.

  20. Nuclear Localization of the Autism Candidate Gene Neurobeachin and Functional Interaction with the NOTCH1 Intracellular Domain Indicate a Role in Regulating Transcription

    PubMed Central

    Tuand, Krizia; Stijnen, Pieter; Volders, Karolien; Declercq, Jeroen; Nuytens, Kim; Meulemans, Sandra; Creemers, John

    2016-01-01

    Background Neurobeachin (NBEA) is an autism spectrum disorders (ASD) candidate gene. NBEA deficiency affects regulated secretion, receptor trafficking, synaptic architecture and protein kinase A (PKA)-mediated phosphorylation. NBEA is a large multidomain scaffolding protein. From N- to C-terminus, NBEA has a concanavalin A-like lectin domain flanked by armadillo repeats (ACA), an A-kinase anchoring protein domain that can bind to PKA, a domain of unknown function (DUF1088) and a BEACH domain, preceded by a pleckstrin homology-like domain and followed by WD40 repeats (PBW). Although most of these domains mediate protein-protein interactions, no interaction screen has yet been performed. Methods Yeast two-hybrid screens with the ACA and PBW domain modules of NBEA gave a list of interaction partners, which were analyzed for Gene Ontology (GO) enrichment. Neuro-2a cells were used for confocal microscopy and nuclear extraction analysis. NOTCH-mediated transcription was studied with luciferase reporter assays and qRT-PCR, combined with NBEA knockdown or overexpression. Results Both domain modules showed a GO enrichment for the nucleus. PBW almost exclusively interacted with transcription regulators, while ACA interacted with a number of PKA substrates. NBEA was partially localized in the nucleus of Neuro-2a cells, albeit much less than in the cytoplasm. A nuclear localization signal was found in the DUF1088 domain, which was shown to contribute to the nuclear localization of an EGFP-DPBW fusion protein. Yeast two-hybrid identified the Notch1 intracellular domain as a physical interactor of the PBW domain and a role for NBEA as a negative regulator in Notch-mediated transcription was demonstrated. Conclusion Defining novel interaction partners of conserved NBEA domain modules identified a role for NBEA as transcriptional regulator in the nucleus. The physical interaction of NBEA with NOTCH1 is most relevant for ASD pathogenesis because NOTCH signaling is essential for

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. Notch and presenilin regulate cellular expansion and cytokine secretion but cannot instruct Th1/Th2 fate acquisition.

    PubMed

    Ong, Chin-Tong; Sedy, John R; Murphy, Kenneth M; Kopan, Raphael

    2008-01-01

    Recent reports suggested that Delta1, 4 and Jagged1, 2 possessed the ability to instruct CD4(+) T cell into selection of Th1 or Th2 fates, respectively, although the underlying mechanism endowing the cleaved Notch receptor with memory of ligand involved in its activation remains elusive. To examine this, we prepared artificial antigen-presenting cells expressing either DLL1 or Jag1. Although both ligands were efficient in inducing Notch2 cleavage and activation in CD4(+) T or reporter cells, the presence of Lunatic Fringe in CD4(+) T cells inhibited Jag1 activation of Notch1 receptor. Neither ligand could induce Th1 or Th2 fate choice independently of cytokines or redirect cytokine-driven Th1 or Th2 development. Instead, we find that Notch ligands only augment cytokine production during T cell differentiation in the presence of polarizing IL-12 and IL-4. Moreover, the differentiation choices of naïve CD4(+) T cells lacking gamma-secretase, RBP-J, or both in response to polarizing cytokines revealed that neither presenilin proteins nor RBP-J were required for cytokine-induced Th1/Th2 fate selection. However, presenilins facilitate cellular proliferation and cytokine secretion in an RBP-J (and thus, Notch) independent manner. The controversies surrounding the role of Notch and presenilins in Th1/Th2 polarization may reflect their role as genetic modifiers of T-helper cells differentiation. PMID:18665263

  9. 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

  10. 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

  11. 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

  12. 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.

  13. Quantitative DNA hypomethylation of ligand Jagged1 and receptor Notch1 signifies occurrence and progression of breast carcinoma

    PubMed Central

    Cao, Yuwen; Li, Yixiao; Zhang, Na; Hu, Jianming; Yin, Liang; Pan, Zemin; Li, Yucong; Du, Xiaoming; Zhang, Wenjie; Li, Feng

    2015-01-01

    Methylation alterations of Jagged1 and Notch1 genes have been reported in non-tumor lesions and a few cancers. However, methylation profiles of Jagged1 promoter and Notch1 exon25 in breast cancer and matched normal tissue and the association of methylation with clinicopathological characteristics still remain unclear. To explore the potential effects of aberrant DNA methylation of Jagged1 and Notch1 on occurrence and progression of breast cancer, we detected the quantitative DNA methylation of Jagged1 and Notch1 in 73 breast cancer (BC) and 20 adjacent normal breast tissues (ANBT) by using MassARRAY spectrometry. The methylation level of overall and majority individual CpG sites of the two genes were synergistically significantly lower in BC than in ANBT. The overall hypomethylation of the two genes, particularly of Jagged1 CpG_8.9.10 and Notch1 CpG_14.15.16 in primary tumors, were markedly associated with lymph node metastasis, advanced stage and high grade. The protein expressions of the both genes were examined by immunohistochemical staining in same cohorts. The expression was significantly inverse correlation with methylation. The two proteins in primary tumor were synergistically up-regulated and dramatically related to lymph node metastasis, advanced stage and high grade. Our findings suggest that the synergetic hypomethylation of Jagged1 and Notch1 genes, especially of Jagged1 CpG_8.9.10 and Notch1 CpG_14.15.16, may involve tumorigenesis and development of breast cancer. The negative relationship between methylation and expression indicates methylation role for expression regulation. The synergetic overexpression of the two proteins further indicates the effects on occurrence and progression of breast cancer. PMID:26175933

  14. Quantitative DNA hypomethylation of ligand Jagged1 and receptor Notch1 signifies occurrence and progression of breast carcinoma

    PubMed Central

    Cao, Yuwen; Li, Yixiao; Zhang, Na; Hu, Jianming; Yin, Liang; Pan, Zemin; Li, Yucong; Du, Xiaoming; Zhang, Wenjie; Li, Feng

    2015-01-01

    Methylation alterations of Jagged1 and Notch1 genes have been reported in non-tumor lesions and a few cancers. However, methylation profiles of Jagged1 promoter and Notch1 exon25 in breast cancer and matched normal tissue and the association of methylation with clinicopathological characteristics still remain unclear. To explore the potential effects of aberrant DNA methylation of Jagged1 and Notch1 on occurrence and progression of breast cancer, we detected the quantitative DNA methylation of Jagged1 and Notch1 in 73 breast cancer (BC) and 20 adjacent normal breast tissues (ANBT) by using MassARRAY spectrometry. The methylation level of overall and majority individual CpG sites of the two genes were synergistically significantly lower in BC than in ANBT. The overall hypomethylation of the two genes, particularly of Jagged1 CpG_8.9.10 and Notch1 CpG_14.15.16 in primary tumors, were markedly associated with lymph node metastasis, advanced stage and high grade. The protein expressions of the both genes were examined by immunohistochemical staining in same cohorts. The expression was significantly inverse correlation with methylation. The two proteins in primary tumor were synergistically up-regulated and dramatically related to lymph node metastasis, advanced stage and high grade. Our findings suggest that the synergetic hypomethylation of Jagged1 and Notch1 genes, especially of Jagged1 CpG_8.9.10 and Notch1 CpG_14.15.16, may involve tumorigenesis and development of breast cancer. The negative relationship between methylation and expression indicates methylation role for expression regulation. The synergetic overexpression of the two proteins further indicates the effects on occurrence and progression of breast cancer. PMID:26269752

  15. Chromatin signatures at Notch-regulated enhancers reveal large-scale changes in H3K56ac upon activation

    PubMed Central

    Skalska, Lenka; Stojnic, Robert; Li, Jinghua; Fischer, Bettina; Cerda-Moya, Gustavo; Sakai, Hiroshi; Tajbakhsh, Shahragim; Russell, Steven; Adryan, Boris; Bray, Sarah J

    2015-01-01

    The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target selection and gene activation in each context. To investigate the influence of chromatin organisation and dynamics on the response to Notch signalling, we partitioned Drosophila chromatin using histone modifications and established the preferred chromatin conditions for binding of Su(H), the Notch pathway transcription factor. By manipulating activity of a co-operating factor, Lozenge/Runx, we showed that it can help facilitate these conditions. While many histone modifications were unchanged by Su(H) binding or Notch activation, we detected rapid changes in acetylation of H3K56 at Notch-regulated enhancers. This modification extended over large regions, required the histone acetyl-transferase CBP and was independent of transcription. Such rapid changes in H3K56 acetylation appear to be a conserved indicator of enhancer activation as they also occurred at the mammalian Notch-regulated Hey1 gene and at Drosophila ecdysone-regulated genes. This intriguing example of a core histone modification increasing over short timescales may therefore underpin changes in chromatin accessibility needed to promote transcription following signalling activation. PMID:26069324

  16. Chromatin signatures at Notch-regulated enhancers reveal large-scale changes in H3K56ac upon activation.

    PubMed

    Skalska, Lenka; Stojnic, Robert; Li, Jinghua; Fischer, Bettina; Cerda-Moya, Gustavo; Sakai, Hiroshi; Tajbakhsh, Shahragim; Russell, Steven; Adryan, Boris; Bray, Sarah J

    2015-07-14

    The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target selection and gene activation in each context. To investigate the influence of chromatin organisation and dynamics on the response to Notch signalling, we partitioned Drosophila chromatin using histone modifications and established the preferred chromatin conditions for binding of Su(H), the Notch pathway transcription factor. By manipulating activity of a co-operating factor, Lozenge/Runx, we showed that it can help facilitate these conditions. While many histone modifications were unchanged by Su(H) binding or Notch activation, we detected rapid changes in acetylation of H3K56 at Notch-regulated enhancers. This modification extended over large regions, required the histone acetyl-transferase CBP and was independent of transcription. Such rapid changes in H3K56 acetylation appear to be a conserved indicator of enhancer activation as they also occurred at the mammalian Notch-regulated Hey1 gene and at Drosophila ecdysone-regulated genes. This intriguing example of a core histone modification increasing over short timescales may therefore underpin changes in chromatin accessibility needed to promote transcription following signalling activation. PMID:26069324

  17. New insights into receptor regulation.

    PubMed

    Poste, G

    1984-11-01

    This review provides a brief summary of certain recent advances in our understanding of receptor regulation, signal transduction, and the diverse pathways by which receptor-ligand complexes are internalized and delivered to specific organelles, together with recycling of receptors back to the cell surface. Emphasis is also given to the importance of methodological advances in receptor isolation, immunologic analysis of receptor structure and function, the development of new instrumentation for microchemical characterization of very small amounts of receptor material, and the increasing use of genetic engineering techniques to isolate the genes for receptors and their regulatory subunits, to transfer such genes between cells, and to study receptor function by creating structurally modified receptors via subtle changes in gene structure. PMID:6151557

  18. Proton myo-inositol cotransporter is a novel γ-secretase associated protein that regulates Aβ production without affecting Notch cleavage.

    PubMed

    Teranishi, Yasuhiro; Inoue, Mitsuhiro; Yamamoto, Natsuko Goto; Kihara, Takahiro; Wiehager, Birgitta; Ishikawa, Taizo; Winblad, Bengt; Schedin-Weiss, Sophia; Frykman, Susanne; Tjernberg, Lars O

    2015-09-01

    γ-Secretase is a transmembrane protease complex that is responsible for the processing of a multitude of type 1 transmembrane proteins, including the amyloid precursor protein and Notch. γ-Secretase processing of amyloid precursor protein results in the release of the amyloid β-peptide (Aβ), which is involved in the pathogenesis in Alzheimer's disease. Processing of Notch leads to the release of its intracellular domain, which is important for cell development. γ-Secretase associated proteins (GSAPs) could be of importance for substrate selection, and we have previously shown that affinity purification of γ-secretase in combination with mass spectrometry can be used for finding such proteins. In the present study, we used this methodology to screen for novel GSAPs from human brain, and studied their effect on Aβ production in a comprehensive gene knockdown approach. Silencing of probable phospholipid-transporting ATPase IIA, brain-derived neurotrophic factor/neurotrophin-3 growth factor receptor precursor and proton myo-inositol cotransporter (SLC2A13) showed a clear reduction of Aβ and these proteins were selected for further studies on Aβ production and Notch cleavage using small interfering RNA-mediated gene silencing, as well as an overexpression approach. Silencing of these reduced Aβ secretion in a small interfering RNA dose-dependent manner. Interestingly, SLC2A13 had a lower effect on Notch processing. Furthermore, overexpression of SLC2A13 increased Aβ40 generation. Finally, the interaction between γ-secretase and SLC2A13 was confirmed using immunoprecipitation and a proximity ligation assay. In summary, SLC2A13 was identified as a novel GSAP that regulates Aβ production without affecting Notch cleavage. We suggest that SLC2A13 could be a target for Aβ lowering therapy aimed at treating Alzheimer's disease. PMID:26094765

  19. 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

  20. 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

  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. Structural basis for Notch1 engagement of Delta-like 4

    SciTech Connect

    Luca, Vincent C.; Jude, Kevin M.; Pierce, Nathan W.; Nachury, Maxence V.; Fischer, Suzanne; Garcia, K. Christopher

    2015-02-20

    Notch receptors guide mammalian cell fate decisions by engaging the proteins Jagged and Delta-like (DLL). The 2.3 angstrom resolution crystal structure of the interacting regions of the Notch1-DLL4 complex reveals a two-site, antiparallel binding orientation assisted by Notch1 O-linked glycosylation. Notch1 epidermal growth factor–like repeats 11 and 12 interact with the DLL4 Delta/Serrate/Lag-2 (DSL) domain and module at the N-terminus of Notch ligands (MNNL) domains, respectively. Threonine and serine residues on Notch1 are functionalized with O-fucose and O-glucose, which act as surrogate amino acids by making specific, and essential, contacts to residues on DLL4. Lastly, the elucidation of a direct chemical role for O-glycans in Notch1 ligand engagement demonstrates how, by relying on posttranslational modifications of their ligand binding sites, Notch proteins have linked their functional capacity to developmentally regulated biosynthetic pathways.

  3. GDE2 regulates subtype-specific motor neuron generation through inhibition of Notch signaling.

    PubMed

    Sabharwal, Priyanka; Lee, Changhee; Park, Sungjin; Rao, Meenakshi; Sockanathan, Shanthini

    2011-09-22

    The specification of spinal interneuron and motor neuron identities initiates within progenitor cells, while motor neuron subtype diversification is regulated by hierarchical transcriptional programs implemented postmitotically. Here we find that mice lacking GDE2, a six-transmembrane protein that triggers motor neuron generation, exhibit selective losses of distinct motor neuron subtypes, specifically in defined subsets of limb-innervating motor pools that correlate with the loss of force-generating alpha motor neurons. Mechanistically, GDE2 is expressed by postmitotic motor neurons but utilizes extracellular glycerophosphodiester phosphodiesterase activity to induce motor neuron generation by inhibiting Notch signaling in neighboring motor neuron progenitors. Thus, neuronal GDE2 controls motor neuron subtype diversity through a non-cell-autonomous feedback mechanism that directly regulates progenitor cell differentiation, implying that subtype specification initiates within motor neuron progenitor populations prior to their differentiation into postmitotic motor neurons.

  4. 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

  5. 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

  6. Dynamic Changes in microRNAs may Regulate Robustness of Wnt/Notch Signaling

    NASA Astrophysics Data System (ADS)

    Gunaratne, Preethi

    2008-03-01

    The mechanisms by which highly reproducible patterns are formed during embryonic development and organismal evolution despite stochasticity at the single cell level is one of the remaining mysteries in Biology. It has been proposed that a hidden layer of regulation formed through the interaction of microRNAs with protein coding gene networks maybe responsible. Recently developed next generation sequencing technologies afford an unprecedented opportunity to uncover novel aspects of miRNA function and evolution. We find extensive heterogeneity in sequences that correspond to mmu-let-7 (targets Wnt1) and mmu-miR-191 (targets Notch1). Approximately 20% of let-7 and miR-191 have undergone modifications to increase stability and binding to the Wnt1 and Notch1 targets and are likely to be destroyed. In contrast, 80% bind the targets with imperfect complementarity and lower stability and are likely to be sequestered and prevented from forming protein. We propose that these two species together form a highly fluid system that is able to absorb stochastic perturbations in gene expression. A gene that goes on to be translated into functional protein therefore must escape both buffers by significantly high expression.

  7. Numb is not a critical regulator of Notch-mediated cell fate decisions in the developing chick inner ear

    PubMed Central

    Eddison, Mark; Weber, Sara J.; Ariza-McNaughton, Linda; Lewis, Julian; Daudet, Nicolas

    2015-01-01

    The Notch signaling pathway controls differentiation of hair cells and supporting cells in the vertebrate inner ear. Here, we have investigated whether Numb, a known regulator of Notch activity in Drosophila, is involved in this process in the embryonic chick. The chicken homolog of Numb is expressed throughout the otocyst at early stages of development and is concentrated at the basal pole of the cells. It is asymmetrically allocated at some cell divisions, as in Drosophila, suggesting that it could act as a determinant inherited by one of the two daughter cells and favoring adoption of a hair-cell fate. To test the implication of Numb in hair cell fate decisions and the regulation of Notch signaling, we used different methods to overexpress Numb at different stages of inner ear development. We found that sustained or late Numb overexpression does not promote hair cell differentiation, and Numb does not prevent the reception of Notch signaling. Surprisingly, none of the Numb-overexpressing cells differentiated into hair cells, suggesting that high levels of Numb protein could interfere with intracellular processes essential for hair cell survival. However, when Numb was overexpressed early and more transiently during ear development, no effect on hair cell formation was seen. These results suggest that in the inner ear at least, Numb does not significantly repress Notch activity and that its asymmetric distribution in dividing precursor cells does not govern the choice between hair cell and supporting cell fates. PMID:25814931

  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. 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

  10. The Notch Ligand Delta-Like 4 Regulates Multiple Stages of Early Hemato-Vascular Development

    PubMed Central

    Neves, Hélia; Gomes, Andreia C.; Saavedra, Pedro; Carvalho, Catarina C.; Duarte, António; Cidadão, António; Parreira, Leonor

    2012-01-01

    Background In mouse embryos, homozygous or heterozygous deletions of the gene encoding the Notch ligand Dll4 result in early embryonic death due to major defects in endothelial remodeling in the yolk sac and embryo. Considering the close developmental relationship between endothelial and hematopoietic cell lineages, which share a common mesoderm-derived precursor, the hemangioblast, and many key regulatory molecules, we investigated whether Dll4 is also involved in the regulation of early embryonic hematopoiesis. Methodology/Principal Findings Using Embryoid Bodies (EBs) derived from embryonic stem cells harboring hetero- or homozygous Dll4 deletions, we observed that EBs from both genotypes exhibit an abnormal endothelial remodeling in the vascular sprouts that arise late during EB differentiation, indicating that this in vitro system recapitulates the angiogenic phenotype of Dll4 mutant embryos. However, analysis of EB development at early time points revealed that the absence of Dll4 delays the emergence of mesoderm and severely reduces the number of blast-colony forming cells (BL-CFCs), the in vitro counterpart of the hemangioblast, and of endothelial cells. Analysis of colony forming units (CFU) in EBs and yolk sacs from Dll4+/− and Dll4−/− embryos, showed that primitive erythropoiesis is specifically affected by Dll4 insufficiency. In Dll4 mutant EBs, smooth muscle cells (SMCs) were seemingly unaffected and cardiomyocyte differentiation was increased, indicating that SMC specification is Dll4-independent while a normal dose of this Notch ligand is essential for the quantitative regulation of cardiomyogenesis. Conclusions/Significance This study highlights a previously unnoticed role for Dll4 in the quantitative regulation of early hemato-vascular precursors, further indicating that it is also involved on the timely emergence of mesoderm in early embryogenesis. PMID:22514637

  11. Receptor tyrosine phosphatase psi is required for Delta/Notch signalling and cyclic gene expression in the presomitic mesoderm.

    PubMed

    Aerne, Birgit; Ish-Horowicz, David

    2004-07-01

    Segmentation in vertebrate embryos is controlled by a biochemical oscillator ('segmentation clock') intrinsic to the cells in the unsegmented presomitic mesoderm, and is manifested in cyclic transcription of genes involved in establishing somite polarity and boundaries. We show that the receptor protein tyrosine phosphatase psi (RPTPpsi) gene is essential for normal functioning of the somitogenesis clock in zebrafish. We show that reduction of RPTPpsi activity using morpholino antisense oligonucleotides results in severe disruption of the segmental pattern of the embryo, and loss of cyclic gene expression in the presomitic mesoderm. Analysis of cyclic genes in RPTPpsi morphant embryos indicates an important requirement for RPTPpsi in the control of the somitogenesis clock upstream of or in parallel with Delta/Notch signalling. Impairing RPTPpsi activity also interferes with convergent extension during gastrulation. We discuss this dual requirement for RPTPpsi in terms of potential functions in Notch and Wnt signalling. PMID:15226256

  12. Notch-ligand expression by NALT dendritic cells regulates mucosal Th1- and Th2-type responses

    SciTech Connect

    Fukuyama, Yoshiko; Tokuhara, Daisuke; Sekine, Shinichi; Kataoka, Kosuke; Markham, Jonathan D.; Irwin, Allyson R.; Moon, Grace H.; Tokuhara, Yuka; Fujihashi, Keiko; Davydova, Julia; Yamamoto, Masato; Gilbert, Rebekah S.; Fujihashi, Kohtaro

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer Nasal Ad-FL effectively up-regulates APC function by CD11c{sup +} DCs in mucosal tissues. Black-Right-Pointing-Pointer Nasal Ad-FL induces Notch ligand (L)-expressing CD11c{sup +} DCs. Black-Right-Pointing-Pointer Notch L-expressing DCs support the induction of Th1- and Th2-type cytokine responses. -- Abstract: Our previous studies showed that an adenovirus (Ad) serotype 5 vector expressing Flt3 ligand (Ad-FL) as nasal adjuvant activates CD11c{sup +} dendritic cells (DCs) for the enhancement of antigen (Ag)-specific IgA antibody (Ab) responses. In this study, we examined the molecular mechanism for activation of CD11c{sup +} DCs and their roles in induction of Ag-specific Th1- and Th2-cell responses. Ad-FL activated CD11c{sup +} DCs expressed increased levels of the Notch ligand (L)-expression and specific mRNA. When CD11c{sup +} DCs from various mucosal and systemic lymphoid tissues of mice given nasal OVA plus Ad-FL were cultured with CD4{sup +} T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice, significantly increased levels of T cell proliferative responses were noted. Furthermore, Ad-FL activated DCs induced IFN-{gamma}, IL-2 and IL-4 producing CD4{sup +} T cells. Of importance, these APC functions by Ad-FL activated DCs were down-regulated by blocking Notch-Notch-L pathway. These results show that Ad-FL induces CD11c{sup +} DCs to the express Notch-ligands and these activated DCs regulate the induction of Ag-specific Th1- and Th2-type cytokine responses.

  13. Notch2 is required for maintaining sustentacular cell function in the adult mouse main olfactory epithelium

    PubMed Central

    Rodriguez, Steve; Sickles, Heather M.; DeLeonardis, Chris; Alcaraz, Ana; Gridley, Thomas; Lin, David M.

    2008-01-01

    Notch receptors are expressed in neurons and glia in the adult nervous system, but why this expression persists is not well-understood. Here we examine the role of the Notch pathway in the postnatal mouse main olfactory system, and show evidence consistent with a model where Notch2 is required for maintaining sustentacular cell function. In the absence of Notch2, the laminar nature of these glial-like cells is disrupted. Hes1, Hey1, and Six1, which are downstream effectors of the Notch pathway, are down-regulated, and cytochrome P450 and Glutathione S-transferase (GST) expression by sustentacular cells is reduced. Functional levels of GST activity are also reduced. These disruptions are associated with increased olfactory sensory neuron degeneration. Surprisingly, expression of Notch3 is also down-regulated. This suggests the existence of a feedback loop where expression of Notch3 is initially independent of Notch2, but requires Notch2 for maintained expression. While the Notch pathway has previously been shown to be important for promoting gliogenesis during development, this is the first demonstration that the persistent expression of Notch receptors is required for maintaining glial function in adult. PMID:18155189

  14. 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

  15. 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

  16. Notch signaling differentially regulates the cell fate of early endocrine precursor cells and their maturing descendants in the mouse pancreas and intestine.

    PubMed

    Li, Hui Joyce; Kapoor, Archana; Giel-Moloney, Maryann; Rindi, Guido; Leiter, Andrew B

    2012-11-15

    Notch signaling inhibits differentiation of endocrine cells in the pancreas and intestine. In a number of cases, the observed inhibition occurred with Notch activation in multipotential cells, prior to the initiation of endocrine differentiation. It has not been established how direct activation of Notch in endocrine precursor cells affects their subsequent cell fate. Using conditional activation of Notch in cells expressing Neurogenin3 or NeuroD1, we examined the effects of Notch in both organs, on cell fate of early endocrine precursors and maturing endocrine-restricted cells, respectively. Notch did not preclude the differentiation of a limited number of endocrine cells in either organ when activated in Ngn3(+) precursor cells. In addition, in the pancreas most Ngn3(+) cells adopted a duct but not acinar cell fate; whereas in intestinal Ngn3(+) cells, Notch favored enterocyte and goblet cell fates, while selecting against endocrine and Paneth cell differentiation. A small fraction of NeuroD1(+) cells in the pancreas retain plasticity to respond to Notch, giving rise to intraislet ductules as well as cells with no detectable pancreatic lineage markers that appear to have limited ultrastructural features of both endocrine and duct cells. These results suggest that Notch directly regulates cell fate decisions in multipotential early endocrine precursor cells. Some maturing endocrine-restricted NeuroD1(+) cells in the pancreas switch to the duct lineage in response to Notch, indicating previously unappreciated plasticity at such a late stage of endocrine differentiation.

  17. 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.

  18. 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.

  19. 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

  20. Glycogen synthase kinase 3 beta positively regulates Notch signaling in vascular smooth muscle cells: role in cell proliferation and survival.

    PubMed

    Guha, Shaunta; Cullen, John P; Morrow, David; Colombo, Alberto; Lally, Caitríona; Walls, Dermot; Redmond, Eileen M; Cahill, Paul A

    2011-09-01

    The role of glycogen synthase kinase 3 beta (GSK-3β) in modulating Notch control of vascular smooth muscle cell (vSMC) growth (proliferation and apoptosis) was examined in vitro under varying conditions of cyclic strain and validated in vivo following changes in medial tension and stress. Modulation of GSK-3β in vSMC following ectopic expression of constitutively active GSK-3β, siRNA knockdown and pharmacological inhibition with SB-216763 demonstrated that GSK-3β positively regulates Notch intracellular domain expression, CBF-1/RBP-Jκ transactivation and downstream target gene mRNA levels, while concomitantly promoting vSMC proliferation and inhibiting apoptosis. In contrast, inhibition of GSK-3β attenuated Notch signaling and decreased vSMC proliferation and survival. Exposure of vSMC to cyclic strain environments in vitro using both a Flexercell™ Tension system and a novel Sylgard™ phantom vessel following bare metal stent implantation revealed that cyclic strain inhibits GSK-3β activity independent of p42/p44 MAPK and p38 activation concomitant with reduced Notch signaling and decreased vSMC proliferation and survival. Exposure of vSMC to changes in medial strain microenvironments in vivo following carotid artery ligation revealed that enhanced GSK-3β activity was predominantly localized to medial and neointimal vSMC concomitant with increased Notch signaling, proliferating nuclear antigen and decreased Bax expression, respectively, as vascular remodeling progressed. GSK-3β is an important modulator of Notch signaling leading to altered vSMC cell growth where low strain/tension microenvironments prevail.

  1. 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

  2. Clinical Impact of De-Regulated Notch-1 and Notch-3 in the Development and Progression of HPV-Associated Different Histological Subtypes of Precancerous and Cancerous Lesions of Human Uterine Cervix

    PubMed Central

    Tripathi, Richa; Rath, Gayatri; Jawanjal, Poonam; Sharma, Shweta; Singhal, Pallavi; Bhambhani, Suresh; Hussain, Showket; Bharadwaj, Mausumi

    2014-01-01

    Background Cervical cancer is the leading cause of cancer related deaths among women in India. Limited reports are available for Notch-1 and Notch-3 protein in cervical carcinoma, which play crucial role in cell proliferation, differentiation, and apoptosis. Methods This study was designed to evaluate the role of Notch-1 and Notch-3 with context to HPV infection in cervical carcinoma. A total of 168 tissue biopsy samples comprising of tumor specimens (n = 98), precancer (n = 30) and non-neoplastic cervical tissues (n = 40) were screened for HPV infection by PCR and expression of Notch-1 and Notch-3 protein by Immunohistochemistry and Immunoblotting. Results 80% (24/30) were found to be positive for HPV in precancer and 86.7% (85/98) in cancer patients. Notch-1 expression of precancer and cancer cases was found to be significantly down-regulated with severity of disease in nuclear (3.43±0.29; 2.04±0.19, p = 0.0001, p = 0.0001) and cytoplasm (3.07±0.29; 2.29±0.17, p = 0.0001, p = 0.0001) obtained from different stages as compared to normal cervix tissue (5.40±0.19, 4.97±0.15; p<0.001; p<0.001). However, Notch-3 expression of above cases was significantly up-regulated with severity of disease and showed intense nuclear (4.17±0.39; 4.74±0.18, p = 0.0001, p = 0.0001) and cytoplasm (3.67±0.36; 4.48±0.18, p = 0.0001, p = 0.0001) of different stages as compared to normal cervix tissue (0.95±0.20, 0.70±0.20; p<0.001; p<0.001) respectively. Conclusions These findings suggest that Notch-1 and Notch-3 may play an important role with synergistic effect of HPV in regulating development and proliferation of cervical cancer through the deregulation of Notch signalling. This study also shows the clinical utility of both proteins which may be used as predictable biomarkers in diagnosing different histological sub-types of HPV associated cervical cancer. Nevertheless, abnormal activation of this pathway may provide legitimate

  3. Strawberry notch homologue 2 regulates osteoclast fusion by enhancing the expression of DC-STAMP.

    PubMed

    Maruyama, Kenta; Uematsu, Satoshi; Kondo, Takeshi; Takeuchi, Osamu; Martino, Mikaël M; Kawasaki, Takumi; Akira, Shizuo

    2013-09-23

    Osteoclasts are multinucleated cells formed by fusion of mononuclear precursors in response to receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). We found that RANKL induced expression of the DExD/H helicase family corepressor strawberry notch homologue 2 (Sbno2). Previous in vitro studies showed that Sbno2 is induced by IL-10 and is involved in NF-κB repression. However, the role of Sbno2 in vivo and its pleiotropic functions are unknown. Sbno2 gene targeting resulted in normal NF-κB activation by TLR ligands. However, Sbno2-deficient mice exhibited increased bone mass due to impaired osteoclast fusion. Expression of dendritic cell-specific transmembrane protein (DC-STAMP), a critical player in osteoclast fusion, was significantly attenuated, and cell fusion of Sbno2-deficient osteoclasts was rescued by DC-STAMP. Sbno2 directly bound to T cell acute lymphocytic leukemia 1 (Tal1) and attenuated its inhibition of DC-STAMP expression, leading to activation of the DC-STAMP promoter by microphthalmia-associated transcription factor (MITF). Thus, Sbno2 plays a pivotal role in bone homeostasis in vivo by fine-tuning osteoclast fusion.

  4. The conserved clusterin gene is expressed in the developing choroid plexus under the regulation of notch but not IGF signaling in zebrafish.

    PubMed

    Jiao, Shuang; Dai, Wei; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng; Li, Yun; Korzh, Vladimir; Duan, Cunming

    2011-05-01

    Recent genome-wide association studies have implicated the clusterin gene in the etiology of Alzheimer's disease. The expression and function of clusterin in the developing brain, however, is poorly understood. In this study, we have characterized the zebrafish clusterin gene and determined its structural conservation, developmental expression, and physiological regulation. The structure of the zebrafish clusterin gene and protein is similar to its human orthologue. Biochemical assays show that zebrafish Clusterin is a secreted protein that cannot bind IGFs. In adult zebrafish, clusterin mRNA is detected in many tissues. In early development, clusterin mRNA becomes detectable at 12 h postfertilization, and its levels gradually increase thereafter. In situ hybridization analysis indicates that clusterin mRNA is specifically expressed in the developing diencephalic and myelencephalic choroid plexus. Among various stresses tested, heat shock, but not hypoxic or ionic stresses, increases the levels of clusterin mRNA. Inhibition of the IGF-I receptor-mediated signaling or overexpression of IGF ligands did not change clusterin mRNA levels. In comparison, inhibition or targeted knockdown of Notch signaling significantly increased clusterin mRNA expression in choroid plexus. These results suggest that clusterin is a marker of choroid plexus in zebrafish, and its expression in the developing choroid plexus is under the regulation of Notch but not IGF signaling.

  5. Notch regulation of progenitor cell behavior in quiescent and regenerating auditory epithelium of mature birds

    PubMed Central

    Daudet, Nicolas; Gibson, Robin; Shang, Jialin; Bernard, Amy; Lewis, Julian; Stone, Jennifer

    2009-01-01

    Unlike mammals, birds regenerate auditory hair cells (HCs) after injury. During regeneration, mature non-sensory supporting cells (SCs) leave quiescence and convert into HCs, through non-mitotic or mitotic mechanisms. During embryogenesis, Notch ligands from nascent HCs exert lateral inhibition, restricting HC production. Here, we examined whether Notch signalling (1) is needed in mature birds to maintain the HC/SC pattern in the undamaged auditory epithelium or (2) governs SC behavior once HCs are injured. We show that Notch pathway genes are transcribed in the mature undamaged epithelium, and after HC injury, their transcription is upregulated in the region of highest mitotic activity. In vitro treatment with DAPT, an inhibitor of Notch activity, had no effect on SCs in the undamaged epithelium. Following HC damage, DAPT had no direct effect on SC division. However, after damage, DAPT caused excessive regeneration of HCs at the expense of SCs, through both mitotic and non-mitotic mechanisms. Conversely, overexpression of activated Notch in SCs after damage caused them to maintain their phenotype and inhibited HC regeneration. Therefore, signalling through Notch is not required for SC quiescence in the healthy epithelium or to initiate HC regeneration after damage. Rather, Notch prevents SCs from regenerating excessive HCs after damage. PMID:19013445

  6. 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

  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. 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. 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

  10. Activation of an endothelial Notch1-Jagged1 circuit induces VCAM1 expression, an effect amplified by interleukin-1β.

    PubMed

    Verginelli, Federica; Adesso, Laura; Limon, Isabelle; Alisi, Anna; Gueguen, Marie; Panera, Nadia; Giorda, Ezio; Raimondi, Lavinia; Ciarapica, Roberta; Campese, Antonio F; Screpanti, Isabella; Stifani, Stefano; Kitajewski, Jan; Miele, Lucio; Rota, Rossella; Locatelli, Franco

    2015-12-22

    The Notch1 and Notch4 signaling pathways regulate endothelial cell homeostasis. Inflammatory cytokines induce the expression of endothelial adhesion molecules, including VCAM1, partly by downregulating Notch4 signaling. We investigated the role of endothelial Notch1 in this IL-1β-mediated process. Brief treatment with IL-1β upregulated endothelial VCAM1 and Notch ligand Jagged1. IL-1β decreased Notch1 mRNA levels, but levels of the active Notch1ICD protein remained constant. IL-1β-mediated VCAM1 induction was downregulated in endothelial cells subjected to pretreatment with a pharmacological inhibitor of the γ-secretase, which activates Notch receptors, producing NotchICD. It was also downregulated in cells in which Notch1 and/or Jagged1 were silenced.Conversely, the forced expression of Notch1ICD in naïve endothelial cells upregulated VCAM1 per se and amplified IL-1β-mediated VCAM1 induction. Jagged1 levels increased and Notch4 signaling was downregulated in parallel. Finally, Notch1ICD and Jagged1 expression was upregulated in the endothelium of the liver in a model of chronic liver inflammation.In conclusion, we describe here a cell-autonomous, pro-inflammatory endothelial Notch1-Jagged1 circuit (i) triggering the expression of VCAM1 even in the absence of inflammatory cytokines and (ii) enhancing the effects of IL-1β. Thus, IL-1β regulates Notch1 and Notch4 activity in opposite directions, consistent with a selective targeting of Notch1 in inflamed endothelium.

  11. Activation of an endothelial Notch1-Jagged1 circuit induces VCAM1 expression, an effect amplified by interleukin-1β.

    PubMed

    Verginelli, Federica; Adesso, Laura; Limon, Isabelle; Alisi, Anna; Gueguen, Marie; Panera, Nadia; Giorda, Ezio; Raimondi, Lavinia; Ciarapica, Roberta; Campese, Antonio F; Screpanti, Isabella; Stifani, Stefano; Kitajewski, Jan; Miele, Lucio; Rota, Rossella; Locatelli, Franco

    2015-12-22

    The Notch1 and Notch4 signaling pathways regulate endothelial cell homeostasis. Inflammatory cytokines induce the expression of endothelial adhesion molecules, including VCAM1, partly by downregulating Notch4 signaling. We investigated the role of endothelial Notch1 in this IL-1β-mediated process. Brief treatment with IL-1β upregulated endothelial VCAM1 and Notch ligand Jagged1. IL-1β decreased Notch1 mRNA levels, but levels of the active Notch1ICD protein remained constant. IL-1β-mediated VCAM1 induction was downregulated in endothelial cells subjected to pretreatment with a pharmacological inhibitor of the γ-secretase, which activates Notch receptors, producing NotchICD. It was also downregulated in cells in which Notch1 and/or Jagged1 were silenced.Conversely, the forced expression of Notch1ICD in naïve endothelial cells upregulated VCAM1 per se and amplified IL-1β-mediated VCAM1 induction. Jagged1 levels increased and Notch4 signaling was downregulated in parallel. Finally, Notch1ICD and Jagged1 expression was upregulated in the endothelium of the liver in a model of chronic liver inflammation.In conclusion, we describe here a cell-autonomous, pro-inflammatory endothelial Notch1-Jagged1 circuit (i) triggering the expression of VCAM1 even in the absence of inflammatory cytokines and (ii) enhancing the effects of IL-1β. Thus, IL-1β regulates Notch1 and Notch4 activity in opposite directions, consistent with a selective targeting of Notch1 in inflamed endothelium. PMID:26646450

  12. VEGF, Notch and TGFβ/BMPs in regulation of sprouting angiogenesis and vascular patterning.

    PubMed

    Jin, Yi; Kaluza, David; Jakobsson, Lars

    2014-12-01

    The blood vasculature is constantly adapting to meet the demand from tissue. In so doing, branches may form, reorganize or regress. These complex processes employ integration of multiple signalling cascades, some of them being restricted to endothelial and mural cells and, hence, suitable for targeting of the vasculature. Both genetic and drug targeting experiments have demonstrated the requirement for the vascular endothelial growth factor (VEGF) system, the Delta-like-Notch system and the transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) cascades in vascular development. Although several of these signalling cascades in part converge into common downstream components, they differ in temporal and spatial regulation and expression. For example, the pro-angiogenic VEGFA is secreted by cells in need of oxygen, presented to the basal side of the endothelium, whereas BMP9 and BMP10 are supplied via the bloodstream in constant interaction with the apical side to suppress angiogenesis. Delta-like 4 (DLL4), on the other hand, is provided as an endothelial membrane bound ligand. In the present article, we discuss recent data on the integration of these pathways in the process of sprouting angiogenesis and vascular patterning and malformation.

  13. Jarid2 (Jumonji, AT rich interactive domain 2) regulates NOTCH1 expression via histone modification in the developing heart.

    PubMed

    Mysliwiec, Matthew R; Carlson, Clayton D; Tietjen, Josh; Hung, Holly; Ansari, Aseem Z; Lee, Youngsook

    2012-01-01

    Jarid2/Jumonji, the founding member of the Jmj factor family, critically regulates various developmental processes, including cardiovascular development. The Jmj family was identified as histone demethylases, indicating epigenetic regulation by Jmj proteins. Deletion of Jarid2 in mice resulted in cardiac malformation and increased endocardial Notch1 expression during development. Although Jarid2 has been shown to occupy the Notch1 locus in the developing heart, the precise molecular role of Jarid2 remains unknown. Here we show that deletion of Jarid2 results in reduced methylation of lysine 9 on histone H3 (H3K9) at the Notch1 genomic locus in embryonic hearts. Interestingly, SETDB1, a histone H3K9 methyltransferase, was identified as a putative cofactor of Jarid2 by yeast two-hybrid screening, and the physical interaction between Jarid2 and SETDB1 was confirmed by coimmunoprecipitation experiments. Concurrently, accumulation of SETDB1 at the site of Jarid2 occupancy was significantly reduced in Jarid2 knock out (KO) hearts. Employing genome-wide approaches, putative Jarid2 target genes regulated by SETDB1 via H3K9 methylation were identified in the developing heart by ChIP-chip. These targets are involved in biological processes that, when dysregulated, could manifest in the phenotypic defects observed in Jarid2 KO mice. Our data demonstrate that Jarid2 functions as a transcriptional repressor of target genes, including Notch1, through a novel process involving the modification of H3K9 methylation via specific interaction with SETDB1 during heart development. Therefore, our study provides new mechanistic insights into epigenetic regulation by Jarid2, which will enhance our understanding of the molecular basis of other organ development and biological processes.

  14. 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

  15. Notch 1 Receptor, Delta 1 Ligand and HES 1 Transcription Factor are Expressed in the Lining Epithelium of Periapical Cysts (Preliminary Study)

    PubMed Central

    Meliou, E; Kerezoudis, NP; Tosios, KI; Kiaris, H

    2010-01-01

    Periapical cyst is a chronic inflammatory disorder of periradicular tissues. The precise pathological mechanisms involved in periapical cyst enlargement remain unclear. Notch signaling is an evolutionarily conserved pathway with a regulatory role in cell fate decisions during development and in carcinogenesis. To date, there are no published data available on the expression of Notch signaling components in periapical cysts or any other jaw cyst. In this immunohistochemical study we have examined the expression of the receptor Notch 1, the ligand Delta 1 and the transcription factor HES 1 in the epithelium of well defined periapical cysts. Immunostaining reaction of Notch 1, Delta 1 and HES 1 was observed in the cytoplasm and/or the cytoplasmic membrane and occasionally in the nucleus in the majority of epithelial cells of all periapical cysts. The present observations indicate that Notch pathway is active in the epithelium of periapical cysts. It can be speculated that activation of epithelial cells of periapical cysts is associated with activation of Notch pathway and imply involvement of this pathway in periapical cyst growth and expansion. PMID:21116324

  16. Notch 1 Receptor, Delta 1 Ligand and HES 1 Transcription Factor are Expressed in the Lining Epithelium of Periapical Cysts (Preliminary Study).

    PubMed

    Meliou, E; Kerezoudis, Np; Tosios, Ki; Kiaris, H

    2010-01-01

    Periapical cyst is a chronic inflammatory disorder of periradicular tissues. The precise pathological mechanisms involved in periapical cyst enlargement remain unclear. Notch signaling is an evolutionarily conserved pathway with a regulatory role in cell fate decisions during development and in carcinogenesis. To date, there are no published data available on the expression of Notch signaling components in periapical cysts or any other jaw cyst. In this immunohistochemical study we have examined the expression of the receptor Notch 1, the ligand Delta 1 and the transcription factor HES 1 in the epithelium of well defined periapical cysts. Immunostaining reaction of Notch 1, Delta 1 and HES 1 was observed in the cytoplasm and/or the cytoplasmic membrane and occasionally in the nucleus in the majority of epithelial cells of all periapical cysts. The present observations indicate that Notch pathway is active in the epithelium of periapical cysts. It can be speculated that activation of epithelial cells of periapical cysts is associated with activation of Notch pathway and imply involvement of this pathway in periapical cyst growth and expansion.

  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. Activated Notch1 Target Genes during Embryonic Cell Differentiation Depend on the Cellular Context and Include Lineage Determinants and Inhibitors

    PubMed Central

    Meier-Stiegen, Franziska; Schwanbeck, Ralf; Bernoth, Kristina; Martini, Simone; Hieronymus, Thomas; Ruau, David; Zenke, Martin; Just, Ursula

    2010-01-01

    Background Notch receptor signaling controls developmental cell fates in a cell-context dependent manner. Although Notch signaling directly regulates transcription via the RBP-J/CSL DNA binding protein, little is known about the target genes that are directly activated by Notch in the respective tissues. Methodology/Principal Findings To analyze how Notch signaling mediates its context dependent function(s), we utilized a Tamoxifen-inducible system to activate Notch1 in murine embryonic stem cells at different stages of mesodermal differentiation and performed global transcriptional analyses. We find that the majority of genes regulated by Notch1 are unique for the cell type and vary widely dependent on other signals. We further show that Notch1 signaling regulates expression of genes playing key roles in cell differentiation, cell cycle control and apoptosis in a context dependent manner. In addition to the known Notch1 targets of the Hes and Hey families of transcriptional repressors, Notch1 activates the expression of regulatory transcription factors such as Sox9, Pax6, Runx1, Myf5 and Id proteins that are critically involved in lineage decisions in the absence of protein synthesis. Conclusion/Significance We suggest that Notch signaling determines lineage decisions and expansion of stem cells by directly activating both key lineage specific transcription factors and their repressors (Id and Hes/Hey proteins) and propose a model by which Notch signaling regulates cell fate commitment and self renewal in dependence of the intrinsic and extrinsic cellular context. PMID:20628604

  20. 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.

  1. Buyang Huanwu decoction up-regulates Notch1 gene expression in injured spinal cord.

    PubMed

    Guo, Zhan-Peng; Huang, Mi-Na; Liu, An-Qi; Yuan, Ya-Jiang; Zhao, Jian-Bo; Mei, Xi-Fan

    2015-08-01

    Expression of genes in the Notch signaling pathway is altered in the injured spinal cord, which indicates that Notch participates in repair after spinal cord injury. Buyang Huanwu decoction, a traditional Chinese herbal preparation, can promote the growth of nerve cells and nerve fibers; however, it is unclear whether Buyang Huanwu decoction affects the Notch signaling pathway in injured spinal cord. In this study, a rat model was established by injuring the T10 spinal cord. At 2 days after injury, rats were intragastrically administered 2 mL of 0.8 g/mL Buyang Huanwu decoction daily until sacrifice. Real-time reverse transcription polymerase chain reaction analysis demonstrated that at 7, 14 and 28 days after injury, the expression of Notch1 was increased in the Buyang Huanwu decoction group compared with controls. These findings confirm that Buyang Huanwu decoction can promote the expression of Notch1 in rats with incomplete spinal cord injury, and may indicate a mechanism to promote the repair of spinal cord injury.

  2. 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.

  3. 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

  4. Adenosine Receptors: Expression, Function and Regulation

    PubMed Central

    Sheth, Sandeep; Brito, Rafael; Mukherjea, Debashree; Rybak, Leonard P.; Ramkumar, Vickram

    2014-01-01

    Adenosine receptors (ARs) comprise a group of G protein-coupled receptors (GPCR) which mediate the physiological actions of adenosine. To date, four AR subtypes have been cloned and identified in different tissues. These receptors have distinct localization, signal transduction pathways and different means of regulation upon exposure to agonists. This review will describe the biochemical characteristics and signaling cascade associated with each receptor and provide insight into how these receptors are regulated in response to agonists. A key property of some of these receptors is their ability to serve as sensors of cellular oxidative stress, which is transmitted by transcription factors, such as nuclear factor (NF)-κB, to regulate the expression of ARs. Recent observations of oligomerization of these receptors into homo- and heterodimers will be discussed. In addition, the importance of these receptors in the regulation of normal and pathological processes such as sleep, the development of cancers and in protection against hearing loss will be examined. PMID:24477263

  5. 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.

  6. Active form Notch4 promotes the proliferation and differentiation of 3T3-L1 preadipocytes

    SciTech Connect

    Lai, Peng-Yeh; Tsai, Chong-Bin; Tseng, Min-Jen

    2013-01-18

    Highlights: ► Notch4IC modulates the ERK pathway and cell cycle to promote 3T3-L1 proliferation. ► Notch4IC facilitates 3T3-L1 differentiation by up-regulating proadipogenic genes. ► Notch4IC promotes proliferation during the early stage of 3T3-L1 adipogenesis. ► Notch4IC enhances differentiation during subsequent stages of 3T3-L1 adipogenesis. -- Abstract: Adipose tissue is composed of adipocytes, which differentiate from precursor cells in a process called adipogenesis. Many signal molecules are involved in the transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCR analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. To elucidate the role of Notch4 in adipogenesis, the human active form Notch4 (N4IC) was transiently transfected into 3T3-L1 cells. The expression of HES1, Hey1, C/EBPδ and PPARγ was up-regulated, and the expression of Pref-1, an adipogenic inhibitor, was down-regulated. To further characterize the effect of N4IC in adipogenesis, stable cells expressing human N4IC were established. The expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. These data suggest that N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through up-regulating adipogenic genes such as C/EBPα, PPARγ, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis.

  7. Α-arrestin 1 (ARRDC1) and β-arrestins cooperate to mediate Notch degradation in mammals.

    PubMed

    Puca, Loredana; Chastagner, Patricia; Meas-Yedid, Vannary; Israël, Alain; Brou, Christel

    2013-10-01

    Notch signaling is a conserved signaling pathway implicated in embryogenesis and adult tissue maintenance. Notch signaling strength is strictly regulated, notably by maintaining a controlled pool of functional receptor at the cell surface. Mammalian non-activated Notch receptor is internalized, ubiquitylated by the Itch E3 ubiquitin ligase and degraded in the lysosomes. Here, we show that β-arrestins are necessary for Itch-Notch interaction and for Itch-driven ubiquitylation and degradation of Notch. Interestingly, β-arrestins do not directly bind Itch but heterodimerize with a member of another subfamily of arrestins called ARRDC1 or α-arrestin 1, which harbors PPxY motifs that allow direct interaction with Itch. Cells transfected with ARRDC1 mutated in PPxY motifs show reduced Itch-mediated Notch ubiquitylation and impaired lysosomal degradation of Notch, as observed in β-arrestin(-/-) or Itch(-/-) cells. Our data show for the first time that ARRDC1 and β-arrestins heterodimerize and cooperate in the same complex to promote non-activated Notch receptor degradation, thus acting as negative regulators of Notch signaling.

  8. 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

  9. 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

  10. 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

  11. 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

  12. Keratin-6 driven ODC expression to hair follicle keratinocytes enhances stemness and tumorigenesis by negatively regulating Notch

    SciTech Connect

    Arumugam, Aadithya; Weng, Zhiping; Chaudhary, Sandeep C.; Afaq, Farrukh; Elmets, Craig A.; Athar, Mohammad

    2014-08-29

    Highlights: • Targeting ODC to hair follicle augments skin carcinogenesis and invasive SCCs. • Hair follicle ODC expands stem cell compartment carrying CD34{sup +}/K15{sup +}/p63{sup +} keratinocytes. • Negatively regulated Notch1 is associated with expansion of stem cell compartment. - Abstract: Over-expression of ornithine decarboxylase (ODC) is known to be involved in the epidermal carcinogenesis. However, the mechanism by which it enhances skin carcinogenesis remains undefined. Recently, role of stem cells localized in various epidermal compartments has been shown in the pathogenesis of skin cancer. To direct ODC expression in distinct epidermal compartments, we have developed keratin 6 (K6)-ODC/SKH-1 and keratin 14 (K14)-ODC/SKH-1 mice and employed them to investigate the role of ODC directed to these epidermal compartments on UVB-induced carcinogenesis. K6-driven ODC over-expression directed to outer root sheath (ORS) of hair follicle was more effective in augmenting tumorigenesis as compared to mice where K14-driven ODC expression was directed to inter-follicular epidermal keratinocytes. Chronically UVB-irradiated K6-ODC/SKH-1 developed 15 ± 2.5 tumors/mouse whereas K14-ODC/SKH-1 developed only 6.8 ± 1.5 tumors/mouse. K6-ODC/SKH-1 showed augmented UVB-induced proliferation and much higher pro-inflammatory responses than K14-ODC/SKH-1 mice. Tumors induced in K6-ODC/SKH-1 were rapidly growing, invasive and ulcerative squamous cell carcinoma (SCC) showing decreased expression of epidermal polarity marker E-cadherin and enhanced mesenchymal marker, fibronectin. Interestingly, the number of CD34/CK15/p63 positive stem-like cells was significantly higher in chronically UVB-irradiated K6-ODC/SKH-1 as compared to K14-ODC/SKH-1 mice. Reduced Notch1 expression was correlated with the expansion of stem cell compartment in these animals. However, other signaling pathways such as DNA damage response or mTOR signaling pathways were not significantly different in

  13. Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs.

    PubMed

    Lai, Eric C; Tam, Bergin; Rubin, Gerald M

    2005-05-01

    Although hundreds of distinct animal microRNAs (miRNAs) are known, the specific biological functions of only a handful are understood at present. Here, we demonstrate that three different families of Drosophila miRNAs directly regulate two large families of Notch target genes, including basic helix-loop-helix (bHLH) repressor and Bearded family genes. These miRNAs regulate Notch target gene activity via GY-box (GUCUUCC), Brd-box (AGCUUUA), and K-box (cUGUGAUa) motifs. These are conserved sites in target 3'-untranslated regions (3'-UTRs) that are complementary to the 5'-ends of miRNAs, or "seed" regions. Collectively, these motifs represent >40 miRNA-binding sites in Notch target genes, and we show all three classes of motif to be necessary and sufficient for miRNA-mediated regulation in vivo. Importantly, many of the validated miRNA-binding sites have limited pairing to miRNAs outside of the "box:seed" region. Consistent with this, we find that seed-related miRNAs that are otherwise quite divergent can regulate the same target sequences. Finally, we demonstrate that ectopic expression of several Notch-regulating miRNAs induces mutant phenotypes that are characteristic of Notch pathway loss of function, including loss of wing margin, thickened wing veins, increased bristle density, and tufted bristles. Collectively, these data establish insights into miRNA target recognition and demonstrate that the Notch signaling pathway is a major target of miRNA-mediated regulation in Drosophila.

  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. 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.

  16. 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.

  17. 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

  18. 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

  19. Regulation of AMPA receptors in spinal nociception

    PubMed Central

    2010-01-01

    The functional properties of α-amino-3-hydroxy-5-methy-4-isoxazole propionate (AMPA) receptors in different brain regions, such as hippocampus and cerebellum, have been well studied in vitro and in vivo. The AMPA receptors present a unique characteristic in the mechanisms of subunit regulation during LTP (long-term potentiation) and LTD (long-term depression), which are involved in the trafficking, altered composition and phosphorylation of AMPA receptor subunits. Accumulated data have demonstrated that spinal AMPA receptors play a critical role in the mechanism of both acute and persistent pain. However, less is known about the biochemical regulation of AMPA receptor subunits in the spinal cord in response to painful stimuli. Recent studies have shown that some important regulatory processes, such as the trafficking of AMPA receptor subunit, subunit compositional changes, phosphorylation of AMPA receptor subunits, and their interaction with partner proteins may contribute to spinal nociceptive transmission. Of all these regulation processes, the phosphorylation of AMPA receptor subunits is the most important since it may trigger or affect other cellular processes. Therefore, these study results may suggest an effective strategy in developing novel analgesics targeting AMPA receptor subunit regulation that may be useful in treating persistent and chronic pain without unacceptable side effects in the clinics. PMID:20092646

  20. Scapular Notching.

    PubMed

    Dare, David; Dines, Joshua S; Tebo, Collin; Edwards, T Bradley; Craig, Edward V; Dines, David M

    2016-01-01

    Developed in 1985, the Grammont-style reverse total shoulder arthroplasty offered a biomechanical advantage for the deltoid muscle as well as predictably reduced pain and improved shoulder function in rotator cuff-deficient shoulders. Despite favorable outcomes, reverse total shoulder arthroplasty is associated with a unique set of complications, one of which is scapular notching. Scapular notching is believed to be a result of mechanical impingement of the humeral component on the lateral scapular pillar. Although it appears that scapular notching progresses with time, its effect on implant survivorship and clinical outcomes is unknown. Factors associated with scapular notching are categorized into several groups, including patient-specific risk factors, surgical approach and technique, and prosthetic design. Surgical strategies to reduce the rate of scapular notching include inferior positioning of the glenosphere, inferior tilting of the glenosphere, and increasing the size of the glenosphere. A lateralized center of rotation and a decreased humeral shaft-neck angle also decrease the incidence of scapular notching. As the indications for reverse total shoulder arthroplasty expand, it is important for orthopaedic surgeons to understand the etiology and incidence, predictive factors, and clinical relevance of scapular notching as well as strategies to avoid it. PMID:27049187

  1. Structure, function, and regulation of adrenergic receptors.

    PubMed Central

    Strosberg, A. D.

    1993-01-01

    Adrenergic receptors for adrenaline and noradrenaline belong to the large multigenic family of receptors coupled to GTP-binding proteins. Three pharmacologic types have been identified: alpha 1-, alpha 2-, and beta-adrenergic receptors. Each of these has three subtypes, characterized by both structural and functional differences. The alpha 2 and beta receptors are coupled negatively and positively, respectively, to adenylyl cyclase via Gi or Gs regulatory proteins, and the alpha 1 receptors modulate phospholipase C via the Go protein. Subtype expression is regulated at the level of the gene, the mRNA, and the protein through various transcriptional and postsynthetic mechanisms. Adrenergic receptors constitute, after rhodopsin, one of the best studied models for the other receptors coupled to G proteins that are likely to display similar structural and functional properties. PMID:8401205

  2. A Disintegrin and Metalloproteinase Domain 17 Regulates Colorectal Cancer Stem Cells and Chemosensitivity Via Notch1 Signaling.

    PubMed

    Wang, Rui; Ye, Xiangcang; Bhattacharya, Rajat; Boulbes, Delphine R; Fan, Fan; Xia, Ling; Ellis, Lee M

    2016-03-01

    Evidence is accumulating for the role of cancer stem cells (CSCs) in mediating chemoresistance in patients with metastatic colorectal cancer (mCRC). A disintegrin and metalloproteinase domain 17 (ADAM17; also known as tumor necrosis factor-α-converting enzyme [TACE]) was shown to be overexpressed and to mediate cell proliferation and chemoresistance in CRC cells. However, its role in mediating the CSC phenotype in CRC has not been well-characterized. The objective of the present study was to determine whether ADAM17 regulates the CSC phenotype in CRC and to elucidate the downstream signaling mechanism that mediates cancer stemness. We treated established CRC cell lines and a newly established human CRC cell line HCP-1 with ADAM17-specific small interfering RNA (siRNA) or the synthetic peptide inhibitor TAPI-2. The effects of ADAM17 inhibition on the CSC phenotype and chemosensitivity to 5-fluorouracil (5-FU) in CRC cells were examined. siRNA knockdown and TAPI-2 decreased the protein levels of cleaved Notch1 (Notch1 intracellular domain) and HES-1 in CRC cells. A decrease in the CSC phenotype was determined by sphere formation and ALDEFLUOR assays. Moreover, TAPI-2 sensitized CRC cells to 5-FU by decreasing cell viability and the median lethal dose of 5-FU and increasing apoptosis. We also showed the cleavage and release of soluble Jagged-1 and -2 by ADAM17 in CRC cells. Our studies have elucidated a role of ADAM17 in regulating the CSC phenotype and chemoresistance in CRC cells. The use of drugs that inhibit ADAM17 activity might increase the therapeutic benefit to patients with mCRC and, potentially, those with other solid malignancies.

  3. GABA(B) receptors in neuroendocrine regulation.

    PubMed

    Lux-Lantos, Victoria A; Bianchi, María S; Catalano, Paolo N; Libertun, Carlos

    2008-09-01

    Gamma-amino butyric acid (GABA), in addition to being a metabolic intermediate and the main inhibitory neurotransmitter in the synaptic cleft, is postulated as a neurohormone, a paracrine signaling molecule, and a trophic factor. It acts through pre- and post-synaptic receptors, named GABA(A) and GABA(C) (ionotropic receptors) and GABA(B) (metabotropic receptor). Here we reviewed the participation of GABA(B) receptors in the regulation of the hypothalamic-pituitary-gonadal axis, using physiological, biochemical, and pharmacological approaches in rats, as well as in GABA(B1) knock-out mice, that lack functional GABA(B) receptors. Our general conclusion indicates that GABA(B )receptors participate in the regulation of pituitary hormone secretion acting both in the central nervous system and directly on the gland. PRL and gonadotropin axes are affected by GABA(B) receptor activation, as demonstrated in the rat and also in the GABA(B1) knock-out mouse. In addition, hypothalamic and pituitary GABA(B) receptor expression is modulated by steroid hormones. GABA participation in the brain control of pituitary secretion through GABA(B) receptors depends on physiological conditions, being age and sex critical factors.These results indicate that patients receiving GABA(B) agonists/antagonists should be monitored for possible endocrine side effects.

  4. 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

  5. RUNX super-enhancer control through the Notch pathway by Epstein-Barr virus transcription factors regulates B cell growth

    PubMed Central

    Gunnell, Andrea; Webb, Helen M.; Wood, C. David; McClellan, Michael J.; Wichaidit, Billy; Kempkes, Bettina; Jenner, Richard G.; Osborne, Cameron; Farrell, Paul J.; West, Michelle J.

    2016-01-01

    In B cells infected by the cancer-associated Epstein-Barr virus (EBV), RUNX3 and RUNX1 transcription is manipulated to control cell growth. The EBV-encoded EBNA2 transcription factor (TF) activates RUNX3 transcription leading to RUNX3-mediated repression of the RUNX1 promoter and the relief of RUNX1-directed growth repression. We show that EBNA2 activates RUNX3 through a specific element within a −97 kb super-enhancer in a manner dependent on the expression of the Notch DNA-binding partner RBP-J. We also reveal that the EBV TFs EBNA3B and EBNA3C contribute to RUNX3 activation in EBV-infected cells by targeting the same element. Uncovering a counter-regulatory feed-forward step, we demonstrate EBNA2 activation of a RUNX1 super-enhancer (−139 to −250 kb) that results in low-level RUNX1 expression in cells refractory to RUNX1-mediated growth inhibition. EBNA2 activation of the RUNX1 super-enhancer is also dependent on RBP-J. Consistent with the context-dependent roles of EBNA3B and EBNA3C as activators or repressors, we find that these proteins negatively regulate the RUNX1 super-enhancer, curbing EBNA2 activation. Taken together our results reveal cell-type-specific exploitation of RUNX gene super-enhancers by multiple EBV TFs via the Notch pathway to fine tune RUNX3 and RUNX1 expression and manipulate B-cell growth. PMID:26883634

  6. RUNX super-enhancer control through the Notch pathway by Epstein-Barr virus transcription factors regulates B cell growth.

    PubMed

    Gunnell, Andrea; Webb, Helen M; Wood, C David; McClellan, Michael J; Wichaidit, Billy; Kempkes, Bettina; Jenner, Richard G; Osborne, Cameron; Farrell, Paul J; West, Michelle J

    2016-06-01

    In B cells infected by the cancer-associated Epstein-Barr virus (EBV), RUNX3 and RUNX1 transcription is manipulated to control cell growth. The EBV-encoded EBNA2 transcription factor (TF) activates RUNX3 transcription leading to RUNX3-mediated repression of the RUNX1 promoter and the relief of RUNX1-directed growth repression. We show that EBNA2 activates RUNX3 through a specific element within a -97 kb super-enhancer in a manner dependent on the expression of the Notch DNA-binding partner RBP-J. We also reveal that the EBV TFs EBNA3B and EBNA3C contribute to RUNX3 activation in EBV-infected cells by targeting the same element. Uncovering a counter-regulatory feed-forward step, we demonstrate EBNA2 activation of a RUNX1 super-enhancer (-139 to -250 kb) that results in low-level RUNX1 expression in cells refractory to RUNX1-mediated growth inhibition. EBNA2 activation of the RUNX1 super-enhancer is also dependent on RBP-J. Consistent with the context-dependent roles of EBNA3B and EBNA3C as activators or repressors, we find that these proteins negatively regulate the RUNX1 super-enhancer, curbing EBNA2 activation. Taken together our results reveal cell-type-specific exploitation of RUNX gene super-enhancers by multiple EBV TFs via the Notch pathway to fine tune RUNX3 and RUNX1 expression and manipulate B-cell growth. PMID:26883634

  7. Function of Integrin-Linked Kinase in Modulating the Stemness of IL-6-Abundant Breast Cancer Cells by Regulating γ-Secretase-Mediated Notch1 Activation in Caveolae.

    PubMed

    Hsu, En-Chi; Kulp, Samuel K; Huang, Han-Li; Tu, Huang-Ju; Salunke, Santosh B; Sullivan, Nicholas J; Sun, Duxin; Wicha, Max S; Shapiro, Charles L; Chen, Ching-Shih

    2015-06-01

    Interleukin-6 (IL-6) and Notch signaling are important regulators of breast cancer stem cells (CSCs), which drive the malignant phenotype through self-renewal, differentiation, and development of therapeutic resistance. We investigated the role of integrin-linked kinase (ILK) in regulating IL-6-driven Notch1 activation and the ability to target breast CSCs through ILK inhibition. Ectopic expression/short hairpin RNA-mediated knockdown of ILK, pharmacological inhibition of ILK with the small molecule T315, Western blot analysis, immunofluorescence, and luciferase reporter assays were used to evaluate the regulation of IL-6-driven Notch1 activation by ILK in IL-6-producing triple-negative breast cancer cell lines (MDA-MB-231, SUM-159) and in MCF-7 and MCF-7(IL-6) cells. The effects of ILK on γ-secretase complex assembly and cellular localization were determined by immunofluorescence, Western blots of membrane fractions, and immunoprecipitation. In vivo effects of T315-induced ILK inhibition on CSCs in SUM-159 xenograft models were assessed by mammosphere assays, flow cytometry, and tumorigenicity assays. Results show that the genetic knockdown or pharmacological inhibition of ILK suppressed Notch1 activation and the abundance of the γ-secretase components presenilin-1, nicastrin, and presenilin enhancer 2 at the posttranscriptional level via inhibition of caveolin-1-dependent membrane assembly of the γ-secretase complex. Accordingly, knockdown of ILK inhibited breast CSC-like properties in vitro and the breast CSC subpopulation in vivo in xenograft tumor models. Based on these findings, we propose a novel function of ILK in regulating γ-secretase-mediated Notch1 activation, which suggests the targeting of ILK as a therapeutic approach to suppress IL-6-induced breast CSCs.

  8. LIN-12/Notch trafficking and regulation of DSL ligand activity during vulval induction in Caenorhabditis elegans.

    PubMed

    Shaye, Daniel D; Greenwald, Iva

    2005-11-01

    A novel mode of crosstalk between the EGFR-Ras-MAPK and LIN-12/Notch pathways occurs during the patterning of a row of vulval precursor cells (VPCs) in Caenorhabditis elegans: activation of the EGFR-Ras-MAPK pathway in the central VPC promotes endocytosis and degradation of LIN-12 protein. LIN-12 downregulation in the central VPC is a prerequisite for the activity of the lateral signal, which activates LIN-12 in neighboring VPCs. Here we characterize cis-acting targeting sequences in the LIN-12 intracellular domain and find that in addition to a di-leucine motif, serine/threonine residues are important for internalization and lysine residues are important for post-internalization trafficking and degradation. We also identify two trans-acting factors that are required for post-internalization trafficking and degradation: ALX-1, a homolog of yeast Bro1p and mammalian Alix and the WWP-1/Su(dx)/Itch ubiquitin ligase. By examining the effects of mutated forms of LIN-12 and reduced wwp-1 or alx-1 activity on subcellular localization and activity of LIN-12, we provide evidence that the lateral signal-inhibiting activity of LIN-12 resides in the extracellular domain and occurs at the apical surface of the VPCs.

  9. 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

  10. 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

  11. Wt1a, Foxc1a, and the Notch mediator Rbpj Physically Interact and Regulate the Formation of Podocytes in Zebrafish

    PubMed Central

    O’Brien, Lori L.; Grimaldi, Michael; Kostun, Zachary; Wingert, Rebecca A.; Selleck, Rori; Davidson, Alan J.

    2011-01-01

    Podocytes help form the glomerular blood filtration barrier in the kidney and their injury or loss leads to renal disease. The Wilms’ tumor suppressor-1 (Wt1) and the FoxC1/2 transcription factors, as well as Notch signaling, have been implicated as important regulators of podocyte fate. It is not known whether these factors work in parallel or sequentially on different gene targets, or as higher-order transcriptional complexes on common genes. Here, we use the zebrafish to demonstrate that embryos treated with morpholinos against wt1a, foxc1a, or the Notch transcriptional mediator rbpj develop fewer podocytes, as determined by wt1b, hey1 and nephrin expression, while embryos deficient in any two of these factors completely lack podocytes. From GST-pull-downs and co-immunoprecipitation experiments we show that Wt1a, Foxc1a, and Rbpj can physically interact with each other, whereas only Rbpj binds to the Notch intracellular domain (NICD). In transactivation assays, combinations of Wt1, FoxC1/2, and NICD synergistically induce the Hey1 promoter, and have additive or repressive effects on the Podocalyxin promoter, depending on dosage. Taken together, these data suggest that Wt1, FoxC1/2, and Notch signaling converge on common target genes where they physically interact to regulate a podocyte-specific gene program. These findings further our understanding of the transcriptional circuitry responsible for podocyte formation and differentiation during kidney development. PMID:21871448

  12. 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.

  13. Mood disorders: regulation by metabotropic glutamate receptors.

    PubMed

    Pilc, Andrzej; Chaki, Shigeyuki; Nowak, Gabriel; Witkin, Jeffrey M

    2008-03-01

    Medicinal therapies for mood disorders neither fully serve the efficacy needs of patients nor are they free of side-effect issues. Although monoamine-based therapies are the primary current treatment approaches, both preclinical and clinical findings have implicated the excitatory neurotransmitter glutamate in the pathogenesis of major depressive disorders. The present commentary focuses on the metabotropic glutamate receptors and their relationship to mood disorders. Metabotropic glutamate (mGlu) receptors regulate glutamate transmission by altering the release of neurotransmitter and/or modulating the post-synaptic responses to glutamate. Convergent biochemical, pharmacological, behavioral, and clinical data will be reviewed that establish glutamatergic neurotransmission via mGlu receptors as a biologically relevant process in the regulation of mood and that these receptors may serve as novel targets for the discovery of small molecule modulators with unique antidepressant properties. Specifically, compounds that antagonize mGlu2, mGlu3, and/or mGlu5 receptors (e.g. LY341495, MGS0039, MPEP, MTEP) exhibit biochemical effects indicative of antidepressant effects as well as in vivo activity in animal models predictive of antidepressant efficacy. Both preclinical and clinical data have previously been presented to define NMDA and AMPA receptors as important targets for the modulation of major depression. In the present review, we present a model suggesting how the interplay of glutamate at the mGlu and at the ionotropic AMPA and NMDA receptors might account for the antidepressant-like effects of glutamatergic- and monoaminergic-based drugs affecting mood in patients. The current data lead to the hypothesis that mGlu-based compounds and conventional antidepressants impact a network of interactive effects that converge upon a down regulation of NMDA receptor function and an enhancement in AMPA receptor signaling. PMID:18164691

  14. 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

  15. Structural basis for Notch1 engagement of Delta-like 4

    DOE PAGES

    Luca, Vincent C.; Jude, Kevin M.; Pierce, Nathan W.; Nachury, Maxence V.; Fischer, Suzanne; Garcia, K. Christopher

    2015-02-20

    Notch receptors guide mammalian cell fate decisions by engaging the proteins Jagged and Delta-like (DLL). The 2.3 angstrom resolution crystal structure of the interacting regions of the Notch1-DLL4 complex reveals a two-site, antiparallel binding orientation assisted by Notch1 O-linked glycosylation. Notch1 epidermal growth factor–like repeats 11 and 12 interact with the DLL4 Delta/Serrate/Lag-2 (DSL) domain and module at the N-terminus of Notch ligands (MNNL) domains, respectively. Threonine and serine residues on Notch1 are functionalized with O-fucose and O-glucose, which act as surrogate amino acids by making specific, and essential, contacts to residues on DLL4. Lastly, the elucidation of a directmore » chemical role for O-glycans in Notch1 ligand engagement demonstrates how, by relying on posttranslational modifications of their ligand binding sites, Notch proteins have linked their functional capacity to developmentally regulated biosynthetic pathways.« less

  16. Developing HSCs become Notch independent by the end of maturation in the AGM region

    PubMed Central

    Souilhol, Céline; Lendinez, Javier G.; Rybtsov, Stanislav; Murphy, Fiona; Wilson, Heather; Hills, David; Batsivari, Antoniana; Binagui-Casas, Anahí; McGarvey, Alison C.; MacDonald, H. Robson; Kageyama, Ryoichiro; Siebel, Christian; Zhao, Suling

    2016-01-01

    The first definitive hematopoietic stem cells (dHSCs) in the mouse emerge in the dorsal aorta of the embryonic day (E) 10.5 to 11 aorta-gonad-mesonephros (AGM) region. Notch signaling is essential for early HSC development but is dispensable for the maintenance of adult bone marrow HSCs. How Notch signaling regulates HSC formation in the embryo is poorly understood. We demonstrate here that Notch signaling is active in E10.5 HSC precursors and involves both Notch1 and Notch2 receptors, but is gradually downregulated while they progress toward dHSCs at E11.5. This downregulation is accompanied by gradual functional loss of Notch dependency. Thus, as early as at final steps in the AGM region, HSCs begin acquiring the Notch independency characteristic of adult bone marrow HSCs as part of the maturation program. Our data indicate that fine stage-dependent tuning of Notch signaling may be required for the generation of definitive HSCs from pluripotent cells. PMID:27421959

  17. Notch Antennas

    NASA Technical Reports Server (NTRS)

    Lee, Richard Q.

    2004-01-01

    Notch antennas, also known as the tapered slot antenna (TSA), have been the topics of research for decades. TSA has demonstrated multi-octave bandwidth, moderate gain (7 to 10 dB), and symmetric E- and H- plane beam patterns and can be used for many different applications. This chapter summarizes the research activities on notch antennas over the past decade with emphasis on their most recent advances and applications. This chapter begins with some discussions on the designs of single TSA; then follows with detailed discussions of issues associated with TSA designs and performance characteristics. To conclude the chapter, some recent developments in TSA arrays and their applications are highlighted.

  18. Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability

    PubMed Central

    Chao, Min-Wu; Chu, Po-Chen; Chuang, Hsiao-Ching; Shen, Fang-Hsiu; Chou, Chih-Chien; Hsu, En-Chi; Himmel, Lauren E.; Huang, Han-Li; Tu, Huang-Ju; Kulp, Samuel K.; Teng, Che-Ming; Chen, Ching-Shih

    2016-01-01

    Here, we report a novel non-epigenetic function of histone deacetylase (HDAC) 8 in activating cancer stem cell (CSC)-like properties in breast cancer cells by enhancing the stability of Notch1 protein. The pan-HDAC inhibitors AR-42 and SAHA, and the class I HDAC inhibitor depsipeptide, suppressed mammosphere formation and other CSC markers by reducing Notch1 expression in MDA-MB-231 and SUM-159 cells. Interrogation of individual class I isoforms (HDAC1–3 and 8) using si/shRNA-mediated knockdown, ectopic expression and/or pharmacological inhibition revealed HDAC8 to be the primary mediator of this drug effect. This suppression of Notch1 in response to HDAC8 inhibition was abrogated by the proteasome inhibitor MG132 and siRNA-induced silencing of Fbwx7, indicating Notch1 suppression occurred through proteasomal degradation. However, co-immunoprecipitation analysis indicated that HDAC8 did not form complexes with Notch1 and HDAC inhibition had no effect on Notch1 acetylation. In a xenograft tumor model, the tumorigenicity of breast cancer cells was decreased by HDAC8 knockdown. These findings suggest the therapeutic potential of HDAC8 inhibition to suppress Notch1 signaling in breast cancer. PMID:26625202

  19. Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability.

    PubMed

    Chao, Min-Wu; Chu, Po-Chen; Chuang, Hsiao-Ching; Shen, Fang-Hsiu; Chou, Chih-Chien; Hsu, En-Chi; Himmel, Lauren E; Huang, Han-Li; Tu, Huang-Ju; Kulp, Samuel K; Teng, Che-Ming; Chen, Ching-Shih

    2016-01-12

    Here, we report a novel non-epigenetic function of histone deacetylase (HDAC) 8 in activating cancer stem cell (CSC)-like properties in breast cancer cells by enhancing the stability of Notch1 protein. The pan-HDAC inhibitors AR-42 and SAHA, and the class I HDAC inhibitor depsipeptide, suppressed mammosphere formation and other CSC markers by reducing Notch1 expression in MDA-MB-231 and SUM-159 cells. Interrogation of individual class I isoforms (HDAC1-3 and 8) using si/shRNA-mediated knockdown, ectopic expression and/or pharmacological inhibition revealed HDAC8 to be the primary mediator of this drug effect. This suppression of Notch1 in response to HDAC8 inhibition was abrogated by the proteasome inhibitor MG132 and siRNA-induced silencing of Fbwx7, indicating Notch1 suppression occurred through proteasomal degradation. However, co-immunoprecipitation analysis indicated that HDAC8 did not form complexes with Notch1 and HDAC inhibition had no effect on Notch1 acetylation. In a xenograft tumor model, the tumorigenicity of breast cancer cells was decreased by HDAC8 knockdown. These findings suggest the therapeutic potential of HDAC8 inhibition to suppress Notch1 signaling in breast cancer.

  20. Notch filter

    NASA Technical Reports Server (NTRS)

    Shelton, G. B. (Inventor)

    1977-01-01

    A notch filter for the selective attenuation of a narrow band of frequencies out of a larger band was developed. A helical resonator is connected to an input circuit and an output circuit through discrete and equal capacitors, and a resistor is connected between the input and the output circuits.

  1. Dissecting the mechanisms of Notch induced hyperplasia

    PubMed Central

    Djiane, Alexandre; Krejci, Alena; Bernard, Frédéric; Fexova, Silvie; Millen, Katherine; Bray, Sarah J

    2013-01-01

    The outcome of the Notch pathway on proliferation depends on cellular context, being growth promotion in some, including several cancers, and growth inhibition in others. Such disparate outcomes are evident in Drosophila wing discs, where Notch overactivation causes hyperplasia despite having localized inhibitory effects on proliferation. To understand the underlying mechanisms, we have used genomic strategies to identify the Notch-CSL target genes directly activated during wing disc hyperplasia. Among them were genes involved in both autonomous and non-autonomous regulation of proliferation, growth and cell death, providing molecular explanations for many characteristics of Notch induced wing disc hyperplasia previously reported. The Notch targets exhibit different response patterns, which are shaped by both positive and negative feed-forward regulation between the Notch targets themselves. We propose, therefore, that both the characteristics of the direct Notch targets and their cross-regulatory relationships are important in coordinating the pattern of hyperplasia. PMID:23232763

  2. Social regulation of cortisol receptor gene expression

    PubMed Central

    Korzan, Wayne J.; Grone, Brian P.; Fernald, Russell D.

    2014-01-01

    In many social species, individuals influence the reproductive capacity of conspecifics. In a well-studied African cichlid fish species, Astatotilapia burtoni, males are either dominant (D) and reproductively competent or non-dominant (ND) and reproductively suppressed as evidenced by reduced gonadotropin releasing hormone (GnRH1) release, regressed gonads, lower levels of androgens and elevated levels of cortisol. Here, we asked whether androgen and cortisol levels might regulate this reproductive suppression. Astatotilapia burtoni has four glucocorticoid receptors (GR1a, GR1b, GR2 and MR), encoded by three genes, and two androgen receptors (ARα and ARβ), encoded by two genes. We previously showed that ARα and ARβ are expressed in GnRH1 neurons in the preoptic area (POA), which regulates reproduction, and that the mRNA levels of these receptors are regulated by social status. Here, we show that GR1, GR2 and MR mRNAs are also expressed in GnRH1 neurons in the POA, revealing potential mechanisms for both androgens and cortisol to influence reproductive capacity. We measured AR, MR and GR mRNA expression levels in a microdissected region of the POA containing GnRH1 neurons, comparing D and ND males. Using quantitative PCR (qPCR), we found D males had higher mRNA levels of ARα, MR, total GR1a and GR2 in the POA compared with ND males. In contrast, ND males had significantly higher levels of GR1b mRNA, a receptor subtype with a reduced transcriptional response to cortisol. Through this novel regulation of receptor type, neurons in the POA of an ND male will be less affected by the higher levels of cortisol typical of low status, suggesting GR receptor type change as a potential adaptive mechanism to mediate high cortisol levels during social suppression. PMID:25013108

  3. Notch-Dependent Pituitary SOX2(+) Stem Cells Exhibit a Timed Functional Extinction in Regulation of the Postnatal Gland.

    PubMed

    Zhu, Xiaoyan; Tollkuhn, Jessica; Taylor, Havilah; Rosenfeld, Michael G

    2015-12-01

    Although SOX2(+) stem cells are present in the postnatal pituitary gland, how they are regulated molecularly and whether they are required for pituitary functions remain unresolved questions. Using a conditional knockout animal model, here we demonstrate that ablation of the canonical Notch signaling in the embryonic pituitary gland leads to progressive depletion of the SOX2(+) stem cells and hypoplastic gland. Furthermore, we show that the SOX2(+) stem cells initially play a significant role in contributing to postnatal pituitary gland expansion by self-renewal and differentiating into distinct lineages in the immediate postnatal period. However, we found that within several weeks postpartum, the SOX2(+) stem cells switch to an essentially dormant state and are no longer required for homeostasis/tissue adaptation. Our results present a dynamic tissue homeostatic model in which stem cells provide an initial contribution to the growth of the neonatal pituitary gland, whereas the mature gland can be maintained in a stem cell-independent fashion.

  4. 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.

  5. 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

  6. Folate receptor {alpha} regulates cell proliferation in mouse gonadotroph {alpha}T3-1 cells

    SciTech Connect

    Yao, Congjun; Evans, Chheng-Orn; Stevens, Victoria L.; Owens, Timothy R.; Oyesiku, Nelson M.

    2009-11-01

    We have previously found that the mRNA and protein levels of the folate receptor alpha (FR{alpha}) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FR{alpha} has not fully been determined. We investigated the effect of FR{alpha} over-expression in the mouse gonadotroph {alpha}T3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FR{alpha} were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNA staining. These observations indicate that over-expression of FR{alpha} promotes cell proliferation. These effects were abrogated in the same {alpha}T3-1 cells when transfected with a mutant FR{alpha} cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FR{alpha} over-expressing cells. In summary, our data suggests that FR{alpha} regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.

  7. Loss of Notch2 and Notch3 in vascular smooth muscle causes patent ductus arteriosus.

    PubMed

    Baeten, Jeremy T; Jackson, Ashley R; McHugh, Kirk M; Lilly, Brenda

    2015-12-01

    The overlapping roles of the predominant Notch receptors in vascular smooth muscle cells, Notch2 and Notch3, have not been clearly defined in vivo. In this study, we use a smooth muscle-specific deletion of Notch2 together with a global Notch3 deletion to produce mice with combinations of mutant and wild-type Notch2/3 alleles in vascular smooth muscle cells. Mice with complete loss of Notch3 and smooth muscle-expressed Notch2 display late embryonic lethality and subcutaneous hemorrhage. Mice without smooth muscle-Notch2 and only one wild-type copy of Notch3 die within one day of birth and present with vascular defects, most notably patent ductus arteriosus (DA) and aortic dilation. These defects were associated with decreased expression of contractile markers in both the DA and aorta. These results demonstrate that Notch2 and Notch3 have overlapping roles in promoting development of vascular smooth muscle cells, and together contribute to functional closure of the DA.

  8. MicroRNA-10b regulates epithelial-mesenchymal transition by modulating KLF4/Notch1/E-cadherin in cisplatin-resistant nasopharyngeal carcinoma cells

    PubMed Central

    Zhang, Pei; Hong, Haiyu; Sun, Xiaojin; Jiang, Hao; Ma, Shiyin; Zhao, Surong; Zhang, Mengxiao; Wang, Zhiwei; Jiang, Chenchen; Liu, Hao

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is an initiating event in tumor cell invasion and metastasis that contributes to therapeutic resistance to compounds including cisplatin. MicroRNAs (miRNAs) have been associated with EMT as well as resistance to standard therapies. However, the underlying mechanisms by which miRNAs control the development of resistance to cisplatin (DDP), and the accompanying EMT-like properties are required to elucidate. Here we show that microRNA-10b (miR-10b) is up-regulated in HNE1/DDP cells, and inhibition of miR-10b expression reversed the EMT phenotype. However, over-expression of miR-10b was able to promote the acquisition of an EMT phenotype in HNE1 cells. Additionally, we identified that miR-10b expression inversely correlates with KLF4, which then controls expression of Notch1. Knock-down of Notch1 inhibited cell migration, invasion, and reversed EMT in HNE1/DDP cells, which was dependent on miR-10b. In summary, our results reveal that miR-10b regulates EMT by modulating KLF4/Notch1/E-cadherin expression, which promotes invasion and migration of nasal pharyngeal carcinoma cells. PMID:27186392

  9. G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors*

    PubMed Central

    Franklin, Jade M.; Carrasco, Gonzalo A.

    2013-01-01

    We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB2 receptors, β-arrestin 2, and ERK1/2 signaling because treatment with CB2 shRNA lentiviral particles, β-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB2 receptors and increased interaction between β-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB2 receptors, which up-regulates 5-HT2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB2 receptors; and the β-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure. PMID:23592773

  10. Assembly of AMPA receptors: mechanisms and regulation

    PubMed Central

    Gan, Quan; Salussolia, Catherine L; Wollmuth, Lonnie P

    2015-01-01

    AMPA receptors (AMPARs) play a critical role in excitatory glutamatergic neurotransmission. The number and subunit composition of AMPARs at synapses determines the dynamics of fast glutamatergic signalling. Functional AMPARs on the cell surface are tetramers. Thus tetrameric assembly of AMPARs represents a promising target for modulating AMPAR-mediated signalling in health and disease. Multiple structural domains within the receptor influence AMPAR assembly. In a proposed model for AMPAR assembly, the amino-terminal domain underlies the formation of a dimer pool. The transmembrane domain facilitates the formation and enhances the stability of the tetramer. The ligand-binding domain influences assembly through a process referred to as ‘domain swapping’. We propose that this core AMPAR assembly process could be regulated by neuronal signals and speculate on possible mechanisms for such regulation. PMID:25556786

  11. 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

  12. CDKN1C/P57 is regulated by the Notch target gene Hes1 and induces senescence in human hepatocellular carcinoma.

    PubMed

    Giovannini, Catia; Gramantieri, Laura; Minguzzi, Manuela; Fornari, Francesca; Chieco, Pasquale; Grazi, Gian Luca; Bolondi, Luigi

    2012-08-01

    CDKN1C/P57 is a cyclin-dependent kinase inhibitor implicated in different human cancers, including hepatocellular carcinoma (HCC); however, little is known regarding the role of CDKN1C/P57 and its regulation in HCC. In this study, we show that the down-regulation of Notch1 and Notch3 in two HCC cell lines resulted in Hes1 down-regulation, CDKN1C/P57 up-regulation, and reduced cell growth. In line with these data, we report that CDKN1C/P57 is a target of transcriptional repression by the Notch effector, Hes1. We found that the up-regulation of CDKN1C/P57 by cDNA transfection decreased tumor growth, as determined by growth curve, flow cytometry analysis, and cyclin D1 down-regulation, without affecting the apoptosis machinery. Indeed, the expression of Bax, Noxa, PUMA, BNIP(3), and cleaved caspase-3 was not affected by CDKN1C/P57 induction. Morphologically CDKN1C/p57-induced HCC cells became flat and lengthened in shape, accumulated the senescence-associated β-galactosidase marker, and increased P16 protein expression. Evaluation of senescence in cells depleted both for Hes1 and CDKN1C/P57 revealed that the senescent state really depends on the accumulation of CDKN1C/p57. Finally, we validated our in vitro results in primary HCCs, showing that Hes1 protein expression inversely correlates with CDKN1C/P57 mRNA levels. In addition, reduced Hes1 protein expression is accompanied by a shorter time to recurrence after curative resection, suggesting that Hes1 may represent a biomarker for prediction of patients with poor prognosis.

  13. 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

  14. 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

  15. 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

  16. Glucocorticoid Regulation of the Vitamin D Receptor

    PubMed Central

    Hidalgo, Alejandro A.; Trump, Donald L.; Johnson, Candace S.

    2010-01-01

    Many studies indicate calcitriol has potent anti-tumor activity in different types of cancers. However, high levels of vitamin D can produce hypercalcemia in some patients. Glucocorticoids are used to ameliorate hypercalcemia and to enhance calcitriol anti-tumor activity. Calcitriol in combination with the glucocorticoid dexamethasone (Dex) increased vitamin D receptor (VDR) protein levels and ligand binding in squamous cell carcinoma VII (SCC). In this study we found that both calcitriol and Dex induce VDR- and glucocorticoid receptor (GR)-mediated transcription respectively, indicating both hormone receptors are active in SCC. Pre-treatment with Dex increases VDR-mediated transcription at the human CYP24A1 promoter. Whereas, pre-treatment with other steroid hormones, including dihydrotestosterone and R1881, has no effect on VDR-mediated transcription. Real-time PCR indicates treatment with Dex increases Vdr transcripts in a time-dependent manner, suggesting Dex may directly regulate expression of Vdr. Numerous putative glucocorticoid response elements (GREs) were found in the Vdr gene. Chromatin immunoprecipitation (ChIP) assay demonstrated GR binding at several putative GREs located within the mouse Vdr gene. However, none of the putative GREs studied increase GR-mediated transcription in luciferase reporter assays. In an attempt to identify the response element responsible for Vdr transcript regulation, future studies will continue to analyze newly identified GREs more distal from the Vdr gene promoter. PMID:20398752

  17. Notch regulation of myogenic versus endothelial fates of cells that migrate from the somite to the limb

    PubMed Central

    Mayeuf-Louchart, Alicia; Lagha, Mounia; Danckaert, Anne; Rocancourt, Didier; Relaix, Frederic; Vincent, Stéphane D.; Buckingham, Margaret

    2014-01-01

    Multipotent Pax3-positive (Pax3+) cells in the somites give rise to skeletal muscle and to cells of the vasculature. We had previously proposed that this cell-fate choice depends on the equilibrium between Pax3 and Foxc2 expression. In this study, we report that the Notch pathway promotes vascular versus skeletal muscle cell fates. Overactivating the Notch pathway specifically in Pax3+ progenitors, via a conditional Pax3NICD allele, results in an increase of the number of smooth muscle and endothelial cells contributing to the aorta. At limb level, Pax3+ cells in the somite give rise to skeletal muscles and to a subpopulation of endothelial cells in blood vessels of the limb. We now demonstrate that in addition to the inhibitory role of Notch signaling on skeletal muscle cell differentiation, the Notch pathway affects the Pax3:Foxc2 balance and promotes the endothelial versus myogenic cell fate, before migration to the limb, in multipotent Pax3+ cells in the somite of the mouse embryo. PMID:24927569

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. Mechanism of regulation of receptor histidine kinases.

    PubMed

    Ferris, Hedda U; Dunin-Horkawicz, Stanislaw; Hornig, Nora; Hulko, Michael; Martin, Jörg; Schultz, Joachim E; Zeth, Kornelius; Lupas, Andrei N; Coles, Murray

    2012-01-11

    Bacterial transmembrane receptors regulate an intracellular catalytic output in response to extracellular sensory input. To investigate the conformational changes that relay the regulatory signal, we have studied the HAMP domain, a ubiquitous intracellular module connecting input to output domains. HAMP forms a parallel, dimeric, four-helical coiled coil, and rational substitutions in our model domain (Af1503 HAMP) induce a transition in its interhelical packing, characterized by axial rotation of all four helices (the gearbox signaling model). We now illustrate how these conformational changes are propagated to a downstream domain by fusing Af1503 HAMP variants to the DHp domain of EnvZ, a bacterial histidine kinase. Structures of wild-type and mutant constructs are correlated with ligand response in vivo, clearly associating them with distinct signaling states. We propose that altered recognition of the catalytic domain by DHp, rather than a shift in position of the phospho-accepting histidine, forms the basis for regulation of kinase activity.

  3. O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1

    SciTech Connect

    Sakaidani, Yuta; Ichiyanagi, Naoki; Saito, Chika; Nomura, Tomoko; Ito, Makiko; Nishio, Yosuke; Nadano, Daita; Matsuda, Tsukasa; Furukawa, Koichi; Okajima, Tetsuya

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer We characterized A130022J15Rik (Eogt1)-a mouse gene homologous to Drosophila Eogt. Black-Right-Pointing-Pointer Eogt1 encodes EGF domain O-GlcNAc transferase. Black-Right-Pointing-Pointer Expression of Eogt1 in Drosophila rescued the cell-adhesion defect in the Eogt mutant. Black-Right-Pointing-Pointer O-GlcNAcylation reaction in the secretory pathway is conserved through evolution. -- Abstract: O-linked-{beta}-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shown whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution.

  4. Melanocortin 1 Receptor: Structure, Function, and Regulation

    PubMed Central

    Wolf Horrell, Erin M.; Boulanger, Mary C.; D’Orazio, John A.

    2016-01-01

    The melanocortin 1 receptor (MC1R) is a melanocytic Gs protein coupled receptor that regulates skin pigmentation, UV responses, and melanoma risk. It is a highly polymorphic gene, and loss of function correlates with a fair, UV-sensitive, and melanoma-prone phenotype due to defective epidermal melanization and sub-optimal DNA repair. MC1R signaling, achieved through adenylyl cyclase activation and generation of the second messenger cAMP, is hormonally controlled by the positive agonist melanocortin, the negative agonist agouti signaling protein, and the neutral antagonist β-defensin 3. Activation of cAMP signaling up-regulates melanin production and deposition in the epidermis which functions to limit UV penetration into the skin and enhances nucleotide excision repair (NER), the genomic stability pathway responsible for clearing UV photolesions from DNA to avoid mutagenesis. Herein we review MC1R structure and function and summarize our laboratory’s findings on the molecular mechanisms by which MC1R signaling impacts NER. PMID:27303435

  5. MicroRNA-101 regulates T-cell acute lymphoblastic leukemia progression and chemotherapeutic sensitivity by targeting Notch1

    PubMed Central

    Qian, Lu; Zhang, Wanggang; Lei, Bo; He, Aili; Ye, Lianhong; Li, Xingzhou; Dong, Xin

    2016-01-01

    The present study aimed to investigate the role of microRNA (miR)-101 in acute lymphoblastic leukemia progression and chemoresistance. Furthermore, a novel target gene of miR-101 was identified. Here, we confirmed that miR-101 was significantly downregulated in the blood samples of patients with T-cell acute lymphoblastic leukemia (T-ALL) compared with the healthy controls, as determined by reverse transcription quantitative polymerase chain reaction (RTqPCR) analysis. The in vitro experiments demonstrated that miR-101 significantly repressed the proliferation and invasion, and induced potent apoptosis in Jurkat cells, as determined by CCK-8, flow cytometer and cell invasion assays. Luciferase assay confirmed that Notch1 was a target gene of miR-101, and western blotting showed that miR-101 suppressed the expression of Notch1 at the protein level. Moreover, functional restoration assays revealed that Notch1 mediates the effects of miR-101 on Jurkat cell proliferation, apoptosis and invasion. miR-101 enhanced the sensitivity of Jurkat cells to the chemotherapeutic agent adriamycin. Taken together, our results show for the first time that miR-101 acts as a tumor suppressor in T-cell acute lymphoblastic leukaemia and it could enhance chemotherapeutic sensitivity. Furthermore, Notch1 was identified to be a novel target of miR-101. This study indicates that miR-101 may represent a potential therapeutic target for T-cell acute lymphoblastic leukemia intervention. PMID:27666896

  6. 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

  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. An Aquaporin 3-Notch1 Axis in Keratinocyte Differentiation and Inflammation

    PubMed Central

    Guo, Liqiong; Chen, Hongxiang; Li, Yongsheng; Zhou, Qixing; Sui, Yang

    2013-01-01

    Aquaporin 3 (AQP3) is an aquaglyceroporin which transports water, glycerol and small solutes across the plasma membrane. Its functions are not limited to fluid transport but also involve the regulation of cell proliferation, migration, skin hydration, wound healing and tumorigenesis. While AQP3 has been reported to play an important role in keratinocyte proliferation, its role in differentiation remains controversial. Our study demonstrated that the expression of AQP3 was regulated during differentiation and that it participated in keratinocyte differentiation control. We further revealed that AQP3 was a transcriptional target of Notch signaling, a critical pathway regulating keratinocyte differentiation and tumor suppression, and it regulated differentiation through a reciprocal negative feedback loop with Notch1. When the expression level of AQP3 was elevated, impaired barrier integrity and increased pro-inflammatory cytokine production ensued, mimicking the pathological conditions in Notch deficient mice and in atopic dermatitis. Dysregulation of AQP3 and Notch receptors has been reported in several skin diseases, including skin cancer. Our discovery of the novel AQP3-Notch1 axis may provide insight into epidermal homeostasis control and possible translational applications, including its potential use as a biomarker for molecular diagnosis in environmental studies. PMID:24260356

  9. An aquaporin 3-notch1 axis in keratinocyte differentiation and inflammation.

    PubMed

    Guo, Liqiong; Chen, Hongxiang; Li, Yongsheng; Zhou, Qixing; Sui, Yang

    2013-01-01

    Aquaporin 3 (AQP3) is an aquaglyceroporin which transports water, glycerol and small solutes across the plasma membrane. Its functions are not limited to fluid transport but also involve the regulation of cell proliferation, migration, skin hydration, wound healing and tumorigenesis. While AQP3 has been reported to play an important role in keratinocyte proliferation, its role in differentiation remains controversial. Our study demonstrated that the expression of AQP3 was regulated during differentiation and that it participated in keratinocyte differentiation control. We further revealed that AQP3 was a transcriptional target of Notch signaling, a critical pathway regulating keratinocyte differentiation and tumor suppression, and it regulated differentiation through a reciprocal negative feedback loop with Notch1. When the expression level of AQP3 was elevated, impaired barrier integrity and increased pro-inflammatory cytokine production ensued, mimicking the pathological conditions in Notch deficient mice and in atopic dermatitis. Dysregulation of AQP3 and Notch receptors has been reported in several skin diseases, including skin cancer. Our discovery of the novel AQP3-Notch1 axis may provide insight into epidermal homeostasis control and possible translational applications, including its potential use as a biomarker for molecular diagnosis in environmental studies. PMID:24260356

  10. Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21Cip1 expression and hampers tumour cell growth in vitro and in vivo

    PubMed Central

    Raimondi, L; Ciarapica, R; De Salvo, M; Verginelli, F; Gueguen, M; Martini, C; De Sio, L; Cortese, G; Locatelli, M; Dang, T P; Carlesso, N; Miele, L; Stifani, S; Limon, I; Locatelli, F; Rota, R

    2012-01-01

    Rhabdomyosarcoma (RMS) is a paediatric soft-tissue sarcoma arising from skeletal muscle precursors coexpressing markers of proliferation and differentiation. Inducers of myogenic differentiation suppress RMS tumourigenic phenotype. The Notch target gene HES1 is upregulated in RMS and prevents tumour cell differentiation in a Notch-dependent manner. However, Notch receptors regulating this phenomenon are unknown. In agreement with data in RMS primary tumours, we show here that the Notch3 receptor is overexpressed in RMS cell lines versus normal myoblasts. Notch3-targeted downregulation in RMS cells induces hyper-phosphorylation of p38 and Akt essential for myogenesis, resulting in the differentiation of tumour cells into multinucleated myotubes expressing Myosin Heavy Chain. These phenomena are associated to a marked decrease in HES1 expression, an increase in p21Cip1 level and the accumulation of RMS cells in the G1 phase. HES1-forced overexpression in RMS cells reverses, at least in part, the pro-differentiative effects of Notch3 downregulation. Notch3 depletion also reduces the tumourigenic potential of RMS cells both in vitro and in vivo. These results indicate that downregulation of Notch3 is sufficient to force RMS cells into completing a correct full myogenic program providing evidence that it contributes, partially through HES1 sustained expression, to their malignant phenotype. Moreover, they suggest Notch3 as a novel potential target in human RMS. PMID:22117196

  11. ADAM10 overexpression shifts lympho- and myelopoiesis by dysregulating site 2/site 3 cleavage products of Notch.

    PubMed

    Gibb, David R; Saleem, Sheinei J; Kang, Dae-Joong; Subler, Mark A; Conrad, Daniel H

    2011-04-01

    Although the physiological consequences of Notch signaling in hematopoiesis have been extensively studied, the differential effects of individual notch cleavage products remain to be elucidated. Given that ADAM10 is a critical regulator of Notch and that its deletion is embryonically lethal, we generated mice that overexpress ADAM10 (ADAM10 transgenic [A10Tg]) at early stages of lympho- and myeloid development. Transgene expression resulted in abrogated B cell development, delayed T cell development in the thymus, and unexpected systemic expansion of CD11b(+)Gr-1(+) cells, also known as myeloid-derived suppressor cells. Mixed bone marrow reconstitution assays demonstrated that transgene expression altered hematopoiesis via a cell-intrinsic mechanism. Consistent with previously reported observations, we hypothesized that ADAM10 overexpression dysregulated Notch by uncoupling the highly regulated proteolysis of Notch receptors. This was confirmed using an in vitro model of hematopoiesis via culturing A10Tg hematopoietic Lineage(-)Sca-1(+)c-Kit(+) cells with OP-9 stromal cells in the presence or absence of Delta-like 1, a primary ligand for Notch. Blockade of the site 2 (S2) and site 3 (S3) cleavage of the Notch receptor demonstrated differential effects on hematopoiesis. OP9-DL1 cultures containing the ADAM10 inhibitor (S2 cleavage site) enhanced and rescued B cell development from wild-type and A10Tg Lineage(-)Sca-1(+)c-Kit(+) cells, respectively. In contrast, blockade of γ-secretase at the S3 cleavage site induced accumulation of the S2 product and consequently prevented B cell development and resulted in myeloid cell accumulation. Collectively, these findings indicate that the differential cleavage of Notch into S2 and S3 products regulated by ADAM10 is critical to hematopoietic cell-fate determination.

  12. 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.

  13. Assembly of a Notch transcriptional activation complex requires multimerization.

    PubMed

    Vasquez-Del Carpio, Rodrigo; Kaplan, Fred M; Weaver, Kelly L; VanWye, Jeffrey D; Alves-Guerra, Marie-Clotilde; Robbins, David J; Capobianco, Anthony J

    2011-04-01

    Notch transmembrane receptors direct essential cellular processes, such as proliferation and differentiation, through direct cell-to-cell interactions. Inappropriate release of the intracellular domain of Notch (N(ICD)) from the plasma membrane results in the accumulation of deregulated nuclear N(ICD) that has been linked to human cancers, notably T-cell acute lymphoblastic leukemia (T-ALL). Nuclear N(ICD) forms a transcriptional activation complex by interacting with the coactivator protein Mastermind-like 1 and the DNA binding protein CSL (for CBF-1/Suppressor of Hairless/Lag-1) to regulate target gene expression. Although it is well understood that N(ICD) forms a transcriptional activation complex, little is known about how the complex is assembled. In this study, we demonstrate that N(ICD) multimerizes and that these multimers function as precursors for the stepwise assembly of the Notch activation complex. Importantly, we demonstrate that the assembly is mediated by N(ICD) multimers interacting with Skip and Mastermind. These interactions form a preactivation complex that is then resolved by CSL to form the Notch transcriptional activation complex on DNA.

  14. Self-Renewal and High Proliferative Colony Forming Capacity of Late-Outgrowth Endothelial Progenitors Is Regulated by Cyclin-Dependent Kinase Inhibitors Driven by Notch Signaling.

    PubMed

    Patel, Jatin; Wong, Ho Yi; Wang, Weili; Alexis, Josue; Shafiee, Abbas; Stevenson, Alexander J; Gabrielli, Brian; Fisk, Nicholas M; Khosrotehrani, Kiarash

    2016-04-01

    Since the discovery of endothelial colony forming cells (ECFC), there has been significant interest in their therapeutic potential to treat vascular injuries. ECFC cultures display significant heterogeneity and a hierarchy among cells able to give rise to high proliferative versus low proliferative colonies. Here we aimed to define molecularly this in vitro hierarchy. Based on flow cytometry, CD34 expression levels distinguished two populations. Only CD34 + ECFC had the capacity to reproduce high proliferative potential (HPP) colonies on replating, whereas CD34- ECFCs formed only small clusters. CD34 + ECFCs were the only ones to self-renew in stringent single-cell cultures and gave rise to both CD34 + and CD34- cells. Upon replating, CD34 + ECFCs were always found at the centre of HPP colonies and were more likely in G0/1 phase of cell cycling. Functionally, CD34 + ECFC were superior at restoring perfusion and better engrafted when injected into ischemic hind limbs. Transcriptomic analysis identified cyclin-dependent kinase (CDK) cell cycle inhibiting genes (p16, p21, and p57), the Notch signaling pathway (dll1, dll4, hes1, and hey1), and the endothelial cytokine il33 as highly expressed in CD34 + ECFC. Blocking the Notch pathway using a γ-secretase inhibitor (DAPT) led to reduced expression of cell cycle inhibitors, increased cell proliferation followed by a loss of self-renewal, and HPP colony formation capacity reflecting progenitor exhaustion. Similarly shRNA knockdown of p57 strongly affected self-renewal of ECFC colonies. ECFC hierarchy is defined by Notch signalling driving cell cycle regulators, progenitor quiescence and self-renewal potential. PMID:26732848

  15. 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

  16. DeltaA/DeltaD regulate multiple and temporally distinct phases of notch signaling during dopaminergic neurogenesis in zebrafish.

    PubMed

    Mahler, Julia; Filippi, Alida; Driever, Wolfgang

    2010-12-01

    Dopaminergic neurons develop at distinct anatomical sites to form some of the major neuromodulatory systems in the vertebrate brain. Despite their relevance in neurodegenerative diseases and the interests in reconstitutive therapies from stem cells, mechanisms of the neurogenic switch from precursor populations to dopaminergic neurons are not well understood. Here, we investigated neurogenesis of different dopaminergic and noradrenergic neuron populations in the zebrafish embryo. Birth-dating analysis by EdU (5-ethynyl-2'-deoxyuridine) incorporation revealed temporal dynamics of catecholaminergic neurogenesis. Analysis of Notch signaling mutants and stage-specific pharmacological inhibition of Notch processing revealed that dopaminergic neurons form by temporally distinct mechanisms: dopaminergic neurons of the posterior tuberculum derive directly from neural plate cells during primary neurogenesis, whereas other dopaminergic groups form in continuous or wavelike neurogenesis phases from proliferating precursor pools. Systematic analysis of Notch ligands revealed that the two zebrafish co-orthologs of mammalian Delta1, DeltaA and DeltaD, control the neurogenic switch of all early developing dopaminergic neurons in a partially redundant manner. DeltaA/D may also be involved in maintenance of dopaminergic precursor pools, as olig2 expression in ventral diencephalic dopaminergic precursors is affected in dla/dld mutants. DeltaA/D act upstream of sim1a and otpa during dopaminergic specification. However, despite the fact that both dopaminergic and corticotropin-releasing hormone neurons derive from sim1a- and otpa-expressing precursors, DeltaA/D does not act as a lineage switch between these two neuronal types. Rather, DeltaA/D limits the size of the sim1a- and otpa-expressing precursor pool from which dopaminergic neurons differentiate. PMID:21148001

  17. Disrupting NOTCH Slows Diffuse Intrinsic Pontine Glioma Growth, Enhances Radiation Sensitivity, and Shows Combinatorial Efficacy with Bromodomain Inhibition

    PubMed Central

    Taylor, Isabella C.; Hütt-Cabezas, Marianne; Brandt, William D.; Kambhampati, Madhuri; Nazarian, Javad; Chang, Howard T.; Warren, Katherine E.; Eberhart, Charles G.; Raabe, Eric H.

    2015-01-01

    NOTCH regulates stem cells during normal development and stem-like cells in cancer but the roles of NOTCH in the lethal pediatric brain tumor diffuse intrinsic pontine glioma (DIPG) remain unknown. Because DIPGs express stem cell factors such as SOX2 and MYCN, we hypothesized that NOTCH activity would be critical for DIPG growth. We determined that primary DIPGs expressed high levels of NOTCH receptors, ligands, and downstream effectors. Treatment of the DIPG cell lines JHH-DIPG1 and SF7761 with the γ-secretase inhibitor MRK003 suppressed the level of the NOTCH effectors HES1, HES4, HES5, inhibited DIPG growth by 75%, and caused a 3-fold induction of apoptosis. Short hairpin RNAs targeting the canonical NOTCH pathway caused similar effects. Pre-treatment of DIPG cells with MRK003 suppressed clonogenic growth by more than 90% and enhanced the efficacy of radiation therapy. The high level of MYCN in DIPG led us to test sequential therapy with the bromodomain inhibitor JQ1 and MRK003, and we found that JQ1 and MRK003 inhibited DIPG growth and induced apoptosis. Together, these results suggest that dual targeting of NOTCH and MYCN in DIPG may be an effective therapeutic strategy in DIPG and that adding a γ-secretase inhibitor during radiation therapy may be efficacious initially or during re-irradiation. PMID:26115193

  18. Notch and the awesome power of genetics.

    PubMed

    Greenwald, Iva

    2012-07-01

    Notch is a receptor that mediates cell-cell interactions in animal development, and aberrations in Notch signal transduction can cause cancer and other human diseases. Here, I describe the major advances in the Notch field from the identification of the first mutant in Drosophila almost a century ago through the elucidation of the unusual mechanism of signal transduction a little over a decade ago. As an essay for the GENETICS Perspectives series, it is my personal and critical commentary as well as an historical account of discovery.

  19. Ganglioside Regulation of AMPA Receptor Trafficking

    PubMed Central

    Prendergast, Jillian; Umanah, George K.E.; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G.; Cole, Robert N.; Huganir, Richard L.; Dawson, Ted M.; Dawson, Valina L.

    2014-01-01

    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p < 10−4), three regulate neurotransmitter receptor trafficking: Thorase (ATPase family AAA domain-containing protein 1), soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP), and the transmembrane protein Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans. PMID:25253868

  20. 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

  1. Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch.

    PubMed

    Harvey, Beth M; Rana, Nadia A; Moss, Hillary; Leonardi, Jessica; Jafar-Nejad, Hamed; Haltiwanger, Robert S

    2016-07-29

    Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the β3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity.

  2. Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch.

    PubMed

    Harvey, Beth M; Rana, Nadia A; Moss, Hillary; Leonardi, Jessica; Jafar-Nejad, Hamed; Haltiwanger, Robert S

    2016-07-29

    Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the β3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity. PMID:27268051

  3. 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

  4. MUC4 is negatively regulated through the Wnt/β-catenin pathway via the Notch effector Hath1 in colorectal cancer.

    PubMed

    Pai, Priya; Rachagani, Satyanarayana; Dhawan, Punita; Sheinin, Yuri M; Macha, Muzafar A; Qazi, Asif Khurshid; Chugh, Seema; Ponnusamy, Moorthy P; Mallya, Kavita; Pothuraju, Ramesh; Batra, Surinder K

    2016-05-01

    MUC4 is a transmembrane mucin lining the normal colonic epithelium. The aberrant/de novo over-expression of MUC4 is well documented in malignancies of the pancreas, ovary and breast. However, studies have reported the loss of MUC4 expression in the majority of colorectal cancers (CRCs). A MUC4 promoter analysis showed the presence of three putative TCF/LEF sites, implying a possible regulation by the Wnt/β-catenin pathway, which has been shown to drive CRC progression. Thus, the objective of our study was to determine whether MUC4 is regulated by β-catenin in CRC. We first knocked down (KD) β-catenin in three CRC cell lines; LS180, HCT-8 and HCT116, which resulted in increased MUC4 transcript and MUC4 protein. Additionally, the overexpression of stabilized mutant β-catenin in LS180 and HCT-8 resulted in a decrease in MUC4 expression. Immunohistochemistry (IHC) of mouse colon tissue harboring tubular adenomas and high grade dysplasia showed dramatically reduced Muc4 in lesions relative to adjacent normal tissue, with increased cytosolic/nuclear β-catenin. Luciferase assays with the complete MUC4 promoter construct p3778 showed increased MUC4 promoter luciferase activity in the absence of β-catenin (KD). Mutation of all three putative TCF/LEF sites showed that MUC4 promoter luciferase activity was increased relative to the un-mutated promoter. Interestingly, it was observed that MUC4 expressing CRC cell lines also expressed high levels of Hath1, a transcription factor repressed by both active Wnt/β-catenin and Notch signaling. The KD of β-catenin and/or treatment with a Notch γ-secretase inhibitor, Dibenzazepine (DBZ) resulted in increased Hath1 and MUC4 in LS180, HCT-8 and HCT116. Furthermore, overexpression of Hath1 in HCT-8 and LS180 caused increased MUC4 transcript and MUC4 protein. Taken together, our results indicate that the Wnt/β-catenin pathway suppresses the Notch pathway effector Hath1, resulting in reduced MUC4 in CRC. PMID:27551331

  5. MUC4 is negatively regulated through the Wnt/β-catenin pathway via the Notch effector Hath1 in colorectal cancer

    PubMed Central

    Pai, Priya; Rachagani, Satyanarayana; Dhawan, Punita; Sheinin, Yuri M.; Mallya, Kavita; Pothuraju, Ramesh; Batra, Surinder K.

    2016-01-01

    MUC4 is a transmembrane mucin lining the normal colonic epithelium. The aberrant/de novo over-expression of MUC4 is well documented in malignancies of the pancreas, ovary and breast. However, studies have reported the loss of MUC4 expression in the majority of colorectal cancers (CRCs). A MUC4 promoter analysis showed the presence of three putative TCF/LEF sites, implying a possible regulation by the Wnt/β-catenin pathway, which has been shown to drive CRC progression. Thus, the objective of our study was to determine whether MUC4 is regulated by β-catenin in CRC. We first knocked down (KD) β-catenin in three CRC cell lines; LS180, HCT-8 and HCT116, which resulted in increased MUC4 transcript and MUC4 protein. Additionally, the overexpression of stabilized mutant β-catenin in LS180 and HCT-8 resulted in a decrease in MUC4 expression. Immunohistochemistry (IHC) of mouse colon tissue harboring tubular adenomas and high grade dysplasia showed dramatically reduced Muc4 in lesions relative to adjacent normal tissue, with increased cytosolic/nuclear β-catenin. Luciferase assays with the complete MUC4 promoter construct p3778 showed increased MUC4 promoter luciferase activity in the absence of β-catenin (KD). Mutation of all three putative TCF/LEF sites showed that MUC4 promoter luciferase activity was increased relative to the un-mutated promoter. Interestingly, it was observed that MUC4 expressing CRC cell lines also expressed high levels of Hath1, a transcription factor repressed by both active Wnt/β-catenin and Notch signaling. The KD of β-catenin and/or treatment with a Notch γ-secretase inhibitor, Dibenzazepine (DBZ) resulted in increased Hath1 and MUC4 in LS180, HCT-8 and HCT116. Furthermore, overexpression of Hath1 in HCT-8 and LS180 caused increased MUC4 transcript and MUC4 protein. Taken together, our results indicate that the Wnt/β-catenin pathway suppresses the Notch pathway effector Hath1, resulting in reduced MUC4 in CRC. PMID:27551331

  6. 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

  7. 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

  8. 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

  9. 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.

  10. Chimeric analysis of Notch2 function: a role for Notch2 in the development of the roof plate of the mouse brain.

    PubMed

    Kadokawa, Yuzo; Marunouchi, Tohru

    2002-10-01

    Notch proteins are transmembrane receptors involved in cell-fate determination throughout development. Targeted disruption of either the Notch1 or Notch2 gene in mice results in embryonic lethality around embryonic day (E) 10.5 with widespread cell death. Although Notch1-deficient mice show disorganized somitogenesis, Notch2 mutants did not show definitive abnormalities in any tissue expressing high levels of the Notch2 gene, including the central nervous system. To study Notch2 function in development beyond the embryonic lethal stage, we performed chimeric analysis between Notch2 mutant and wild-type mouse embryos. Chimeric embryos developed normally and homozygous Notch2 mutant-specific cell death was not observed. Although chimeric embryos showed normal mosaicism until E9.5 in all tissues studied to date, Notch2 homozygous mutant cells failed to contribute to formation of the roof plate of the diencephalon and mesencephalon at later developmental stages, when Notch2 is normally expressed at high levels at there. Furthermore, Notch2 heterozygous mutant cells were also excluded from the roof plate of the chimera, however, Notch2 heterozygous mutant mice developed normally. We also showed that Wnt-1 and Mash1 expression patterns at the roof plate were disorganized in Notch2 homozygous mutant embryos. These results indicate that Notch2 plays an important role in development of the roof plate of the diencephalon and mesencephalon, and suggest that cellular rearrangement is involved in this process.

  11. 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

  12. Regulation of the cytosolic sulfotransferases by nuclear receptors

    PubMed Central

    Runge-Morris, Melissa; Kocarek, Thomas A.; Falany, Charles N.

    2013-01-01

    The cytosolic sulfotransferases (SULTs) are a multigene family of enzymes that catalyze the transfer of a sulfonate group from the physiologic sulfate donor, 3′-phosphoadenosine-5′-phosphosulfate, to a nucleophilic substrate to generate a polar product that is more amenable to elimination from the body. As catalysts of both xenobiotic and endogenous metabolism, the SULTs are major points of contact between the external and physiological environments, and modulation of SULT-catalyzed metabolism can not only affect xenobiotic disposition, but it can also alter endogenous metabolic processes. Therefore, it is not surprising that SULT expression is regulated by numerous members of the nuclear receptor (NR) superfamily that function as sensors of xenobiotics as well as endogenous molecules, such as fatty acids, bile acids, and oxysterols. These NRs include the peroxisome proliferator-activated receptors, pregnane X receptor, constitutive androstane receptor, vitamin D receptor, liver X receptors, farnesoid X receptor, retinoid-related orphan receptors, and estrogen-related receptors. This review summarizes current information about NR regulation of SULT expression. Because species differences in SULT subfamily composition and tissue-, sex-, development-, and inducer-dependent regulation are prominent, these differences will be emphasized throughout the review. In addition, because of the central role of the SULTs in cellular physiology, the effect of NR-mediated SULT regulation on physiological and pathophysiological processes will be discussed. Gaps in current knowledge that require further investigation are also highlighted. PMID:23330539

  13. 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

  14. 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.

  15. 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

  16. The immunohistochemical localization of notch receptors and ligands in human articular cartilage, chondroprogenitor culture and ultrastructural characteristics of these progenitor cells.

    PubMed

    Ustunel, Ismail; Ozenci, Alpay Merter; Sahin, Zeliha; Ozbey, Ozlem; Acar, Nuray; Tanriover, Gamze; Celik-Ozenci, Ciler; Demir, Ramazan

    2008-01-01

    The presence of progenitor/stem cells in human articular cartilage remains controversial. Therefore, we attempted to isolate and culture progenitor/stem cells and to investigate their phenotypic characteristics. Biopsies were obtained (with consent) from patients undergoing arthroscopic surgery. Full depth explants were fixed and cryosectioned or enzymatically digested and the resulting cells cultured and plated on fibronectin-coated dishes. Chondrocytes were cultured until colonies of >32 cells were present. Colonies were trypsinized and expanded in monolayer for pellet culture. Immunolocalization of Notch and its ligands were detected in vivo and in vitro using immunocytochemistry. In vitro studies investigated differences in immunolocalization of Notch and its associated ligands in colony-forming cells and small clusters of non-colony-forming cells. The ultrastructure of the chondroprogenitors was examined by scanning and transmission electron microscopy. Results revealed that the immunolocalization of Notch-1 and its ligand Delta were concentrated in regions closest to the articular surface. Notch-1 was also densely localized in the deeper zone of articular cartilage. Notch-2 immunolabeling was densely localized in all zones of articular cartilage. Jagged-1 was concentrated in the deeper regions of articular cartilage. Notch-1, Delta and Jagged-1 were more abundant in colony-forming cells than non-colony-forming chondrocytes in vitro. Notch-3, Notch-4 and Jagged-2 were absent from all regions of the articular cartilage tissues and cultured cartilage cells in vitro. Ultrastructurally, chondrocytes cultured in monolayer dedifferentiated to fibroblast-like cells with cell surface processes of varying lengths, pellet cultured cells varied in morphology, as flattened and rounded. In conclusion, we propose that adult human articular cartilage may contain cells having progenitor cell features. PMID:18272209

  17. 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

  18. Function of Integrin-Linked Kinase in Modulating the Stemness of IL-6–Abundant Breast Cancer Cells by Regulating γ-Secretase–Mediated Notch1 Activation in Caveolae12

    PubMed Central

    Hsu, En-Chi; Kulp, Samuel K.; Huang, Han-Li; Tu, Huang-Ju; Salunke, Santosh B.; Sullivan, Nicholas J.; Sun, Duxin; Wicha, Max S.; Shapiro, Charles L.; Chen, Ching-Shih

    2015-01-01

    Interleukin-6 (IL-6) and Notch signaling are important regulators of breast cancer stem cells (CSCs), which drive the malignant phenotype through self-renewal, differentiation, and development of therapeutic resistance. We investigated the role of integrin-linked kinase (ILK) in regulating IL-6–driven Notch1 activation and the ability to target breast CSCs through ILK inhibition. Ectopic expression/short hairpin RNA-mediated knockdown of ILK, pharmacological inhibition of ILK with the small molecule T315, Western blot analysis, immunofluorescence, and luciferase reporter assays were used to evaluate the regulation of IL-6–driven Notch1 activation by ILK in IL-6–producing triple-negative breast cancer cell lines (MDA-MB-231, SUM-159) and in MCF-7 and MCF-7IL-6 cells. The effects of ILK on γ-secretase complex assembly and cellular localization were determined by immunofluorescence, Western blots of membrane fractions, and immunoprecipitation. In vivo effects of T315-induced ILK inhibition on CSCs in SUM-159 xenograft models were assessed by mammosphere assays, flow cytometry, and tumorigenicity assays. Results show that the genetic knockdown or pharmacological inhibition of ILK suppressed Notch1 activation and the abundance of the γ-secretase components presenilin-1, nicastrin, and presenilin enhancer 2 at the posttranscriptional level via inhibition of caveolin-1-dependent membrane assembly of the γ-secretase complex. Accordingly, knockdown of ILK inhibited breast CSC-like properties in vitro and the breast CSC subpopulation in vivo in xenograft tumor models. Based on these findings, we propose a novel function of ILK in regulating γ-secretase–mediated Notch1 activation, which suggests the targeting of ILK as a therapeutic approach to suppress IL-6–induced breast CSCs. PMID:26152358

  19. 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

  20. Metabotropic glutamate receptors: beyond the regulation of synaptic transmission.

    PubMed

    Nicoletti, Ferdinando; Battaglia, Giuseppe; Storto, Marianna; Ngomba, Richard T; Iacovelli, Luisa; Arcella, Antonietta; Gradini, Roberto; Sale, Patrizio; Rampello, Liborio; De Vita, Teresa; Di Marco, Roberto; Melchiorri, Daniela; Bruno, Valeria

    2007-08-01

    Metabotropic glutamate (mGlu) receptors are G-protein coupled receptors activated by glutamate, the major excitatory neurotransmitter of the CNS. A growing body of evidence suggests that the function of mGlu receptors is not restricted to the regulation of synaptic transmission. mGlu receptors are expressed in a variety of peripheral cells, including inter alia hepatocytes, pancreatic cells, osteoblasts and immune cells. Within the immunological synapses, mGlu receptors expressed by T cells might contribute to the vast array of signals generated by the antigen-presenting cells. mGlu receptors are also found in embryonic and neural stem cells. This suggests their involvement in the pathophysiology of brain tumors, which likely originates from cancer stem cells similar to neural stem cells. Ligands of mGlu3 and mGlu4 receptors are potential candidates for the experimental treatment of malignant gliomas and medulloblastomas, respectively. PMID:17651904

  1. 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.

  2. Temporal Notch activation through Notch1a and Notch3 is required for maintaining zebrafish rhombomere boundaries.

    PubMed

    Qiu, Xuehui; Lim, Chiaw-Hwee; Ho, Steven Hao-Kee; Lee, Kian-Hong; Jiang, Yun-Jin

    2009-07-01

    In vertebrates, hindbrain is subdivided into seven segments termed rhombomeres and the interface between each rhombomere forms the boundary. Similar to the D/V boundary formation in Drosophila, Notch activation has been shown to regulate the segregation of rhombomere boundary cells. Here we further explored the function of Notch signaling in the formation of rhombomere boundaries. By using bodipy ceramide cell-labeling technique, we found that the hindbrain boundary is formed initially in mib mutants but lost after 24 hours post-fertilization (hpf). This phenotype was more severe in mib(ta52b) allele than in mib(tfi91) allele. Similarly, injection of su(h)-MO led to boundary defects in a dosage-dependent manner. Boundary cells were recovered in mib(ta52b) mutants in the hdac1-deficient background, where neurogenesis is inhibited. Furthermore, boundary cells lost sensitivity to reduced Notch activation from 15 somite stage onwards. We also showed that knockdown of notch3 function in notch1a mutants leads to the loss of rhombomere boundary cells and causes neuronal hyperplasia, indicating that Notch1a and Notch3 play a redundant role in the maintenance of rhombomere boundary.

  3. Nuclear receptors and microRNAs: Who regulates the regulators in neural stem cells?

    PubMed

    Eendebak, Robert J A H; Lucassen, Paul J; Fitzsimons, Carlos P

    2011-03-01

    In this mini-review, we focus on regulatory loops between nuclear receptors and microRNAs, an emerging class of small RNA regulators of gene expression. Evidence supporting interactions between microRNAs and nuclear receptors in the regulation of gene expression networks is discussed in relation to its possible role in neural stem cell self renewal and differentiation. Furthermore, we discuss possible disturbances of the regulatory loops between microRNAs and nuclear receptors in human neurodegenerative disease. Finally, we discuss the possible use of nuclear receptors as pharmacological entry points to regulate neural stem cell self-renewal and differentiation.

  4. Notch3 is important for TGF-β-induced epithelial-mesenchymal transition in non-small cell lung cancer bone metastasis by regulating ZEB-1.

    PubMed

    Liu, L; Chen, X; Wang, Y; Qu, Z; Lu, Q; Zhao, J; Yan, X; Zhang, H; Zhou, Y

    2014-09-01

    The Notch signaling pathway plays an important role in the bone metastasis microenvironment. Although recent evidence suggests that Notch signaling contributes to bone metastasis in breast and prostate cancer, its role and possible mechanisms in non-small cell lung cancer (NSCLC) bone metastasis are not yet clear. Here, we show that Notch3 is overexpressed in NSCLC bone metastases. The inhibition of Notch3 by small interfering RNA transfection decreased the invasion ability of NSCLC cells and transforming growth factor (TGF)-induced interleukin (IL)-6 and parathyroid hormone-related protein (pTHrP) expression in vitro. We also observed that Notch3 induced a strong morphological transformation, promoting the epithelial-mesenchymal transition (EMT). Western blotting and real-time polymerase chain reaction assays revealed that the forced overexpression of Notch3 induced the expression and activity of ZEB-1 and subsequent suppression of E-cadherin and upregulation of fibronectin, contributing to EMT and invasion. Western blotting and immunofluorescence assays showed that RNA interference-mediated ZEB-1 suppression blocked Notch-induced EMT-like transformation and subsequently reversed the observed effects on E-cadherin and downregulated fibronectin. A luciferase reporter system showed that Notch-induced ZEB-1 requires a functional binding site in the ZEB-1 promoter. In vitro invasion assays showed that the inhibition of ZEB-1 can decrease Notch3-promoted invasion and the expression of pTHrP and IL-6. Our results demonstrated that Notch upregulates ZEB-1, which contributes to TGF-β-induced EMT-like transformation and bone metastasis in NSCLC.

  5. Transcriptional regulation through glutamate receptors: Involvement of tyrosine kinases.

    PubMed

    López-Bayghen, Esther; Aguirre, Adán; Ortega, Arturo

    2003-12-01

    Glutamate receptors play a key role in neuronal plasticity, learning and memory, and in several neuropathologies. Short-term and long-term changes in synaptic efficacy are triggered by glutamate. Although an enhanced glutamate-dependent tyrosine phosphorylation has been described in several systems, its role in membrane-to-nuclei signaling is unclear. Taking advantage of the fact that the gene encoding the chick kainate-binding protein undergoes a glutamate-dependent transcriptional regulation via an activator protein-1 (AP-1) site, we evaluated the involvement of tyrosine kinases in this process. We describe here the participation of receptor and non-receptor tyrosine kinases in the signaling cascade triggered by glutamate. Our results suggest that in Bergmann glia cells, glutamate receptors transactivate receptor tyrosine kinases, favoring the idea of a complex network of signals activated by this excitatory neurotransmitter that results in regulation of gene expression.

  6. TARP phosphorylation regulates synaptic AMPA receptors through lipid bilayers

    PubMed Central

    Sumioka, Akio; Yan, Dan; Tomita, Susumu

    2010-01-01

    Summary Neurons use neurotransmitters to communicate across synapses, constructing neural circuits in the brain. AMPA-type glutamate receptors are the predominant excitatory neurotransmitter receptors mediating fast synaptic transmission. AMPA receptors localize at synapses by forming protein complexes with transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95-like MAGUKs. Among the three classes of ionotropic glutamate receptors (AMPA-, NMDA, kainate-type), AMPA receptor activity is most regulatable by neuronal activity to adjust synaptic strength. Here, we mutated the prototypical TARP, stargazin, and found that TARP phosphorylation regulates synaptic AMPA receptor activity in vivo. We also found that stargazin interacts with negatively-charged lipid bilayers in its phosphorylation dependent manner, and that the lipid interaction inhibited stargazin binding to PSD-95. Cationic lipids dissociated stargazin from lipid bilayers and enhanced synaptic AMPA receptor activity in a stargazin phosphorylation-dependent manner. Thus, TARP phosphorylation plays a critical role in regulating AMPA receptor-mediated synaptic transmission via a lipid bilayer interaction. PMID:20547132

  7. Aurora-B and HDAC synergistically regulate survival and proliferation of lymphoma cell via AKT, mTOR and Notch pathways.

    PubMed

    Wang, Chong; Chen, Jing; Cao, Weijie; Sun, Ling; Sun, Hui; Liu, Yanfang

    2016-05-15

    Aurora-B is a protein kinase that functions mainly in the attachment of the mitotic spindle to the centromere. Overexpression of Aurora-B causes unequal distribution of genetic information, creating aneuploidy cells, a hallmark of cancer. Histone deacetylases (HDACs) are a class of enzymes that remove acetyl groups from a ε-N-acetyl lysine amino acid on a histone, allowing the histones to wrap the DNA more tightly, thus globally regulating gene transcription. Additionally, these HDACs can also modify non-histone proteins. Inhibition of HDACs is a potent strategy for cancer treatment. Here, we report that inhibition of Aurora-B and HDAC exerts similar tumor suppressive effects in cells. Knockdown of Aurora-B or inhibition of HDAC achieved the same effect on repression of cell proliferation. Furthermore, we found that the tumor suppressive effect of Aurora-B and HDAC inhibition is due to the induction of cell cycle arrest and/or apoptosis. Mechanistically, we demonstrated that Aurora-B and HDAC can cooperatively regulate AKT, mTOR and Notch pathways.

  8. A novel SCFA receptor, the microbiota, and blood pressure regulation

    PubMed Central

    Pluznick, Jennifer

    2014-01-01

    The maintenance of blood pressure homeostasis is a complex process which is carefully regulated by a variety of inputs. We recently identified two sensory receptors (Olfactory receptor 78 and G protein couple receptor 41) as novel regulators of blood pressure. Both Olfr78 and Gpr41 are receptors for short chain fatty acids (SCFAs), and we showed that propionate (a SCFA) modifies blood pressure in a manner which is differentially modulated by the absence of either Olfr78 or Gpr41. In addition, propionate modifies renin release in an Olfr78-dependent manner. Our study also demonstrated that antibiotic treatment modulates blood pressure in Olfr78 null mice, indicating that SCFAs produced by the gut microbiota likely influence blood pressure regulation. In this addendum, we summarize the findings of our recent study and provide a perspective on the implications of the interactions between the gut microbiota and blood pressure control. PMID:24429443

  9. Identification of novel Notch target genes in T cell leukaemia

    PubMed Central

    Chadwick, Nicholas; Zeef, Leo; Portillo, Virginia; Fennessy, Carl; Warrander, Fiona; Hoyle, Sarah; Buckle, Anne-Marie

    2009-01-01

    Background Dysregulated Notch signalling is believed to play an important role in the development and maintenance of T cell leukaemia. At a cellular level, Notch signalling promotes proliferation and inhibits apoptosis of T cell acute lymphoblastic leukaemia (T-ALL) cells. In this study we aimed to identify novel transcriptional targets of Notch signalling in the T-ALL cell line, Jurkat. Results RNA was prepared from Jurkat cells retrovirally transduced with an empty vector (GFP-alone) or vectors containing constitutively active forms of Notch (N1ΔE or N3ΔE), and used for Affymetrix microarray analysis. A subset of genes found to be regulated by Notch was chosen for real-time PCR validation and in some cases, validation at the protein level, using several Notch-transduced T-ALL and non-T-ALL leukaemic cell lines. As expected, several known transcriptional target of Notch, such as HES1 and Deltex, were found to be overexpressed in Notch-transduced cells, however, many novel transcriptional targets of Notch signalling were identified using this approach. These included the T cell costimulatory molecule CD28, the anti-apoptotic protein GIMAP5, and inhibitor of DNA binding 1 (1D1). Conclusion The identification of such downstream Notch target genes provides insights into the mechanisms of Notch function in T cell leukaemia, and may help identify novel therapeutic targets in this disease. PMID:19508709

  10. Prostate tumor OVerexpressed-1 (PTOV1) down-regulates HES1 and HEY1 notch targets genes and promotes prostate cancer progression

    PubMed Central

    2014-01-01

    Background PTOV1 is an adaptor protein with functions in diverse processes, including gene transcription and protein translation, whose overexpression is associated with a higher proliferation index and tumor grade in prostate cancer (PC) and other neoplasms. Here we report its interaction with the Notch pathway and its involvement in PC progression. Methods Stable PTOV1 knockdown or overexpression were performed by lentiviral transduction. Protein interactions were analyzed by co-immunoprecipitation, pull-down and/or immunofluorescence. Endogenous gene expression was analyzed by real time RT-PCR and/or Western blotting. Exogenous promoter activities were studied by luciferase assays. Gene promoter interactions were analyzed by chromatin immunoprecipitation assays (ChIP). In vivo studies were performed in the Drosophila melanogaster wing, the SCID-Beige mouse model, and human prostate cancer tissues and metastasis. The Excel package was used for statistical analysis. Results Knockdown of PTOV1 in prostate epithelial cells and HaCaT skin keratinocytes caused the upregulation, and overexpression of PTOV1 the downregulation, of the Notch target genes HEY1 and HES1, suggesting that PTOV1 counteracts Notch signaling. Under conditions of inactive Notch signaling, endogenous PTOV1 associated with the HEY1 and HES1 promoters, together with components of the Notch repressor complex. Conversely, expression of active Notch1 provoked the dismissal of PTOV1 from these promoters. The antagonist role of PTOV1 on Notch activity was corroborated in the Drosophila melanogaster wing, where human PTOV1 exacerbated Notch deletion mutant phenotypes and suppressed the effects of constitutively active Notch. PTOV1 was required for optimal in vitro invasiveness and anchorage-independent growth of PC-3 cells, activities counteracted by Notch, and for their efficient growth and metastatic spread in vivo. In prostate tumors, the overexpression of PTOV1 was associated with decreased expression

  11. A Structural Perspective on the Regulation of the EGF Receptor

    PubMed Central

    Kovacs, Erika; Zorn, Julie Anne; Huang, Yongjian; Barros, Tiago; Kuriyan, John

    2015-01-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a critical role in the pathogenesis of many cancers. EGFR is unique in that its ligand-induced dimerization is established solely by contacts between regions of the receptor that are occluded within the monomeric, unliganded state. Activation of EGFR depends on the formation of an asymmetric dimer of the intracellular module of two receptor molecules, a configuration observed in crystal structures of the EGFR kinase domain in the active state. Coupling between the extracellular and intracellular modules is achieved by a switch between alternative geometries of the transmembrane and juxtamembrane segments within the receptor dimer. As the structure of the full-length receptor is yet to be determined, here we review recent structural studies on isolated modules of EGFR and molecular dynamics simulations that have provided much of our current understanding of its signaling mechanism, including how its regulation is compromised by oncogenic mutations. PMID:25621509

  12. 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.

  13. Receptor regulation of osmolyte homeostasis in neural cells.

    PubMed

    Fisher, Stephen K; Heacock, Anne M; Keep, Richard F; Foster, Daniel J

    2010-09-15

    The capacity of cells to correct their volume in response to hyposmotic stress via the efflux of inorganic and organic osmolytes is well documented. However, the ability of cell-surface receptors, in particular G-protein-coupled receptors (GPCRs), to regulate this homeostatic mechanism has received much less attention. Mechanisms that underlie the regulation of cell volume are of particular importance to cells in the central nervous system because of the physical restrictions of the skull and the adverse impact that even small increases in cell volume can have on their function. Increases in brain volume are seen in hyponatraemia, which can arise from a variety of aetiologies and is the most frequently diagnosed electrolyte disorder in clinical practice. In this review we summarize recent evidence that the activation of GPCRs facilitates the volume-dependent efflux of osmolytes from neural cells and permits them to more efficiently respond to small, physiologically relevant, reductions in osmolarity. The characteristics of receptor-regulated osmolyte efflux, the signalling pathways involved and the physiological significance of receptor activation are discussed. In addition, we propose that GPCRs may also regulate the re-uptake of osmolytes into neural cells, but that the influx of organic and inorganic osmolytes is differentially regulated. The ability of neural cells to closely regulate osmolyte homeostasis through receptor-mediated alterations in both efflux and influx mechanisms may explain, in part at least, why the brain selectively retains its complement of inorganic osmolytes during chronic hyponatraemia, whereas its organic osmolytes are depleted. PMID:20498228

  14. Prolactin receptor in regulation of neuronal excitability and channels.

    PubMed

    Patil, Mayur J; Henry, Michael A; Akopian, Armen N

    2014-01-01

    Prolactin (PRL) activates PRL receptor isoforms to exert regulation of specific neuronal circuitries, and to control numerous physiological and clinically-relevant functions including; maternal behavior, energy balance and food intake, stress and trauma responses, anxiety, neurogenesis, migraine and pain. PRL controls these critical functions by regulating receptor potential thresholds, neuronal excitability and/or neurotransmission efficiency. PRL also influences neuronal functions via activation of certain neurons, resulting in Ca(2+) influx and/or electrical firing with subsequent release of neurotransmitters. Although PRL was identified almost a century ago, very little specific information is known about how PRL regulates neuronal functions. Nevertheless, important initial steps have recently been made including the identification of PRL-induced transient signaling pathways in neurons and the modulation of neuronal transient receptor potential (TRP) and Ca(2+) -dependent K(+) channels by PRL. In this review, we summarize current knowledge and recent progress in understanding the regulation of neuronal excitability and channels by PRL.

  15. A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans-activation and cis-inhibition.

    PubMed

    Cordle, Jemima; Johnson, Steven; Tay, Joyce Zi Yan; Roversi, Pietro; Wilkin, Marian B; de Madrid, Beatriz Hernández; Shimizu, Hideyuki; Jensen, Sacha; Whiteman, Pat; Jin, Boquan; Redfield, Christina; Baron, Martin; Lea, Susan M; Handford, Penny A

    2008-08-01

    The Notch receptor and its ligands are key components in a core metazoan signaling pathway that regulates the spatial patterning, timing and outcome of many cell-fate decisions. Ligands contain a disulfide-rich Delta/Serrate/LAG-2 (DSL) domain required for Notch trans-activation or cis-inhibition. Here we report the X-ray structure of a receptor binding region of a Notch ligand, the DSL-EGF3 domains of human Jagged-1 (J-1(DSL-EGF3)). The structure reveals a highly conserved face of the DSL domain, and we show, by functional analysis of Drosophila melanogster ligand mutants, that this surface is required for both cis- and trans-regulatory interactions with Notch. We also identify, using NMR, a surface of Notch-1 involved in J-1(DSL-EGF3) binding. Our data imply that cis- and trans-regulation may occur through the formation of structurally distinct complexes that, unexpectedly, involve the same surfaces on both ligand and receptor.

  16. Regulation of atrial natriuretic peptide receptors in the rat brain

    SciTech Connect

    Saavedra, J.M.

    1987-06-01

    We have studied the localization, kinetics, and regulation of receptors for the circulating form of the atrial natriuretic peptide (ANP; 99-126) in the rat brain. Quantitative autoradiographic techniques and a /sup 125/I-labeled ligand, /sup 125/I-ANP (99-126), were employed. After in vitro autoradiography, quantification was achieved by computerized microdensitometry followed by comparison with /sup 125/I-standards. ANP receptors were discretely localized in the rat brain, with the highest concentrations in circumventricular organs, the choroid plexus, and selected hypothalamic nuclei involved in the production of the antidiuretic hormone vasopressin and in blood-pressure control. Spontaneously (genetic) hypertensive rats showed much lower numbers of ANP receptors than normotensive controls in the subfornical organ, the area postrema, the nucleus of the solitary tract, and the choroid plexus. These changes are in contrast to those observed for receptors of angiotensin II, another circulating peptide with actions opposite to those of ANP. Under conditions of acute dehydration after water deprivation, as well as under conditions of chronic dehydration such as those present in homozygous Brattleboro rats, there was an up-regulation of ANP receptors in the subfornical organ. Our results indicate that in the brain, circumventricular organs contain ANP receptors which could respond to variations in the concentration of circulating ANP. In addition, brain areas inside the blood-brain barrier contain ANP receptors probably related to the endogenous, central ANP system. The localization of ANP receptors and the alterations in their regulation present in genetically hypertensive rats and after dehydration indicate that brain ANP receptors are probably related to fluid regulation, including the secretion of vasopressin, and to cardiovascular function.

  17. Sex Hormones and Their Receptors Regulate Liver Energy Homeostasis

    PubMed Central

    Shen, Minqian; Shi, Haifei

    2015-01-01

    The liver is one of the most essential organs involved in the regulation of energy homeostasis. Hepatic steatosis, a major manifestation of metabolic syndrome, is associated with imbalance between lipid formation and breakdown, glucose production and catabolism, and cholesterol synthesis and secretion. Epidemiological studies show sex difference in the prevalence in fatty liver disease and suggest that sex hormones may play vital roles in regulating hepatic steatosis. In this review, we summarize current literature and discuss the role of estrogens and androgens and the mechanisms through which estrogen receptors and androgen receptors regulate lipid and glucose metabolism in the liver. In females, estradiol regulates liver metabolism via estrogen receptors by decreasing lipogenesis, gluconeogenesis, and fatty acid uptake, while enhancing lipolysis, cholesterol secretion, and glucose catabolism. In males, testosterone works via androgen receptors to increase insulin receptor expression and glycogen synthesis, decrease glucose uptake and lipogenesis, and promote cholesterol storage in the liver. These recent integrated concepts suggest that sex hormone receptors could be potential promising targets for the prevention of hepatic steatosis. PMID:26491440

  18. G protein-coupled receptors in regulation of body weight.

    PubMed

    Schiöth, Helgi B

    2006-06-01

    In this issue of CNS & Neurological Disorders-Drug Targets, we focus on G protein-coupled receptors (GPCRs) that are involved in regulating body weight. In six reviews, the melanocortins system (including MC4 and MC3 receptors, Agrp, MSH), the NPY receptors (including NPY-Y1, NPY-Y2, and NPY-Y5, PYY3-36), the cannabinoid system (including the development of rimonabant), the ghrelin (GHS, growth hormone secretagogue) system, the monoamine GPCRs (including serotonin, adrenergic and histamine receptors), orexin (hypocretin) system and the galanin receptors are covered. In this overview, an introduction to the GPCRs and the field of central regulation of food intake is provided together with brief mentioning of some other GPCRs that are also implicated in regulation of body weight, such as the melanin-concentrating hormone (MCH), neuromedin U, prolactin-releasing peptide (PrRP), bombesin, cholecystokinin (CCK), Glucagon-like peptide-1 (GLP-1) (and oxyntomodulin), neuropeptide B (NPB) and neuropeptide W (NPW), opioids peptides, free fatty acid (FFA) receptors (GPR40, GPR41). In total over 40 GPCRs are listed that have been implicated to affect regulation of body weight.

  19. 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

  20. Nature and regulation of the insulin receptor: structure and function

    SciTech Connect

    Czech, M.P.

    1985-01-01

    Native, cell-surface insulin receptor consists of two glycoprotein subunit types with apparent masses of about 125,000 daltons (alpha subunit) and 90,000 daltons (beta subunit). The alpha and beta insulin-receptor subunits seem to have distinct functions such that alpha appears to bind hormone whereas beta appears to possess intrinsic tyrosine kinase activity. In detergent extracts, insulin activates receptor autophosphorylation of tyrosine residues on its beta subunit, whereas in the presence of reductant, the alpha subunit is also phosphorylated. In intact cells, insulin activates serine/threonine phosphorylation of insulin receptor beta subunit as well as tyrosine phosphorylation. The biological role of the receptor-associated tyrosine kinase is not known. The insulin receptor kinase is regulated by beta-adrenergic agonists and other agents that elevate cAMP in adipocytes, presumably via the cAMP-dependent protein kinase. Such agents decrease receptor affinity for insulin and partially uncouple receptor tyrosine kinase activity from activation by insulin. These effects appear to contribute to the biological antagonism between insulin and beta-agonists. These data suggest the hypothesis that a complex network of tyrosine and serine/threonine phosphorylations on the insulin receptor modulate its binding and kinase activities in an antagonistic manner.

  1. Stress regulates endocannabinoid-CB1 receptor signaling.

    PubMed

    Hillard, Cecilia J

    2014-10-01

    The CB1 cannabinoid receptor is a G protein coupled receptor that is widely expressed throughout the brain. The endogenous ligands for the CB1 receptor (endocannabinoids) are N-arachidonylethanolamine and 2-arachidonoylglycerol; together the endocannabinoids and CB1R subserve activity dependent, retrograde inhibition of neurotransmitter release in the brain. Deficiency of CB1 receptor signaling is associated with anhedonia, anxiety, and persistence of negative memories. CB1 receptor-endocannabinoid signaling is activated by stress and functions to buffer or dampen the behavioral and endocrine effects of acute stress. Its role in regulation of neuronal responses is more complex. Chronic variable stress exposure reduces endocannabinoid-CB1 receptor signaling and it is hypothesized that the resultant deficiency in endocannabinoid signaling contributes to the negative consequences of chronic stress. On the other hand, repeated exposure to the same stress can sensitize CB1 receptor signaling, resulting in dampening of the stress response. Data are reviewed that support the hypothesis that CB1 receptor signaling is stress responsive and that maintaining robust endocannabinoid/CB1 receptor signaling provides resilience against the development of stress-related pathologies.

  2. Dynamic Regulation of the GABAA Receptor Function by Redox Mechanisms.

    PubMed

    Calvo, Daniel J; González, Andrea N Beltrán

    2016-09-01

    Oxidizing and reducing agents, which are currently involved in cell metabolism and signaling pathways, can regulate fast inhibitory neurotransmission mediated by GABA receptors in the nervous system. A number of in vitro studies have shown that diverse redox compounds, including redox metabolites and reactive oxygen and nitrogen species, modulate phasic and tonic responses mediated by neuronal GABAA receptors through both presynaptic and postsynaptic mechanisms. We review experimental data showing that many redox agents, which are normally present in neurons and glia or are endogenously generated in these cells under physiologic states or during oxidative stress (e.g., hydrogen peroxide, superoxide and hydroxyl radicals, nitric oxide, ascorbic acid, and glutathione), induce potentiating or inhibiting actions on different native and recombinant GABAA receptor subtypes. Based on these results, it is thought that redox signaling might represent a homeostatic mechanism that regulates the function of synaptic and extrasynaptic GABAA receptors in physiologic and pathologic conditions.

  3. Flavonoids as dietary regulators of nuclear receptor activity

    PubMed Central

    Avior, Yishai; Bomze, David; Ramon, Ory

    2013-01-01

    Metabolic diseases such as obesity, type II diabetes, and dyslipidemia are a rising cause of mortality worldwide. The progression of many metabolic diseases is fundamentally regulated on the transcriptional level by a family of ligand-activated transcription factors, called nuclear receptors, which detect and respond to metabolic changes. Their role in maintaining metabolic homeostasis makes nuclear receptors an important pharmaceutical and dietary target. This review will present the growing evidence that flavonoids, natural secondary plant metabolites, are important regulators of nuclear receptor activity. Structural similarities between flavonoids and cholesterol derivatives combined with the promiscuous nature of most nuclear receptors provide a wealth of possibilities for pharmaceutical and dietary modulation of metabolism. While the challenges of bringing flavonoid-derived therapeutics to the market are significant, we consider this rapidly growing field to be an essential aspect of the functional food initiative and an important mine for pharmaceutical compounds. PMID:23598551

  4. The Sorting Receptor SorCS1 Regulates Trafficking of Neurexin and AMPA Receptors.

    PubMed

    Savas, Jeffrey N; Ribeiro, Luís F; Wierda, Keimpe D; Wright, Rebecca; DeNardo-Wilke, Laura A; Rice, Heather C; Chamma, Ingrid; Wang, Yi-Zhi; Zemla, Roland; Lavallée-Adam, Mathieu; Vennekens, Kristel M; O'Sullivan, Matthew L; Antonios, Joseph K; Hall, Elizabeth A; Thoumine, Olivier; Attie, Alan D; Yates, John R; Ghosh, Anirvan; de Wit, Joris

    2015-08-19

    The formation, function, and plasticity of synapses require dynamic changes in synaptic receptor composition. Here, we identify the sorting receptor SorCS1 as a key regulator of synaptic receptor trafficking. Four independent proteomic analyses identify the synaptic adhesion molecule neurexin and the AMPA glutamate receptor (AMPAR) as major proteins sorted by SorCS1. SorCS1 localizes to early and recycling endosomes and regulates neurexin and AMPAR surface trafficking. Surface proteome analysis of SorCS1-deficient neurons shows decreased surface levels of these, and additional, receptors. Quantitative in vivo analysis of SorCS1-knockout synaptic proteomes identifies SorCS1 as a global trafficking regulator and reveals decreased levels of receptors regulating adhesion and neurotransmission, including neurexins and AMPARs. Consequently, glutamatergic transmission at SorCS1-deficient synapses is reduced due to impaired AMPAR surface expression. SORCS1 mutations have been associated with autism and Alzheimer disease, suggesting that perturbed receptor trafficking contributes to synaptic-composition and -function defects underlying synaptopathies.

  5. The Sorting Receptor SorCS1 Regulates Trafficking of Neurexin and AMPA Receptors

    PubMed Central

    Savas, Jeffrey N.; Ribeiro, Luís F.; Wierda, Keimpe D.; Wright, Rebecca; DeNardo, Laura A.; Rice, Heather C.; Chamma, Ingrid; Wang, Yi-Zhi; Zemla, Roland; Lavallée-Adam, Mathieu; Vennekens, Kristel M.; O'Sullivan, Matthew L.; Antonios, Joseph K.; Hall, Elizabeth A.; Thoumine, Olivier; Attie, Alan D.; Ghosh, Anirvan; Yates, John R.; de Wit, Joris

    2015-01-01

    The formation, function, and plasticity of synapses require dynamic changes in synaptic receptor composition. Here we identify the sorting receptor SorCS1 as a key regulator of synaptic receptor trafficking. Four independent proteomic analyses identify the synaptic adhesion molecule neurexin and the AMPA glutamate receptor (AMPAR) as major proteins sorted by SorCS1. SorCS1 localizes to early and recycling endosomes and regulates neurexin and AMPAR surface trafficking. Surface proteome analysis of SorCS1-deficient neurons shows decreased surface levels of these, and additional, receptors. Quantitative in vivo analysis of SorCS1 knockout synaptic proteomes identifies SorCS1 as a global trafficking regulator and reveals decreased levels of receptors regulating adhesion and neurotransmission, including neurexins and AMPARs. Consequently, glutamatergic transmission at SorCS1–deficient synapses is reduced due to impaired AMPAR surface expression. SORCS1 mutations have been associated with autism and Alzheimer's disease, suggesting that perturbed receptor trafficking contributes to defects in synaptic composition and function underlying synaptopathies. PMID:26291160

  6. ESCRT-0 is not required for ectopic Notch activation and tumor suppression in Drosophila.

    PubMed

    Tognon, Emiliana; Wollscheid, Nadine; Cortese, Katia; Tacchetti, Carlo; Vaccari, Thomas

    2014-01-01

    Multivesicular endosome (MVE) sorting depends on proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) family. These are organized in four complexes (ESCRT-0, -I, -II, -III) that act in a sequential fashion to deliver ubiquitylated cargoes into the internal luminal vesicles (ILVs) of the MVE. Drosophila genes encoding ESCRT-I, -II, -III components function in sorting signaling receptors, including Notch and the JAK/STAT signaling receptor Domeless. Loss of ESCRT-I, -II, -III in Drosophila epithelia causes altered signaling and cell polarity, suggesting that ESCRTs genes are tumor suppressors. However, the nature of the tumor suppressive function of ESCRTs, and whether tumor suppression is linked to receptor sorting is unclear. Unexpectedly, a null mutant in Hrs, encoding one of the components of the ESCRT-0 complex, which acts upstream of ESCRT-I, -II, -III in MVE sorting is dispensable for tumor suppression. Here, we report that two Drosophila epithelia lacking activity of Stam, the other known components of the ESCRT-0 complex, or of both Hrs and Stam, accumulate the signaling receptors Notch and Dome in endosomes. However, mutant tissue surprisingly maintains normal apico-basal polarity and proliferation control and does not display ectopic Notch signaling activation, unlike cells that lack ESCRT-I, -II, -III activity. Overall, our in vivo data confirm previous evidence indicating that the ESCRT-0 complex plays no crucial role in regulation of tumor suppression, and suggest re-evaluation of the relationship of signaling modulation in endosomes and tumorigenesis. PMID:24718108

  7. Affinity Regulates Spatial Range of EGF Receptor Autocrine Ligand Binding

    SciTech Connect

    Dewitt, Ann; Iida, Tomoko; Lam, Ho-Yan; Hill, Virginia; Wiley, H S.; Lauffenburger, Douglas A.

    2002-08-08

    Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. The authors employ a computational model to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and provide experimental test by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGF{sup L47M}. The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. The experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling.

  8. Orphan receptor IL-17RD regulates Toll-like receptor signalling via SEFIR/TIR interactions.

    PubMed

    Mellett, Mark; Atzei, Paola; Bergin, Ronan; Horgan, Alan; Floss, Thomas; Wurst, Wolfgang; Callanan, John J; Moynagh, Paul N

    2015-01-01

    Receptor families of the innate immune response engage in 'cross-talk' to tailor optimal immune responses against invading pathogens. However, these responses are subject to multiple levels of regulation to keep in check aberrant inflammatory signals. Here, we describe a role for the orphan receptor interleukin-17 receptor D (IL-17RD) in negatively regulating Toll-like receptor (TLR)-induced responses. Deficiency of IL-17RD expression in cells leads to enhanced pro-inflammatory signalling and gene expression in response to TLR stimulation, and Il17rd(-/-) mice are more susceptible to TLR-induced septic shock. We demonstrate that the intracellular Sef/IL-17R (SEFIR) domain of IL-17RD targets TIR adaptor proteins to inhibit TLR downstream signalling thus revealing a paradigm involving cross-regulation of members of the IL-17R and TLR families.

  9. Notch Signaling Activates Stem Cell Properties of Müller Glia through Transcriptional Regulation and Skp2-mediated Degradation of p27Kip1

    PubMed Central

    Parameswaran, Sowmya; Mathews, Saumi; Xia, Xiaohuan; Zheng, Li; Neville, Andrew J.; Ahmad, Iqbal

    2016-01-01

    Müller glia (MG), the sole glial cells generated by retinal progenitors, have emerged as a viable cellular target for therapeutic regeneration in degenerative blinding diseases, as they possess dormant stem cell properties. However, the mammalian MG does not display the neurogenic potential of their lower vertebrate counterparts, precluding their practical clinical use. The answer to this barrier may be found in two interlinked processes underlying the neurogenic potential, i.e., the activation of the dormant stem cell properties of MG and their differentiation along the neuronal lineage. Here, we have focused on the former and examined Notch signaling-mediated activation of MG. We demonstrate that one of the targets of Notch signaling is the cyclin-dependent kinase inhibitor (CKI), p27Kip1, which is highly expressed in quiescent MG. Notch signaling facilitates the activation of MG by inhibiting p27Kip1 expression. This is likely achieved through the Notch- p27Kip1 and Notch-Skp2-p27Kip1 axes, the former inhibiting the expression of p27Kip1 transcripts and the latter levels of p27Kip1 proteins by Skp2-mediated proteasomal degradation. Thus, Notch signaling may facilitate re-entry of MG into the cell cycle by inhibiting p27Kip1 expression both transcriptionally and post-translationally. PMID:27011052

  10. Notch Signaling Activates Stem Cell Properties of Müller Glia through Transcriptional Regulation and Skp2-mediated Degradation of p27Kip1.

    PubMed

    Del Debbio, Carolina Beltrame; Mir, Qulsum; Parameswaran, Sowmya; Mathews, Saumi; Xia, Xiaohuan; Zheng, Li; Neville, Andrew J; Ahmad, Iqbal

    2016-01-01

    Müller glia (MG), the sole glial cells generated by retinal progenitors, have emerged as a viable cellular target for therapeutic regeneration in degenerative blinding diseases, as they possess dormant stem cell properties. However, the mammalian MG does not display the neurogenic potential of their lower vertebrate counterparts, precluding their practical clinical use. The answer to this barrier may be found in two interlinked processes underlying the neurogenic potential, i.e., the activation of the dormant stem cell properties of MG and their differentiation along the neuronal lineage. Here, we have focused on the former and examined Notch signaling-mediated activation of MG. We demonstrate that one of the targets of Notch signaling is the cyclin-dependent kinase inhibitor (CKI), p27Kip1, which is highly expressed in quiescent MG. Notch signaling facilitates the activation of MG by inhibiting p27Kip1 expression. This is likely achieved through the Notch- p27Kip1 and Notch-Skp2-p27Kip1 axes, the former inhibiting the expression of p27Kip1 transcripts and the latter levels of p27Kip1 proteins by Skp2-mediated proteasomal degradation. Thus, Notch signaling may facilitate re-entry of MG into the cell cycle by inhibiting p27Kip1 expression both transcriptionally and post-translationally. PMID:27011052

  11. Agonist induced constitutive receptor activation as a novel regulatory mechanism. Mu receptor regulation.

    PubMed

    Sadée, W; Wang, Z

    1995-01-01

    We propose the hypothesis that certain G protein coupled receptors can become constitutively activated during agonist stimulation so that the receptor remains active even after the agonist is removed. This new paradigm of receptor regulation may account for some long term effects of neurotransmitters and hormones. We have tested the hypothesis that constitutive mu receptor activation represents a crucial step driving narcotic tolerance and dependence. Our results indeed support the conversion of mu to a constitutively active state, mu*, observed in neuroblastoma SK-N-SH and SH-SY5Y tissue culture, in U293 cells transfected with the mu receptor gene, and in vivo. Constitutive mu activation may result from receptor phosphorylation to yield mu*, and further, in vivo studies indicate that formation of mu* could account for narcotic tolerance and dependence.

  12. Notch Is Required in Adult Drosophila Sensory Neurons for Morphological and Functional Plasticity of the Olfactory Circuit

    PubMed Central

    Struhl, Gary

    2015-01-01

    Olfactory receptor neurons (ORNs) convey odor information to the central brain, but like other sensory neurons were thought to play a passive role in memory formation and storage. Here we show that Notch, part of an evolutionarily conserved intercellular signaling pathway, is required in adult Drosophila ORNs for the structural and functional plasticity of olfactory glomeruli that is induced by chronic odor exposure. Specifically, we show that Notch activity in ORNs is necessary for the odor specific increase in the volume of glomeruli that occurs as a consequence of prolonged odor exposure. Calcium imaging experiments indicate that Notch in ORNs is also required for the chronic odor induced changes in the physiology of ORNs and the ensuing changes in the physiological response of their second order projection neurons (PNs). We further show that Notch in ORNs acts by both canonical cleavage-dependent and non-canonical cleavage-independent pathways. The Notch ligand Delta (Dl) in PNs switches the balance between the pathways. These data define a circuit whereby, in conjunction with odor, N activity in the periphery regulates the activity of neurons in the central brain and Dl in the central brain regulates N activity in the periphery. Our work highlights the importance of experience dependent plasticity at the first olfactory synapse. PMID:26011623

  13. Regulation of plant immune receptors by ubiquitination.

    PubMed

    Furlan, Giulia; Klinkenberg, Jörn; Trujillo, Marco

    2012-01-01

    From pathogen perception and the activation of signal transduction cascades to the deployment of defense responses, protein ubiquitination plays a key role in the modulation of plant immunity. Ubiquitination is mediated by three enzymes, of which the E3 ubiquitin ligases, the substrate determinants, have been the major focus of attention. Accumulating evidence suggests that ubiquitination modulates signaling mediated by pattern recognition receptors and is important for the accumulation of nucleotide-binding leucine-rich repeat type intracellular immune sensors. Recent studies also indicate that ubiquitination directs vesicle trafficking, a function that has been clearly established for immune signaling in animals. In this mini review, we discuss these and other recent advances and highlight important open questions. PMID:23109936

  14. Regulation of carbohydrate metabolism by the farnesoid X receptor.

    PubMed

    Stayrook, Keith R; Bramlett, Kelli S; Savkur, Rajesh S; Ficorilli, James; Cook, Todd; Christe, Michael E; Michael, Laura F; Burris, Thomas P

    2005-03-01

    The farnesoid X receptor (FXR; NR1H4) is a nuclear hormone receptor that functions as the bile acid receptor. In addition to the critical role FXR plays in bile acid metabolism and transport, it regulates a variety of genes important in lipoprotein metabolism. We demonstrate that FXR also plays a role in carbohydrate metabolism via regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression. Treatment of either H4IIE or MH1C1 rat hepatoma cell lines as well as primary rat or human hepatocytes with FXR agonists led to stimulation of PEPCK mRNA expression to levels comparable to those obtained with glucocorticoid receptor agonists. We examined the physiological significance of FXR agonist-induced enhancement of PEPCK expression in primary rat hepatocytes. In addition to inducing PEPCK expression in primary hepatocytes, FXR agonists stimulated glucose output to levels comparable to those observed with a glucocorticoid receptor agonist. Consistent with these observations, treatment of C57BL6 mice with GW4064 significantly increased hepatic PEPCK expression. Activation of FXR initiated a cascade involving induction of peroxisome proliferator-activated receptor alpha and TRB3 expression that is consistent with stimulation of PEPCK gene expression via interference with a pathway that may involve Akt-dependent phosphorylation of Forkhead/winged helix transcription factor (FOXO1). The FXR-peroxisome proliferator-activated receptor alpha-TRB3 pathway was conserved in rat hepatoma cell lines, mice, as well as primary human hepatocytes. Thus, in addition to its role in the regulation of lipid metabolism, FXR regulates carbohydrate metabolism.

  15. Identification of Phosphorylation Sites Regulating sst3 Somatostatin Receptor Trafficking.

    PubMed

    Lehmann, Andreas; Kliewer, Andrea; Günther, Thomas; Nagel, Falko; Schulz, Stefan

    2016-06-01

    The human somatostatin receptor 3 (sst3) is expressed in about 50% of all neuroendocrine tumors and hence a promising target for multireceptor somatostatin analogs. The sst3 receptor is unique among ssts in that it exhibits a very long intracellular C-terminal tail containing a huge number of potential phosphate acceptor sites. Consequently, our knowledge about the functional role of the C-terminal tail in sst3 receptor regulation is very limited. Here, we have generated a series of phosphorylation-deficient mutants that enabled us to determine crucial sites for its agonist-induced β-arrestin mobilization, internalization, and down-regulation. Based on this information, we generated phosphosite-specific antibodies for C-terminal Ser(337)/Thr(341), Thr(348), and Ser(361) that enabled us to investigate the temporal patterns of sst3 phosphorylation and dephosphorylation. We found that the endogenous ligand somatostatin induced a rapid and robust phosphorylation that was completely blocked by the sst3 antagonist NVP-ACQ090. The stable somatostatin analogs pasireotide and octreotide promoted clearly less phosphorylation compared with somatostatin. We also show that sst3 phosphorylation occurred within seconds to minutes, whereas dephosphorylation of the sst3 receptor occurred at a considerable slower rate. In addition, we also identified G protein-coupled receptor kinases 2 and 3 and protein phosphatase 1α and 1β as key regulators of sst3 phosphorylation and dephosphorylation, respectively. Thus, we here define the C-terminal phosphorylation motif of the human sst3 receptor that regulates its agonist-promoted phosphorylation, β-arrestin recruitment, and internalization of this clinically relevant receptor.

  16. 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

  17. Postsynaptic nigrostriatal dopamine receptors and their role in movement regulation

    PubMed Central

    Meyer, Michael F.; Krasnianski, Michael

    2010-01-01

    The article presents the hypothesis that nigrostriatal dopamine may regulate movement by modulation of tone and contraction in skeletal muscles through a concentration-dependent influence on the postsynaptic D1 and D2 receptors on the follow manner: nigrostriatal axons innervate both receptor types within the striatal locus somatotopically responsible for motor control in agonist/antagonist muscle pair around a given joint. D1 receptors interact with lower and D2 receptors with higher dopamine concentrations. Synaptic dopamine concentration increases immediately before movement starts. We hypothesize that increasing dopamine concentrations stimulate first the D1 receptors and reduce muscle tone in the antagonist muscle and than stimulate D2 receptors and induce contraction in the agonist muscle. The preceded muscle tone reduction in the antagonist muscle eases the efficient contraction of the agonist. Our hypothesis is applicable for an explanation of physiological movement regulation, different forms of movement pathology and therapeutic drug effects. Further, this hypothesis provides a theoretical basis for experimental investigation of dopaminergic motor control and development of new strategies for treatment of movement disorders. PMID:21076988

  18. Notch2 genetic fate mapping reveals two previously unrecognized mammary epithelial lineages

    PubMed Central

    Šale, Sanja; Lafkas, Daniel; Artavanis-Tsakonas, Spyros

    2013-01-01

    Notch signalling is implicated in stem and progenitor cell fate control in numerous organs. Using conditional in vivo genetic labelling we traced the fate of cells expressing the Notch2 receptor paralogue and uncovered the existence of two previously unrecognized mammary epithelial cell lineages that we term S (Small) and L (Large). S cells appear in a bead-on-a-string formation and are embedded between the luminal and basal/myoepithelial layers in a unique reiterative pattern, whereas single or paired L cells appear among ductal and alveolar cells. Long-term lineage tracing and functional studies indicate that S and L cells regulate ipsi- and contralateral spatial placement of tertiary branches and formation of alveolar clusters. Our findings revise present models of mammary epithelial cell hierarchy, reveal a hitherto undescribed mechanism regulating branching morphogenesis and may have important implications for identification of the cell-of-origin of distinct breast cancer subtypes. PMID:23604318

  19. Notch2 activation ameliorates nephrosis

    NASA Astrophysics Data System (ADS)

    Tanaka, Eriko; Asanuma, Katsuhiko; Kim, Eunhee; Sasaki, Yu; Trejo, Juan Alejandro Oliva; Seki, Takuto; Nonaka, Kanae; Asao, Rin; Nagai-Hosoe, Yoshiko; Akiba-Takagi, Miyuki; Hidaka, Teruo; Takagi, Masatoshi; Koyanagi, Akemi; Mizutani, Shuki; Yagita, Hideo; Tomino, Yasuhiko

    2014-02-01

    Activation of Notch1 and Notch2 has been recently implicated in human glomerular diseases. Here we show that Notch2 prevents podocyte loss and nephrosis. Administration of a Notch2 agonistic monoclonal antibody ameliorates proteinuria and glomerulosclerosis in a mouse model of nephrosis and focal segmental glomerulosclerosis. In vitro, the specific knockdown of Notch2 increases apoptosis in damaged podocytes, while Notch2 agonistic antibodies enhance activation of Akt and protect damaged podocytes from apoptosis. Treatment with triciribine, an inhibitor of Akt pathway, abolishes the protective effect of the Notch2 agonistic antibody. We find a positive linear correlation between the number of podocytes expressing activated Notch2 and the number of residual podocytes in human nephrotic specimens. Hence, specific activation of Notch2 rescues damaged podocytes and activating Notch2 may represent a novel clinical strategy for the amelioration of nephrosis and glomerulosclerosis.

  20. Specific regulation of male rat liver cytosolic estrogen receptor by the modulator of the glucocorticoid receptor.

    PubMed

    Celiker, M Y; Haas, A; Saunders, D; Litwack, G

    1993-08-31

    Modulator is a novel low-molecular-weight organic compound that regulates activities of glucocorticoid and mineralocorticoid receptors as well as protein kinase C. In this study we show that male rat liver cytosolic estrogen receptor activation is inhibited by modulator in a dose-dependent manner. Fifty percent inhibition is obtained with 1 unit/ml modulator purified from bovine liver which is within the physiological concentration for modulator. However, sheep uterine cytosolic estrogen and androgen receptors are insensitive to regulation by modulator. Exogenous sodium molybdate treatment inhibits activation of all of these receptors of liver or uterus origin in an identical manner, further differentiating the effects of modulator and the molybdate anion. PMID:8363596

  1. Analysis of Dominant Enhancers and Suppressors of Activated Notch in Drosophila

    PubMed Central

    Verheyen, E. M.; Purcell, K. J.; Fortini, M. E.; Artavanis-Tsakonas, S.

    1996-01-01

    The Notch receptor controls cell fate decisions throughout Drosophila development. Truncated, ligand-independent forms of this protein delay or block differentiation. We have previously shown that expression of the intracellular domain of the receptor under the control of the sevenless enhancer/promoter induces a rough eye phenotype in the adult fly. Analysis of the resultant cellular transformations suggested that this form of Notch acts as a constitutively activated receptor. To identify gene products that interact with Notch, a second-site mutagenesis screen was performed to isolate enhancers and suppressors of the eye phenotype caused by expression of these activated Notch molecules. We screened 137,000 mutagenized flies and recovered 290 dominant modifiers. Many new alleles of previously identified genes were isolated, as were mutations defining novel loci that may function in the Notch signaling pathway. We discuss the data with respect to known features of Notch receptor signaling and Drosophila eye development. PMID:8913755

  2. Mitochondrial benzodiazepine receptors regulate steroid biosynthesis

    SciTech Connect

    Mukhin, A.G.; Papadopoulos, V.; Costa, E.; Krueger, K.E. )

    1989-12-01

    Recent observations on the steroid synthetic capability within the brain open the possibility that benzodiazepines may influence steroid synthesis in nervous tissue through interactions with peripheral-type benzodiazepine recognition sites, which are highly expressed in steroidogenic cells and associated with the outer mitochondrial membrane. To examine this possibility nine molecules that exhibit a greater than 10,000-fold difference in their affinities for peripheral-type benzodiazepine binding sites were tested for their effects on a well-established steroidogenic model system, the Y-1 mouse adrenal tumor cell line. 4{prime}-Chlorodiazepam, PK 11195, and PK 14067 stimulated steroid production by 2-fold in Y-1 cells, whereas diazepam, flunitrazepam, zolpidem, and PK 14068 displayed a lower (1.2- to 1.5-fold) maximal stimulation. In contrast, clonazepam and flumazenil did not stimulate steroid synthesis. The potencies of these compounds to inhibit {sup 3}H-labeled PK 11195 binding to peripheral-type benzodiazepine recognition sites correlated with their potencies to stimulate steroid production. Similar findings were observed in bovine and rat adrenocortical cell preparations. These results suggest that ligands of the peripheral-type benzodiazepine recognition site acting on this mitochondrial receptor can enhance steroid production. This action may contribute specificity to the pharmacological profile of drugs preferentially acting on the benzodiazepine recognition site associated with the outer membrane of certain mitochondrial populations.

  3. Ligand regulation of a constitutively dimeric EGF receptor

    NASA Astrophysics Data System (ADS)

    Freed, Daniel M.; Alvarado, Diego; Lemmon, Mark A.

    2015-06-01

    Ligand-induced receptor dimerization has traditionally been viewed as the key event in transmembrane signalling by epidermal growth factor receptors (EGFRs). Here we show that the Caenorhabditis elegans EGFR orthologue LET-23 is constitutively dimeric, yet responds to its ligand LIN-3 without changing oligomerization state. SAXS and mutational analyses further reveal that the preformed dimer of the LET-23 extracellular region is mediated by its domain II dimerization arm and resembles other EGFR extracellular dimers seen in structural studies. Binding of LIN-3 induces only minor structural rearrangements in the LET-23 dimer to promote signalling. Our results therefore argue that EGFR can be regulated by allosteric changes within an existing receptor dimer--resembling signalling by insulin receptor family members, which share similar extracellular domain compositions but form covalent dimers.

  4. Leptin regulates gonadotropins and steroid receptors in the rats ovary.

    PubMed

    Silveira Cavalcante, Fernanda; Aiceles, Verónica; da Fonte Ramos, Cristiane

    2013-01-01

    The leptin hormone is important to satiety and an important link between the nutritional status and reproductive processes. Owing to the contradictory effects of leptin on the ovary and the failure to clarify the precise mechanism by which leptin affects the ovary, our aim was to contribute to evaluation if leptin can directly regulate the gene expression of leptin itself and its receptors, and the expression of several genes related to the ovary function by a model of tissue culture. Ovaries from Wistar dams were used at 90 days of age and were submitted to medium with presence and absence of leptin. The results can demonstrate that leptin regulates gonadotropins and steroid receptors, which could suggest that the ovarian leptin role could be secondary to the changes in these receptors expression in rats.

  5. Glutamate Receptor Ion Channels: Structure, Regulation, and Function

    PubMed Central

    Wollmuth, Lonnie P.; McBain, Chris J.; Menniti, Frank S.; Vance, Katie M.; Ogden, Kevin K.; Hansen, Kasper B.; Yuan, Hongjie; Myers, Scott J.; Dingledine, Ray

    2010-01-01

    The mammalian ionotropic glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels containing an agonist recognition site, a transmembrane ion permeation pathway, and gating elements that couple agonist-induced conformational changes to the opening or closing of the permeation pore. Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and are localized on neuronal and non-neuronal cells. These receptors regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. Glutamate receptors are postulated to play important roles in numerous neurological diseases and have attracted intense scrutiny. The description of glutamate receptor structure, including its transmembrane elements, reveals a complex assembly of multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. In this review we discuss International Union of Basic and Clinical Pharmacology glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, as well as the potential therapeutic use of pharmacological agents acting at glutamate receptors. PMID:20716669

  6. Notch-EGFR/HER2 Bidirectional Crosstalk in Breast Cancer

    PubMed Central

    Baker, Andrew T.; Zlobin, Andrei; Osipo, Clodia

    2014-01-01

    The Notch pathway is a well-established mediator of cell–cell communication that plays a critical role in stem cell survival, self-renewal, cell fate decisions, tumorigenesis, invasion, metastasis, and drug resistance in a variety of cancers. An interesting form of crosstalk exists between the Notch receptor and the Epidermal Growth Factor Receptor Tyrosine Kinase family, which consists of HER-1, -2, -3, and -4. Overexpression of HER and/or Notch occurs in several human cancers including brain, lung, breast, ovary, and skin making them potent oncogenes capable of advancing malignant disease. Continued assessment of interplay between these two critical signaling networks uncovers new insight into mechanisms used by HER-driven cancer cells to exploit Notch as a compensatory pathway. The compensatory Notch pathway maintains HER-induced downstream signals transmitted to pathways such as Mitogen Activated Protein Kinase and Phosphatidylinositol 3-Kinase (PI3K), thereby allowing cancer cells to survive molecular targeted therapies, undergo epithelial to mesenchymal transitioning, and increase cellular invasion. Uncovering the critical crosstalk between the HER and Notch pathways can lead to improved screening for the expression of these oncogenes enabling patients to optimize their personal treatment options and predict potential treatment resistance. This review will focus on the current state of crosstalk between the HER and Notch receptors and the effectiveness of current therapies targeting HER-driven cancers. PMID:25566499

  7. 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.

  8. HNF1B controls proximal-intermediate nephron segment identity in vertebrates by regulating Notch signalling components and Irx1/2.

    PubMed

    Heliot, Claire; Desgrange, Audrey; Buisson, Isabelle; Prunskaite-Hyyryläinen, Renata; Shan, Jingdong; Vainio, Seppo; Umbhauer, Muriel; Cereghini, Silvia

    2013-02-01

    The nephron is a highly specialised segmented structure that provides essential filtration and resorption renal functions. It arises by formation of a polarised renal vesicle that differentiates into a comma-shaped body and then a regionalised S-shaped body (SSB), with the main prospective segments mapped to discrete domains. The regulatory circuits involved in initial nephron patterning are poorly understood. We report here that HNF1B, a transcription factor known to be involved in ureteric bud branching and initiation of nephrogenesis, has an additional role in segment fate acquisition. Hnf1b conditional inactivation in murine nephron progenitors results in rudimentary nephrons comprising a glomerulus connected to the collecting system by a short tubule displaying distal fates. Renal vesicles develop and polarise normally but fail to progress to correctly patterned SSBs. Major defects are evident at late SSBs, with altered morphology, reduction of a proximo-medial subdomain and increased apoptosis. This is preceded by strong downregulation of the Notch pathway components Lfng, Dll1 and Jag1 and the Irx1/2 factors, which are potential regulators of proximal and Henle's loop segment fates. Moreover, HNF1B is recruited to the regulatory sequences of most of these genes. Overexpression of a HNF1B dominant-negative construct in Xenopus embryos causes downregulation specifically of proximal and intermediate pronephric segment markers. These results show that HNF1B is required for the acquisition of a proximo-intermediate segment fate in vertebrates, thus uncovering a previously unappreciated function of a novel SSB subcompartment in global nephron segmentation and further differentiation.

  9. Delta-Like 4 Differentially Regulates Murine CD4+ T Cell Expansion via BMI1

    PubMed Central

    Schaller, Matthew A.; Logue, Hannah; Mukherjee, Sumanta; Lindell, Dennis M.; Coelho, Ana Lucia; Lincoln, Pamela; Carson, William F.; Ito, Toshihiro; Cavassani, Karen A.; Chensue, Stephen W.; Hogaboam, Cory M.; Lukacs, Nicholas W.; Kunkel, Steven L.

    2010-01-01

    Background Studies have shown that Notch is essential for the maintenance of a T cell Th2 phenotype in vivo. It has also been shown that Notch ligands have diverse functions during T cell activation. We chose to investigate the role of Notch ligands during the Th2 response. Principal Findings We studied the relationship of two Notch ligands, delta-like 4 and jagged-1, to T cell proliferation in C57 Bl/6 mice. Our findings indicate that jagged-1 does not affect the rate of T cell proliferation in any subset examined. However, delta-like 4 causes an increase in the expansion of Th2 memory cells and a decrease in effector cell proliferation. Our in vivo studies indicate that the Notch system is dynamically regulated, and that blocking one Notch ligand increases the effective concentration of other Notch ligands, thus altering the response. Examination of genes related to the Notch pathway revealed that the Notch receptors were increased in memory T cells. Expression of BMI1, a gene involved in T cell proliferation, was also higher in memory T cells. Further experiments demonstrated that Notch directly regulates the expression of the BMI1 gene in T cells and may govern T cell proliferation through this pathway. Conclusions From these experiments we can make several novel conclusions about the role of Notch ligands in T cell biology. The first is that delta-like 4 suppresses effector cell proliferation and enhances Th2 memory cell proliferation. The second is that blocking one Notch ligand in vivo effectively increases the concentration of other Notch ligands, which can then alter the response. PMID:20808960

  10. Local overexpression of Su(H)-MAPK variants affects Notch target gene expression and adult phenotypes in Drosophila.

    PubMed

    Auer, Jasmin S; Nagel, Anja C; Schulz, Adriana; Wahl, Vanessa; Preiss, Anette

    2015-12-01

    In Drosophila, Notch and EGFR signalling pathways are closely intertwined. Their relationship is mostly antagonistic, and may in part be based on the phosphorylation of the Notch signal transducer Suppressor of Hairless [Su(H)] by MAPK. Su(H) is a transcription factor that together with several cofactors regulates the expression of Notch target genes. Here we address the consequences of a local induction of three Su(H) variants on Notch target gene expression. To this end, wild-type Su(H), a phospho-deficient Su(H) (MAPK-) (ko) and a phospho-mimetic Su(H) (MAPK-ac) isoform were overexpressed in the central domain of the wing anlagen. The expression of the Notch target genes cut, wingless, E(spl)m8-HLH and vestigial, was monitored. For the latter two, reporter genes were used (E(spl)m8-lacZ, vg (BE) -lacZ). In general, Su(H) (MAPK-) (ko) induced a stronger response than wild-type Su(H), whereas the response to Su(H) (MAPK-ac) was very weak. Notch target genes cut, wingless and vg (BE) -lacZ were ectopically activated, whereas E(spl)m8-lacZ was repressed by overexpression of Su(H) proteins. In addition, in epistasis experiments an activated form of the EGF-receptor (DER (act) ) or the MAPK (rl (SEM) ) and individual Su(H) variants were co-overexpressed locally, to compare the resultant phenotypes in adult flies (thorax, wings and eyes) as well as to assay the response of the Notch target gene cut in cell clones.

  11. MOLECULAR MECHANISMS INVOLVED IN PROGESTERONE RECEPTOR REGULATION OF UTERINE FUNCTION

    PubMed Central

    Lee, K.; Jeong, J.; Tsai, M.-J.; Tsai, S.; Lydon, J. P.; DeMayo, F. J.

    2007-01-01

    The ovarian steroid hormone progesterone is a major regulator of uterine function. The actions of this hormone is mediated through its cognate receptor, the progesterone receptor, Pgr. Ablation of the Pgr has shown that this receptor is critical for all female reproductive functions including the ability of the uterus to support and maintain the development of the implanting mouse embryo. High density DNA microarray analysis has identified direct and indirect targets of Pgr action. One of the targets of Pgr action is a member of the Hedgehog morphogen Indian hedgehog, Ihh. Ihh and members of the Hh signaling cascade show a coordinate expression pattern in the mouse uterus during the preimplantation period of pregnancy. The expression of Ihh and its receptor Patched-1, Ptc1, as well as, down stream targets of Ihh-Ptch1 signaling, such as the orphan nuclear receptor COUP-TF II show that this morphogen pathway mediates communication between the uterine epithelial and stromal compartments. The members of the Ihh signaling axis may function to coordinate the proliferation, vascularization and differentiation of the uterine stroma during pregnancy. This analysis demonstrates that progesterone regulates uterine function in the mouse by coordinating the signals from the uterine epithelium to stroma in the preimplantation mouse uterus. PMID:17067792

  12. Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism

    SciTech Connect

    Yu, Hongjun; Li, Huilin; Takeuchi, Megumi; LeBarron, Jamie; Kantharia, Joshua; London, Erwin; Bakker, Hans; Haltiwanger, Robert S.; Takeuchi, Hideyuki

    2015-09-28

    A major question remaining in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xyloside α-1,3-xylosyltransferase (XXYLT1) is a retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi-like reaction as the retaining mechanism for XXYLT1. Unexpectedly, the epidermal growth factor–like repeat acceptor substrate undergoes a large conformational change upon binding to the active site, providing a structural basis for substrate specificity. As a result, our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations in which Notch dysregulation is known to cause cancer or developmental disorders.

  13. 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

  14. Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism

    DOE PAGES

    Yu, Hongjun; Li, Huilin; Takeuchi, Megumi; LeBarron, Jamie; Kantharia, Joshua; London, Erwin; Bakker, Hans; Haltiwanger, Robert S.; Takeuchi, Hideyuki

    2015-09-28

    A major question remaining in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xyloside α-1,3-xylosyltransferase (XXYLT1) is a retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi-like reaction as the retaining mechanism for XXYLT1. Unexpectedly, the epidermal growth factor–like repeat acceptor substrate undergoes a largemore » conformational change upon binding to the active site, providing a structural basis for substrate specificity. As a result, our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations in which Notch dysregulation is known to cause cancer or developmental disorders.« less

  15. Local receptors as novel regulators for peripheral clock expression

    PubMed Central

    Wu, Changhao; Sui, Guiping; Archer, Simon N.; Sassone-Corsi, Paolo; Aitken, Karen; Bagli, Darius; Chen, Ying

    2014-01-01

    Mammalian circadian control is determined by a central clock in the brain suprachiasmatic nucleus (SCN) and synchronized peripheral clocks in other tissues. Increasing evidence suggests that SCN-independent regulation of peripheral clocks also occurs. We examined how activation of excitatory receptors influences the clock protein PERIOD 2 (PER2) in a contractile organ, the urinary bladder. PERIOD2::LUCIFERASE-knock-in mice were used to report real-time PER2 circadian dynamics in the bladder tissue. Rhythmic PER2 activities occurred in the bladder wall with a cycle of ∼24 h and peak at ∼12 h. Activation of the muscarinic and purinergic receptors by agonists shifted the peak to an earlier time (7.2±2.0 and 7.2±0.9 h, respectively). PER2 expression was also sensitive to mechanical stimulation. Aging significantly dampened PER2 expression and its response to the agonists. Finally, muscarinic agonist-induced smooth muscle contraction also exhibited circadian rhythm. These data identified novel regulators, endogenous receptors, in determining local clock activity, in addition to mediating the central control. Furthermore, the local clock appears to reciprocally align receptor activity to circadian rhythm for muscle contraction. The interaction between receptors and peripheral clock represents an important mechanism for maintaining physiological functions and its dysregulation may contribute to age-related organ disorders.—Wu, C., Sui, G., Archer, S. N., Sassone-Corsi, P., Aitken, K., Bagli, D., Chen, Y. Local receptors as novel regulators for peripheral clock expression. PMID:25145629

  16. GABAB(1) receptor subunit isoforms differentially regulate stress resilience.

    PubMed

    O'Leary, Olivia F; Felice, Daniela; Galimberti, Stefano; Savignac, Hélène M; Bravo, Javier A; Crowley, Tadhg; El Yacoubi, Malika; Vaugeois, Jean-Marie; Gassmann, Martin; Bettler, Bernhard; Dinan, Timothy G; Cryan, John F

    2014-10-21

    Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(1b)(-/-) mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression.

  17. GABAB(1) receptor subunit isoforms differentially regulate stress resilience

    PubMed Central

    O’Leary, Olivia F.; Felice, Daniela; Galimberti, Stefano; Savignac, Hélène M.; Bravo, Javier A.; Crowley, Tadhg; El Yacoubi, Malika; Vaugeois, Jean-Marie; Gassmann, Martin; Bettler, Bernhard; Dinan, Timothy G.; Cryan, John F.

    2014-01-01

    Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(1b)−/− mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression. PMID:25288769

  18. Disruption of Notch signaling aggravates irradiation-induced bone marrow injury, which is ameliorated by a soluble Dll1 ligand through Csf2rb2 upregulation

    PubMed Central

    Chen, Juan-Juan; Gao, Xiao-Tong; Yang, Lan; Fu, Wei; Liang, Liang; Li, Jun-Chang; Hu, Bin; Sun, Zhi-Jian; Huang, Si-Yong; Zhang, Yi-Zhe; Liang, Ying-Min; Qin, Hong-Yan; Han, Hua

    2016-01-01

    Physical and chemical insult-induced bone marrow (BM) damage often leads to lethality resulting from the depletion of hematopoietic stem and progenitor cells (HSPCs) and/or a deteriorated BM stroma. Notch signaling plays an important role in hematopoiesis, but whether it is involved in BM damage remains unclear. In this study, we found that conditional disruption of RBP-J, the transcription factor of canonical Notch signaling, increased irradiation sensitivity in mice. Activation of Notch signaling with the endothelial cell (EC)-targeted soluble Dll1 Notch ligand mD1R promoted BM recovery after irradiation. mD1R treatment resulted in a significant increase in myeloid progenitors and monocytes in the BM, spleen and peripheral blood after irradiation. mD1R also enhanced hematopoiesis in mice treated with cyclophosphamide, a chemotherapeutic drug that induces BM suppression. Mechanistically, mD1R increased the proliferation and reduced the apoptosis of myeloid cells in the BM after irradiation. The β chain cytokine receptor Csf2rb2 was identified as a downstream molecule of Notch signaling in hematopoietic cells. mD1R improved hematopoietic recovery through up-regulation of the hematopoietic expression of Csf2rb2. Our findings reveal the role of Notch signaling in irradiation- and drug-induced BM suppression and establish a new potential therapy of BM- and myelo-suppression induced by radiotherapy and chemotherapy. PMID:27188577

  19. 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

  20. GABAA receptor regulation of voluntary ethanol drinking requires PKCepsilon.

    PubMed

    Besheer, Joyce; Lepoutre, Veronique; Mole, Beth; Hodge, Clyde W

    2006-11-01

    Protein kinase C (PKC) regulates a variety of neural functions, including ion channel activity, neurotransmitter release, receptor desensitization and differentiation. We have shown previously that mice lacking the epsilon-isoform of PKC (PKCepsilon) self-administer 75% less ethanol and exhibit supersensitivity to acute ethanol and allosteric positive modulators of GABA(A) receptors when compared with wild-type controls. The purpose of the present study was to examine involvement of PKCepsilon in GABA(A) receptor regulation of voluntary ethanol drinking. To address this question, PKCepsilon null-mutant and wild-type control mice were allowed to drink ethanol (10% v/v) vs. water on a two-bottle continuous access protocol. The effects of diazepam (nonselective GABA(A) BZ positive modulator), zolpidem (GABA(A) alpha1 agonist), L-655,708 (BZ-sensitive GABA(A) alpha5 inverse agonist), and flumazenil (BZ antagonist) were then tested on ethanol drinking. Ethanol intake (grams/kg/day) by wild-type mice decreased significantly after diazepam or zolpidem but increased after L-655,708 administration. Flumazenil antagonized diazepam-induced reductions in ethanol drinking in wild-type mice. However, ethanol intake by PKCepsilon null mice was not altered by any of the GABAergic compounds even though effects were seen on water drinking in these mice. Increased acute sensitivity to ethanol and diazepam, which was previously reported, was confirmed in PKCepsilon null mice. Thus, results of the present study show that PKCepsilon null mice do not respond to doses of GABA(A) BZ receptor ligands that regulate ethanol drinking by wild-type control mice. This suggests that PKCepsilon may be required for GABA(A) receptor regulation of chronic ethanol drinking.

  1. Nuclear Receptor Regulation of Aquaporin-2 in the Kidney

    PubMed Central

    Zhang, Xiao-Yan; Wang, Bing; Guan, You-Fei

    2016-01-01

    Aquaporin-2 (AQP2) is a vasopressin-regulated water channel responsible for regulating water reabsorption through the apical plasma membrane of the principal cells of renal collecting ducts. It has been found that dysregulation and dysfunction of AQP2 cause many disorders related to water balance in people and animals, including polyuria and dilutional hyponatremia. Classically, AQP2 mRNA and protein expression and its membrane translocation are regulated by systemic vasopressin involving short-term regulation of AQP2 trafficking to and from the apical plasma membrane and long-term regulation of the total amount of the AQP2 protein in the cell. Recently, increasing evidence has demonstrated that collecting duct AQP2 expression and membrane translocation are also under the control of many other local factors, especially nuclear receptors. Here, we briefly review the progress of studies in this area and discuss the role of nuclear receptors in the regulation of water reabsorption via affecting AQP2 expression and function. PMID:27409611

  2. Nuclear Receptor Regulation of Aquaporin-2 in the Kidney.

    PubMed

    Zhang, Xiao-Yan; Wang, Bing; Guan, You-Fei

    2016-01-01

    Aquaporin-2 (AQP2) is a vasopressin-regulated water channel responsible for regulating water reabsorption through the apical plasma membrane of the principal cells of renal collecting ducts. It has been found that dysregulation and dysfunction of AQP2 cause many disorders related to water balance in people and animals, including polyuria and dilutional hyponatremia. Classically, AQP2 mRNA and protein expression and its membrane translocation are regulated by systemic vasopressin involving short-term regulation of AQP2 trafficking to and from the apical plasma membrane and long-term regulation of the total amount of the AQP2 protein in the cell. Recently, increasing evidence has demonstrated that collecting duct AQP2 expression and membrane translocation are also under the control of many other local factors, especially nuclear receptors. Here, we briefly review the progress of studies in this area and discuss the role of nuclear receptors in the regulation of water reabsorption via affecting AQP2 expression and function. PMID:27409611

  3. 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

  4. Opioid receptors regulate the extinction of Pavlovian fear conditioning.

    PubMed

    McNally, Gavan P; Westbrook, R Frederick

    2003-12-01

    Rats received a single pairing of an auditory conditioned stimulus (CS) with a footshock unconditioned stimulus (US). The fear (freezing) that had accrued to the CS was then extinguished. Injection of naloxone prior to this extinction significantly impaired the development of extinction. This impairment was mediated by opioid receptors in the brain and was not observed when naloxone was injected after extinction training. Finally, an injection of naloxone on test failed to reinstate extinguished responding that had already accrued to the CS. These experiments show that opioid receptors regulate the development, but not the expression, of fear extinction and are discussed with reference to the roles of opioid receptors in US processing, memory, and appetitive motivation.

  5. Regulation of G Protein-Coupled Receptors by Allosteric Ligands

    PubMed Central

    2013-01-01

    Topographically distinct, druggable, allosteric sites may be present on all G protein-coupled receptors (GPCRs). As such, targeting these sites with synthetic small molecules offers an attractive approach to develop receptor-subtype selective chemical leads for the development of novel therapies. A crucial part of drug development is to understand the acute and chronic effects of such allosteric modulators at their corresponding GPCR target. Key regulatory processes including cell-surface delivery, endocytosis, recycling, and down-regulation tightly control the number of receptors at the surface of the cell. As many GPCR therapeutics will be administered chronically, understanding how such ligands modulate these regulatory pathways forms an essential part of the characterization of novel GPCR ligands. This is true for both orthosteric and allosteric ligands. In this Review, we summarize our current understanding of GPCR regulatory processes with a particular focus on the effects and implications of allosteric targeting of GPCRs. PMID:23398684

  6. 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

  7. 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.

  8. New Insights into How Trafficking Regulates T Cell Receptor Signaling

    PubMed Central

    Lou, Jieqiong; Rossy, Jérémie; Deng, Qiji; Pageon, Sophie V.; Gaus, Katharina

    2016-01-01

    There is emerging evidence that exocytosis plays an important role in regulating T cell receptor (TCR) signaling. The trafficking molecules involved in lytic granule (LG) secretion in cytotoxic T lymphocytes (CTL) have been well-studied due to the immune disorder known as familial hemophagocytic lymphohistiocytosis (FHLH). However, the knowledge of trafficking machineries regulating the exocytosis of receptors and signaling molecules remains quite limited. In this review, we summarize the reported trafficking molecules involved in the transport of the TCR and downstream signaling molecules to the cell surface. By combining this information with the known knowledge of LG exocytosis and general exocytic trafficking machinery, we attempt to draw a more complete picture of how the TCR signaling network and exocytic trafficking matrix are interconnected to facilitate T cell activation. This also highlights how membrane compartmentalization facilitates the spatiotemporal organization of cellular responses that are essential for immune functions. PMID:27508206

  9. On the regulative role of the glutamate receptor in mitochondria.

    PubMed

    Selin, Alexey A; Lobysheva, Natalia V; Nesterov, Semen V; Skorobogatova, Yulia A; Byvshev, Ivan M; Pavlik, Lyubov L; Mikheeva, Irina B; Moshkov, Dmitry A; Yaguzhinsky, Lev S; Nartsissov, Yaroslav R

    2016-05-01

    The purpose of this work was to study the regulative role of the glutamate receptor found earlier in the brain mitochondria. In the present work a glutamate-dependent signaling system with similar features was detected in mitochondria of the heart. The glutamate-dependent signaling system in the heart mitochondria was shown to be suppressed by γ-aminobutyric acid (GABA). The GABA receptor presence in the heart mitochondria was shown by golding with the use of antibodies to α- and β-subunits of the receptor. The activity of glutamate receptor was assessed according to the rate of synthesis of hydrogen peroxide. The glutamate receptor in mitochondria could be activated only under conditions of hypoxic stress, which in model experiments was imitated by blocking Complex I by rotenone or fatty acids. The glutamate signal in mitochondria was shown to be calcium- and potential-dependent and the activation of the glutamate cascade was shown to be accompanied by production of hydrogen peroxide. It was discovered that H2O2 synthesis involves two complexes of the mitochondrial electron transfer system - succinate dehydrogenase (SDH) and fatty acid dehydrogenase (ETF:QO). Thus, functions of the glutamate signaling system are associated with the system of respiration-glycolysis switching (the Pasteur-Crabtree) under conditions of hypoxia. PMID:26812870

  10. H-Ras regulation of TRAIL death receptor mediated apoptosis

    PubMed Central

    Chen, Jun-Jie; Bozza, William P.; Di, Xu; Zhang, Yaqin; Hallett, William; Zhang, Baolin

    2014-01-01

    TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors (DRs) 4 and/or 5 expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. Here we provide evidence demonstrating the role of H-Ras in TRAIL receptor mediated apoptosis. By analyzing the genome wide mRNA expression data of the NCI60 cancer cell lines, we found that H-Ras expression was consistently upregulated in TRAIL-resistant cell lines. By contrast, no correlation was found between TRAIL sensitivity and K-Ras expression levels or their mutational profiles. Notably, H-Ras upregulation associated with a surface deficiency of TRAIL death receptors. Selective inhibition of H-Ras activity in TRAIL-resistant cells restored the surface expression of both DR4 and DR5 without changing their total protein levels. The resulting cells became highly susceptible to both TRAIL and agonistic DR5 antibody, whereas K-Ras inhibition had little or no effect on TRAIL-induced apoptosis, indicating H-Ras plays a distinct role in the regulation of TRAIL death receptors. Further studies are warranted to determine the therapeutic potential of H-Ras-specific inhibitors in combination with TRAIL receptor agonists. PMID:25026275

  11. Adrenal medullary regulation of rat renal cortical adrenergic receptors

    SciTech Connect

    Sundaresan, P.R.; Guarnaccia, M.M.; Izzo, J.L. Jr. )

    1987-11-01

    The role of the adrenal medulla in the regulation of renal cortical adrenergic receptors was investigated in renal cortical particular fractions from control rats and rats 6 wk after adrenal demedullation. The specific binding of ({sup 3}H)prazosin, ({sup 3}H)rauwolscine, and ({sup 125}I)iodocyanopindolol were used to quantitate {alpha}{sub 1}-, {alpha}{sub 2}-, and {beta}-adrenergic receptors, respectively. Adrenal demedullation increased the concentration of all three groups of renal adrenergic receptors; maximal number of binding sites (B{sub max}, per milligram membrane protein) for {alpha}{sub 1}-, and {alpha}{sub 2}-, and {beta}-adrenergic receptors were increased by 22, 18.5, and 25%, respectively. No differences were found in the equilibrium dissociation constants (K{sub D}) for any of the radioligands. Plasma corticosterone and plasma and renal norepinephrine levels were unchanged, whereas plasma epinephrine was decreased 72% by adrenal demedullation, renal cortical epinephrine was not detectable in control or demedullated animals. The results suggest that, in the physiological state, the adrenal medulla modulates the number of renal cortical adrenergic receptors, presumably through the actions of a circulating factor such as epinephrine.

  12. Nuclear receptor corepressor complexes in cancer: mechanism, function and regulation

    PubMed Central

    Wong, Madeline M; Guo, Chun; Zhang, Jinsong

    2014-01-01

    Nuclear receptor corepressor (NCoR) and silencing mediator for retinoid and thyroid hormone receptors (SMRT) function as corepressors for diverse transcription factors including nuclear receptors such as estrogen receptors and androgen receptors. Deregulated functions of NCoR and SMRT have been observed in many types of cancers and leukemias. NCoR and SMRT directly bind to transcription factors and nucleate the formation of stable complexes that include histone deacetylase 3, transducin b-like protein 1/TBL1-related protein 1, and G-protein pathway suppressor 2. These NCoR/SMRT-interacting proteins also show deregulated functions in cancers. In this review, we summarize the literature on the mechanism, regulation, and function of the core components of NCoR/SMRT complexes in the context of their involvement in cancers and leukemias. While the current studies support the view that the corepressors are promising targets for cancer treatment, elucidation of the mechanisms of corepressors involved in individual types of cancers is likely required for effective therapy. PMID:25374920

  13. Insulin Growth Factor 1 Receptor Expression Is Associated with NOTCH1 Mutation, Trisomy 12 and Aggressive Clinical Course in Chronic Lymphocytic Leukaemia

    PubMed Central

    Maura, Francesco; Mosca, Laura; Fabris, Sonia; Cutrona, Giovanna; Matis, Serena; Lionetti, Marta; Agnelli, Luca; Barbieri, Marzia; D’Anca, Marianna; Manzoni, Martina; Colombo, Monica; Massucco, Carlotta; Reverberi, Daniele; Gentile, Massimo; Recchia, Anna Grazia; Bossio, Sabrina; Ilariucci, Fiorella; Musolino, Caterina; Di Raimondo, Francesco; Cortelezzi, Agostino; Morabito, Fortunato; Ferrarini, Manlio; Neri, Antonino

    2015-01-01

    IGF1R is emerging as an important gene in the pathogenesis of many solid and haematological cancers and its over-expression has been reported as frequently associated with aggressive disease and chemotherapy resistance. In this study we performed an investigation of the role of IGF1R expression in a large and representative prospective series of 217 chronic lymphocytic leukaemia (CLL) patients enrolled in the multicentre O-CLL1 protocol (clinicaltrial.gov #NCT00917540). High IGF1R gene expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high CD38 expression (p<0.0001), trisomy 12 (p<0.0001), and del(11)(q23) (p=0.014). Interestingly, higher IGF1R expression (p=0.002) characterized patients with NOTCH1 mutation (c.7541_7542delCT), identified in 15.5% of cases of our series by next generation sequencing and ARMS-PCR. Furthermore, IGF1R expression has been proven as an independent prognostic factor associated with time to first treatment in our CLL prospective cohort. These data suggest that IGF1R may play an important role in CLL biology, in particular in aggressive CLL clones characterized by IGHV-UM, trisomy 12 and NOTCH1 mutation. PMID:25786252

  14. G protein-coupled receptors and the regulation of autophagy

    PubMed Central

    Wauson, Eric M.; Dbouk, Hashem A.; Ghosh, Anwesha B.; Cobb, Melanie H.

    2014-01-01

    Autophagy is an important catabolic cellular process that eliminates damaged and unnecessary cytoplasmic proteins and organelles. Basal autophagy occurs during normal physiological conditions, but the activity of this process can be significantly altered in human diseases. Thus, defining the regulatory inputs and signals that control autophagy is essential. Nutrients are key modulators of autophagy. While autophagy is generally accepted to be regulated in a cell autonomous fashion, recent studies suggest nutrients can modulate autophagy in a systemic manner by inducing the secretion of hormones and neurotransmitters that regulate G protein-coupled receptors (GPCRs). Emerging studies show that GPCRs also regulate autophagy by directly detecting extracellular nutrients. We review the role of GPCRs in autophagy regulation, highlighting their potential as therapeutic drug targets. PMID:24751357

  15. 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

  16. Post-transcriptional regulation of GABAB receptor and GIRK1 channels by Nogo receptor 1

    PubMed Central

    2013-01-01

    Background Type B GABA receptors (GABA Rs) play a critical role in synaptic transmission. We carried out studies to determine whether neuronal cell surface expression of GABAB-Rs might be regulated by the Nogo receptor 1 (NgR1). Results siRNA knock-down of NgR1 resulted in a selective increase of GABAB R1 and GABAB R2 protein without altering the expression of GABAA receptor or GAD65. The increase in GABAB receptor subunits was unaccompanied by a change in mRNA, but inhibition of mTOR by rapamycin blocked the increase in GABAB protein. NgR1 siRNA also caused an increase in G protein coupled inwardly rectifying potassium channel (GIRK1). The increase in GABAB receptor and GIRK1 channel proteins was in the plasma membrane, determined by cell surface biotinylation. In NgR1 knockout mice, the amount of GABAB R2 and GIRK1 in hippocampus-derived synaptosomes was increased. Conclusions Together these findings suggest that NgR1 mediated modulation of synaptic transmission may be accomplished, at least in part, through modulation of G protein coupled receptors and channels. PMID:23829864

  17. Histamine and histamine receptor regulation of gastrointestinal cancers

    PubMed Central

    Kennedy, Lindsey; Hodges, Kyle; Meng, Fanyin; Alpini, Gianfranco; Francis, Heather

    2012-01-01

    Histamine is a neurotransmitter released throughout the body that regulates multiple physiological responses. Primarily histamine is acknowledged for its role in inflammatory reactions to foreign pathogens that enter the body. Aside from inflammatory responses, histamine expression and synthesis has been detected in various cancer cell lines and multiple malignancies. Through experimentation histamine has demonstrated its ability to manage proliferation and angiogenesis in these cancerous cells, in either a positive or inhibitory manner. Regulation of angiogenesis and proliferation have been proven to be carried out by the stimulation or inhibition of numerous pathways and secondary response elements, such as VEGFA/C, IP3/Ca2+, G-proteins, cAMP, and many more. The activation of these different response pathways is linked to the binding of ligands to the histamine receptors H1-H4HR. These receptors exhibit various effects dependent on whether it binds an agonist, antagonist, or its specific ligand, histamine. In cancer cell lines and different tumor cells the binding of these different compounds has shown to be one of the main components in exerting proliferative or antiproliferative changes in the microenvironment. It is also known that the histamine receptors have varying degrees of expression in different forms of cancer, and this expression can impact the tumor in various ways. This clearly indicates the significance of histamine receptors in cancer formation, and one of the aims of this review is to cover this topic concisely and in depth. Histamine is produced from numerous cells such as basophils and mast cells and is synthesized from the enzyme histidine decarboxylase (HDC). In this review we will prominently discuss the function of mast cells and HDC in histamine expression in various gastrointestinal carcinomas. We also briefly discuss current studies to support these claims. In this review we hope to give the reader a clear and comprehensible overview of

  18. Molecular mechanisms of gonadotropin-releasing hormone receptor gene regulation.

    PubMed

    Norwitz, E R; Jeong, K H; Chin, W W

    1999-01-01

    GnRH plays a critical role in regulating mammalian reproductive development and function. At the level of the anterior pituitary, GnRH binds to the GnRH receptor (GnRHR) on the cell surface of pituitary gonadotropes. Here, it activates intracellular signal transduction pathways to effect both the synthesis and intermittent release of the gonadotropins LH and FSH. These hormones then enter the systemic circulation to regulate gonadal function, including steroid hormone synthesis and gametogenesis. The response of pituitary gonadotropes to GnRH correlates directly with the concentration of GnRHR on the cell surface, which is mediated, at least in part, at the level of gene expression. A number of endocrine, paracrine, and autocrine factors are known to regulate GnRHR gene expression. This article reviews in detail the role of the GnRHR in the hypothalamic-pituitary-gonadal axis and the factors mediating expression of this gene. A better understanding of the molecular mechanisms that regulate transcription of the GnRHR gene will further our knowledge about the role of this receptor in mammalian reproductive physiology in health and disease.

  19. Regulation of death receptors-Relevance in cancer therapies

    SciTech Connect

    Thonel, A. de; Eriksson, J.E. . E-mail: john.eriksson@btk.fi

    2005-09-01

    Apoptosis is an essential non-inflammatory mechanism for cell removal, which occurs during both physiological and pathological conditions. Apoptosis is characteristically executed by cysteine proteases, termed caspases. The most specific way to activate the caspases machinery is through death receptors (DRs), such as the tumor necrosis factor (TNFR), Fas receptor (FasR), and TRAIL (TRAIL-R). The apoptotic signaling is tightly regulated by the balance of pro-apoptotic and anti-apoptotic proteins and an imbalance between cell death and proliferation may cause numerous diseases, including cancers. The intensive research during the past decade has delineated the basic mechanisms of apoptosis and outlined many important molecular mechanisms underlying the regulation of apoptosis. There is also a better understanding of how the regulation of apoptosis can be disturbed in human cancer cells. The interplay between DRs signaling and anticancer drugs has offered new concepts for the development of highly specific therapeutical agents. Here we review the current understanding of the different molecular mechanisms that regulate DR-mediated apoptosis and the defects in apoptotic signaling discovered in cancer cells. In light of this knowledge, new promising target-based agents for future cancer therapies have been developed.

  20. Beclin 1 regulates growth factor receptor signaling in breast cancer.

    PubMed

    Rohatgi, R A; Janusis, J; Leonard, D; Bellvé, K D; Fogarty, K E; Baehrecke, E H; Corvera, S; Shaw, L M

    2015-10-16

    Beclin 1 is a haploinsufficient tumor suppressor that is decreased in many human tumors. The function of beclin 1 in cancer has been attributed primarily to its role in the degradative process of macroautophagy. However, beclin 1 is a core component of the vacuolar protein sorting 34 (Vps34)/class III phosphatidylinositoI-3 kinase (PI3KC3) and Vps15/p150 complex that regulates multiple membrane-trafficking events. In the current study, we describe an alternative mechanism of action for beclin 1 in breast cancer involving its control of growth factor receptor signaling. We identify a specific stage of early endosome maturation that is regulated by beclin 1, the transition of APPL1-containing phosphatidyIinositol 3-phosphate-negative (PI3P(-)) endosomes to PI3P(+) endosomes. Beclin 1 regulates PI3P production in response to growth factor stimulation to control the residency time of growth factor receptors in the PI3P(-)/APPL(+)-signaling-competent compartment. As a result, suppression of BECN1 sustains growth factor-stimulated AKT and ERK activation resulting in increased breast carcinoma cell invasion. In human breast tumors, beclin 1 expression is inversely correlated with AKT and ERK phosphorylation. Our data identify a novel role for beclin 1 in regulating growth factor signaling and reveal a mechanism by which loss of beclin 1 expression would enhance breast cancer progression.

  1. Neuropeptides, via specific receptors, regulate T cell adhesion to fibronectin.

    PubMed

    Levite, M; Cahalon, L; Hershkoviz, R; Steinman, L; Lider, O

    1998-01-15

    The ability of T cells to adhere to and interact with components of the blood vessel walls and the extracellular matrix is essential for their extravasation and migration into inflamed sites. We have found that the beta1 integrin-mediated adhesion of resting human T cells to fibronectin, a major glycoprotein component of the extracellular matrix, is induced by physiologic concentrations of three neuropeptides: calcitonin gene-related protein (CGRP), neuropeptide Y, and somatostatin; each acts via its own specific receptor on the T cell membrane. In contrast, substance P (SP), which coexists with CGRP in the majority of peripheral endings of sensory nerves, including those innervating the lymphoid organs, blocks T cell adhesion to fibronectin when induced by CGRP, neuropeptide Y, somatostatin, macrophage inflammatory protein-1beta, and PMA. Inhibition of T cell adhesion was obtained both by the intact SP peptide and by its 1-4 N-terminal and its 4-11, 5-11, and 6-11 C-terminal fragments, used at similar nanomolar concentrations. The inhibitory effects of the parent SP peptide and its fragments were abrogated by an SP NK-1 receptor antagonist, suggesting they all act through the same SP NK-1 receptor. These findings suggest that neuropeptides, by activating their specific T cell-expressed receptors, can provide the T cells with both positive (proadhesive) and negative (antiadhesive) signals and thereby regulate their function. Thus, neuropeptides may influence diverse physiologic processes involving integrins, including leukocyte-mediated migration and inflammation. PMID:9551939

  2. Self-renewal of CD133(hi) cells by IL6/Notch3 signalling regulates endocrine resistance in metastatic breast cancer.

    PubMed

    Sansone, Pasquale; Ceccarelli, Claudio; Berishaj, Marjan; Chang, Qing; Rajasekhar, Vinagolu K; Perna, Fabiana; Bowman, Robert L; Vidone, Michele; Daly, Laura; Nnoli, Jennifer; Santini, Donatella; Taffurelli, Mario; Shih, Natalie N C; Feldman, Michael; Mao, Jun J; Colameco, Christopher; Chen, Jinbo; DeMichele, Angela; Fabbri, Nicola; Healey, John H; Cricca, Monica; Gasparre, Giuseppe; Lyden, David; Bonafé, Massimiliano; Bromberg, Jacqueline

    2016-02-09

    The mechanisms of metastatic progression from hormonal therapy (HT) are largely unknown in luminal breast cancer. Here we demonstrate the enrichment of CD133(hi)/ER(lo) cancer cells in clinical specimens following neoadjuvant endocrine therapy and in HT refractory metastatic disease. We develop experimental models of metastatic luminal breast cancer and demonstrate that HT can promote the generation of HT-resistant, self-renewing CD133(hi)/ER(lo)/IL6(hi) cancer stem cells (CSCs). HT initially abrogates oxidative phosphorylation (OXPHOS) generating self-renewal-deficient cancer cells, CD133(hi)/ER(lo)/OXPHOS(lo). These cells exit metabolic dormancy via an IL6-driven feed-forward ER(lo)-IL6(hi)-Notch(hi) loop, activating OXPHOS, in the absence of ER activity. The inhibition of IL6R/IL6-Notch pathways switches the self-renewal of CD133(hi) CSCs, from an IL6/Notch-dependent one to an ER-dependent one, through the re-expression of ER. Thus, HT induces an OXPHOS metabolic editing of luminal breast cancers, paradoxically establishing HT-driven self-renewal of dormant CD133(hi)/ER(lo) cells mediating metastatic progression, which is sensitive to dual targeted therapy.

  3. Neural regulation of acetylcholine receptors in rat neonatal muscle.

    PubMed Central

    Bambrick, L L; Gordon, T

    1992-01-01

    1. The neuronal regulation of the developmental decline in skeletal muscle acetylcholine (ACh) receptors was studied by comparing the effects of sciatic nerve section or of neuromuscular blockade with botulinum toxin (BoTX) on this decline in neonatal and adult rats, using 125I-alpha-bungarotoxin (125I-BTX) as a ligand for the receptor alpha-subunit. 2. The decline in 125I-BTX binding site concentration in neonatal rat triceps surae muscle homogenates towards low, adult levels followed a simple exponential with a time constant of 8 days. This decline occurred while the muscle is still rapidly growing, before the postnatal increase in numbers of sodium channels. It also preceded the decline in muscle ACh receptor alpha-subunit mRNA, reported in other studies, suggesting that subunit levels are not regulated only by mRNA availability. 3. Muscle denervation in the first two weeks of life prevented this developmental decline. Denervation increased the concentration of 125I-BTX binding sites but the magnitude of this increase became progressively smaller as the muscle matured, showing that removal of innervation during adult life does not revert the muscle, in toto, to its pre-innervation state. 4. Blockade of neuromuscular activity with BoTX increased 125I-BTX binding sites to a lesser extent than muscle denervation during neonatal life. This lesser effect of BoTX blockade contrasts with the equal effects of BoTX blockade and denervation in the adult. PMID:1522519

  4. Regulation of hepatic energy metabolism by the nuclear receptor PXR.

    PubMed

    Hakkola, Jukka; Rysä, Jaana; Hukkanen, Janne

    2016-09-01

    The pregnane X receptor (PXR) is a nuclear receptor that is traditionally thought to be specialized for sensing xenobiotic exposure. In concurrence with this feature PXR was originally identified to regulate drug-metabolizing enzymes and transporters. During the last ten years it has become clear that PXR harbors broader functions. Evidence obtained both in experimental animals and humans indicate that ligand-activated PXR regulates hepatic glucose and lipid metabolism and affects whole body metabolic homeostasis. Currently, the consequences of PXR activation on overall metabolic health are not yet fully understood and varying results on the effect of PXR activation or knockout on metabolic disorders and weight gain have been published in mouse models. Rifampicin and St. John's wort, the prototypical human PXR agonists, impair glucose tolerance in healthy volunteers. Chronic exposure to PXR agonists could potentially represent a risk factor for diabetes and metabolic syndrome. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

  5. Circadian Regulation of Olfactory Receptor Neurons in the Cockroach Antenna

    PubMed Central

    Saifullah, A.S.M.; Page, Terry L.

    2013-01-01

    In the cockroach, olfactory sensitivity as measured by the amplitude of the electroantennogram (EAG) is regulated by the circadian system. We wished to determine how this rhythm in antennal response was reflected in the activity of individual olfactory receptor neurons. The amplitude of the electroantennogram (EAG) and the activity of olfactory receptor neurons (ORNs) in single olfactory sensilla were recorded simultaneously for 3–5 days in constant darkness from an antenna of the cockroach Leucophaea maderae. Both EAG amplitude and the spike frequency of the ORNs exhibited circadian rhythms with peak amplitude/activity occurring in the subjective day. The phases of the rhythms were dependent on the phase of the prior light cycle and thus were entrainable by light. Ablation of the optic lobes abolished the rhythm in EAG amplitude as has been previously reported. In contrast, the rhythm in ORN response persisted following surgery. These results indicated that a circadian clock outside the optic lobes can regulate the responses of olfactory receptor neurons and further that this modulation of the ORN response is not dependent on the circadian rhythm in EAG amplitude. PMID:19346451

  6. TAM receptors regulate multiple features of microglial physiology.

    PubMed

    Fourgeaud, Lawrence; Través, Paqui G; Tufail, Yusuf; Leal-Bailey, Humberto; Lew, Erin D; Burrola, Patrick G; Callaway, Perri; Zagórska, Anna; Rothlin, Carla V; Nimmerjahn, Axel; Lemke, Greg

    2016-04-14

    Microglia are damage sensors for the central nervous system (CNS), and the phagocytes responsible for routine non-inflammatory clearance of dead brain cells. Here we show that the TAM receptor tyrosine kinases Mer and Axl regulate these microglial functions. We find that adult mice deficient in microglial Mer and Axl exhibit a marked accumulation of apoptotic cells specifically in neurogenic regions of the CNS, and that microglial phagocytosis of the apoptotic cells generated during adult neurogenesis is normally driven by both TAM receptor ligands Gas6 and protein S. Using live two-photon imaging, we demonstrate that the microglial response to brain damage is also TAM-regulated, as TAM-deficient microglia display reduced process motility and delayed convergence to sites of injury. Finally, we show that microglial expression of Axl is prominently upregulated in the inflammatory environment that develops in a mouse model of Parkinson's disease. Together, these results establish TAM receptors as both controllers of microglial physiology and potential targets for therapeutic intervention in CNS disease. PMID:27049947

  7. Transcriptional regulation of farnesyl pyrophosphate synthase by liver X receptors.

    PubMed

    Fukuchi, Junichi; Song, Ching; Ko, Andrew L; Liao, Shutsung

    2003-09-01

    Liver X receptors (LXRs) are members of the nuclear receptor superfamily that are involved in cholesterol and lipid metabolism. In addition to liver, the brain is another site where LXRs may control cholesterol homeostasis. In the brain, the regulation of cholesterol homeostasis is independent from other parts of the body, and its disturbance is associated with neurodegenerative disorders, such as Alzheimer's disease. We have used PCR-based suppressive subtractive cloning to identify new LXR target genes in brain cells. In this report, we show that farnesyl pyrophosphate synthase (FPPS) is a new target gene for LXR in astrocytes and neurons. Farnesyl pyrophosphate is an obligate intermediate for de novo cholesterol synthesis and a substrate for protein farnesylation. Stimulation of FPPS mRNA synthesis by an LXR agonist, Hypocholamide, was observed in several cell lines from the central nervous system. We identified a single putative direct repeat 4 (DR4) LXR response element in the FPPS promoter. In a reporter gene assay, LXR transactivated a reporter gene bearing a truncated FPPS promoter containing this DR4 cis-element but not if the DR4 element was mutated. Using gel-mobility shift assay, we further demonstrated the direct interaction between the LXR/retinoid X receptor (RXR) heterodimer and the response element. Taken together, our results indicate that LXRs directly regulate FPPS gene expression, and thus may play a role in modulating cholesterol synthesis in the brain. PMID:12957674

  8. Dual Roles of O-Glucose Glycans Redundant with Monosaccharide O-Fucose on Notch in Notch Trafficking.

    PubMed

    Matsumoto, Kenjiroo; Ayukawa, Tomonori; Ishio, Akira; Sasamura, Takeshi; Yamakawa, Tomoko; Matsuno, Kenji

    2016-06-24

    Notch is a transmembrane receptor that mediates cell-cell interactions and controls various cell-fate specifications in metazoans. The extracellular domain of Notch contains multiple epidermal growth factor (EGF)-like repeats. At least five different glycans are found in distinct sites within these EGF-like repeats. The function of these individual glycans in Notch signaling has been investigated, primarily by disrupting their individual glycosyltransferases. However, we are just beginning to understand the potential functional interactions between these glycans. Monosaccharide O-fucose and O-glucose trisaccharide (O-glucose-xylose-xylose) are added to many of the Notch EGF-like repeats. In Drosophila, Shams adds a xylose specifically to the monosaccharide O-glucose. We found that loss of the terminal dixylose of O-glucose-linked saccharides had little effect on Notch signaling. However, our analyses of double mutants of shams and other genes required for glycan modifications revealed that both the monosaccharide O-glucose and the terminal dixylose of O-glucose-linked saccharides function redundantly with the monosaccharide O-fucose in Notch activation and trafficking. The terminal dixylose of O-glucose-linked saccharides and the monosaccharide O-glucose were required in distinct Notch trafficking processes: Notch transport from the apical plasma membrane to adherens junctions, and Notch export from the endoplasmic reticulum, respectively. Therefore, the monosaccharide O-glucose and terminal dixylose of O-glucose-linked saccharides have distinct activities in Notch trafficking, although a loss of these activities is compensated for by the presence of monosaccharide O-fucose. Given that various glycans attached to a protein motif may have redundant functions, our results suggest that these potential redundancies may lead to a serious underestimation of glycan functions. PMID:27129198

  9. Dual Roles of O-Glucose Glycans Redundant with Monosaccharide O-Fucose on Notch in Notch Trafficking.

    PubMed

    Matsumoto, Kenjiroo; Ayukawa, Tomonori; Ishio, Akira; Sasamura, Takeshi; Yamakawa, Tomoko; Matsuno, Kenji

    2016-06-24

    Notch is a transmembrane receptor that mediates cell-cell interactions and controls various cell-fate specifications in metazoans. The extracellular domain of Notch contains multiple epidermal growth factor (EGF)-like repeats. At least five different glycans are found in distinct sites within these EGF-like repeats. The function of these individual glycans in Notch signaling has been investigated, primarily by disrupting their individual glycosyltransferases. However, we are just beginning to understand the potential functional interactions between these glycans. Monosaccharide O-fucose and O-glucose trisaccharide (O-glucose-xylose-xylose) are added to many of the Notch EGF-like repeats. In Drosophila, Shams adds a xylose specifically to the monosaccharide O-glucose. We found that loss of the terminal dixylose of O-glucose-linked saccharides had little effect on Notch signaling. However, our analyses of double mutants of shams and other genes required for glycan modifications revealed that both the monosaccharide O-glucose and the terminal dixylose of O-glucose-linked saccharides function redundantly with the monosaccharide O-fucose in Notch activation and trafficking. The terminal dixylose of O-glucose-linked saccharides and the monosaccharide O-glucose were required in distinct Notch trafficking processes: Notch transport from the apical plasma membrane to adherens junctions, and Notch export from the endoplasmic reticulum, respectively. Therefore, the monosaccharide O-glucose and terminal dixylose of O-glucose-linked saccharides have distinct activities in Notch trafficking, although a loss of these activities is compensated for by the presence of monosaccharide O-fucose. Given that various glycans attached to a protein motif may have redundant functions, our results suggest that these potential redundancies may lead to a serious underestimation of glycan functions.

  10. Notch strength of composites

    NASA Technical Reports Server (NTRS)

    Whitney, J. M.

    1983-01-01

    The notch strength of composites is discussed. The point stress and average stress criteria relate the notched strength of a laminate to the average strength of a relatively long tensile coupon. Tests of notched specimens in which microstrain gages have been placed at or near the edges of the holes have measured strains much larger that those measured in an unnotched tensile coupon. Orthotropic stress concentration analyses of failed notched laminates have also indicated that failure occurred at strains much larger than those experienced on tensile coupons with normal gage lengths. This suggests that the high strains at the edge of a hole can be related to the very short length of fiber subjected to these strains. Lockheed has attempted to correlate a series of tests of several laminates with holes ranging from 0.19 to 0.50 in. Although the average stress criterion correlated well with test results for hole sizes equal to or greater than 0.50 in., it over-estimated the laminate strength in the range of hole sizes from 0.19 to 0.38 in. It thus appears that a theory is needed that is based on the mechanics of failure and is more generally applicable to the range of hole sizes and the varieties of laminates found in aircraft construction.

  11. Electrochemical polishing of notches

    DOEpatents

    Kephart, A.R.; Alberts, A.H.

    1989-02-21

    An apparatus and method are disclosed for the selective electrochemical polishing of a lateral tip of a deep longitudinal notch in a work piece used to test crack initiation properties of materials. A DC power source is connected to the work piece and to an electrode disposed laterally along the distal end of an insulated body which is inserted in the longitudinal notch. The electrode and distal end of the body are disposed along the tip of the notch, but are spaced from the notch so as to provide a lateral passage for an electrolyte. The electrolyte is circulated through the passage so that the electrolyte only contacts the work piece adjacent the passage. Conveniently, the electrolyte is circulated by use of an inlet tube and an outlet tube provided at opposite ends of the passage. These tubes are preferably detachably located adjacent the ends of the passage and suitable seals are provided. A holding device including arms to which the tubes are attached is conveniently used to rapidly and easily locate the test specimen with the passage aligned with the tubes. The electrode is preferably a wire which is located in grooves along the distal end of the insulated body and up one side of the body or a plastic sheath insulated thin metal strip. 4 figs.

  12. Electrochemical polishing of notches

    DOEpatents

    Kephart, Alan R.; Alberts, Alfred H.

    1989-01-01

    An apparatus and method are disclosed for the selective electrochemical polishing of a lateral tip of a deep longitudinal notch in a work piece used to test crack initiation properties of materials. A DC power source is connected to the work piece and to an electrode disposed laterally along the distal end of an insulated body which is inserted in the longitudinal notch. The electrode and distal end of the body are disposed along the tip of the notch, but are spaced from the notch so as to provide a lateral passage for an electrolyte. The electrolyte is circulated through the passage so that the electrolyte only contacts the work piece adjacent the passage. Conveniently, the electrolyte is circulated by use of an inlet tube and an outlet tube provided at opposite ends of the passage. These tubes are preferably detachably located adjacent the ends of the passage and suitable seals are provided. A holding device including arms to which the tubes are attached is conveniently used to rapidly and easily locate the test specimen with the passage aligned with the tubes. The electrode is preferably a wire which is located in grooves along the distal end of the insulated body and up one side of the body or a plastic sheath insulated thin metal strip.

  13. Notch-Mediated Cell Adhesion

    PubMed Central

    Murata, Akihiko; Hayashi, Shin-Ichi

    2016-01-01

    Notch family members are generally recognized as signaling molecules that control various cellular responses in metazoan organisms. Early fly studies and our mammalian studies demonstrated that Notch family members are also cell adhesion molecules; however, information on the physiological roles of this function and its origin is limited. In this review, we discuss the potential present and ancestral roles of Notch-mediated cell adhesion in order to explore its origin and the initial roles of Notch family members dating back to metazoan evolution. We hypothesize that Notch family members may have initially emerged as cell adhesion molecules in order to mediate multicellularity in the last common ancestor of metazoan organisms. PMID:26784245

  14. Emerging EPO and EPO receptor regulators and signal transducers

    PubMed Central

    Kuhrt, David

    2015-01-01

    As essential mediators of red cell production, erythropoietin (EPO) and its cell surface receptor (EPO receptor [EPOR]) have been intensely studied. Early investigations defined basic mechanisms for hypoxia-inducible factor induction of EPO expression, and within erythroid progenitors EPOR engagement of canonical Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5), rat sarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAS/MEK/ERK), and phosphatidylinositol 3-kinase (PI3K) pathways. Contemporary genetic, bioinformatic, and proteomic approaches continue to uncover new clinically relevant modulators of EPO and EPOR expression, and EPO’s biological effects. This Spotlight review highlights such factors and their emerging roles during erythropoiesis and anemia. PMID:25887776

  15. Receptor Coactivators: Master Regulators of Human Health and Disease

    PubMed Central

    Dasgupta, Subhamoy; Lonard, David M.; O’Malley, Bert W.

    2015-01-01

    Transcriptional coregulators (coactivators and corepressors) have emerged as the principal modulators of the functions of nuclear receptors and other transcription factors. During the decade since the discovery of steroid receptor coactivator-1 (SRC-1), the first authentic coregulator, more than 400 coregulators have been identified and characterized, and deciphering their function has contributed significantly to our understanding of their role in human physiology. Deregulated expression of coregulators has been implicated in diverse disease states and related pathologies. The advancement of molecular technologies has enabled us to better characterize the molecular associations of the SRC family of coactivators with other protein complexes in the context of gene regulation. These continuing discoveries not only expand our knowledge of the roles of coactivators in various human diseases but allow us to discover novel coactivator-targeting strategies for therapeutic intervention in these diseases. PMID:24111892

  16. Emerging EPO and EPO receptor regulators and signal transducers.

    PubMed

    Kuhrt, David; Wojchowski, Don M

    2015-06-01

    As essential mediators of red cell production, erythropoietin (EPO) and its cell surface receptor (EPO receptor [EPOR]) have been intensely studied. Early investigations defined basic mechanisms for hypoxia-inducible factor induction of EPO expression, and within erythroid progenitors EPOR engagement of canonical Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5), rat sarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAS/MEK/ERK), and phosphatidylinositol 3-kinase (PI3K) pathways. Contemporary genetic, bioinformatic, and proteomic approaches continue to uncover new clinically relevant modulators of EPO and EPOR expression, and EPO's biological effects. This Spotlight review highlights such factors and their emerging roles during erythropoiesis and anemia. PMID:25887776

  17. Peripheral cannabinoid receptor, CB2, regulates bone mass

    PubMed Central

    Ofek, Orr; Karsak, Meliha; Leclerc, Nathalie; Fogel, Meirav; Frenkel, Baruch; Wright, Karen; Tam, Joseph; Attar-Namdar, Malka; Kram, Vardit; Shohami, Esther; Mechoulam, Raphael; Zimmer, Andreas; Bab, Itai

    2006-01-01

    The endogenous cannabinoids bind to and activate two G protein-coupled receptors, the predominantly central cannabinoid receptor type 1 (CB1) and peripheral cannabinoid receptor type 2 (CB2). Whereas CB1 mediates the cannabinoid psychotropic, analgesic, and orectic effects, CB2 has been implicated recently in the regulation of liver fibrosis and atherosclerosis. Here we show that CB2-deficient mice have a markedly accelerated age-related trabecular bone loss and cortical expansion, although cortical thickness remains unaltered. These changes are reminiscent of human osteoporosis and may result from differential regulation of trabecular and cortical bone remodeling. The CB2–/– phenotype is also characterized by increased activity of trabecular osteoblasts (bone-forming cells), increased osteoclast (the bone-resorbing cell) number, and a markedly decreased number of diaphyseal osteoblast precursors. CB2 is expressed in osteoblasts, osteocytes, and osteoclasts. A CB2-specific agonist that does not have any psychotropic effects enhances endocortical osteoblast number and activity and restrains trabecular osteoclastogenesis, apparently by inhibiting proliferation of osteoclast precursors and receptor activator of NF-κB ligand expression in bone marrow-derived osteoblasts/stromal cells. The same agonist attenuates ovariectomy-induced bone loss and markedly stimulates cortical thickness through the respective suppression of osteoclast number and stimulation of endocortical bone formation. These results demonstrate that the endocannabinoid system is essential for the maintenance of normal bone mass by osteoblastic and osteoclastic CB2 signaling. Hence, CB2 offers a molecular target for the diagnosis and treatment of osteoporosis, the most prevalent degenerative disease in developed countries. PMID:16407142

  18. Regulation of vitamin D receptor expression by retinoic acid receptor alpha in acute myeloid leukemia cells.

    PubMed

    Marchwicka, Aleksandra; Cebrat, Małgorzata; Łaszkiewicz, Agnieszka; Śnieżewski, Łukasz; Brown, Geoffrey; Marcinkowska, Ewa

    2016-05-01

    Acute myeloid leukemia (AML) is the predominant acute leukemia among adults, characterized by an accumulation of malignant immature myeloid precursors. A very promising way to treat AML is differentiation therapy using either all-trans-retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3 (1,25D), or the use of both these differentiation-inducing agents. However, the effect of combination treatment varies in different AML cell lines, and this is due to ATRA either down- or up-regulating transcription of vitamin D receptor (VDR) in the cells examined. The mechanism of transcriptional regulation of VDR in response to ATRA has not been fully elucidated. Here, we show that the retinoic acid receptor α (RARα) is responsible for regulating VDR transcription in AML cells. We have shown that a VDR transcriptional variant, originating in exon 1a, is regulated by RARα agonists in AML cells. Moreover, in cells with a high basal level of RARα protein, the VDR gene is transcriptionally repressed as long as RARα agonist is absent. In these cells down-regulation of the level of RARα leads to increased expression of VDR. We consider that our findings provide a mechanistic background to explain the different outcomes from treating AML cell lines with a combination of ATRA and 1,25D. PMID:26969398

  19. 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

  20. Cannabinoid receptor signaling regulates liver development and metabolism.

    PubMed

    Liu, Leah Y; Alexa, Kristen; Cortes, Mauricio; Schatzman-Bone, Stephanie; Kim, Andrew J; Mukhopadhyay, Bani; Cinar, Resat; Kunos, George; North, Trista E; Goessling, Wolfram

    2016-02-15

    Endocannabinoid (EC) signaling mediates psychotropic effects and regulates appetite. By contrast, potential roles in organ development and embryonic energy consumption remain unknown. Here, we demonstrate that genetic or chemical inhibition of cannabinoid receptor (Cnr) activity disrupts liver development and metabolic function in zebrafish (Danio rerio), impacting hepatic differentiation, but not endodermal specification: loss of cannabinoid receptor 1 (cnr1) and cnr2 activity leads to smaller livers with fewer hepatocytes, reduced liver-specific gene expression and proliferation. Functional assays reveal abnormal biliary anatomy and lipid handling. Adult cnr2 mutants are susceptible to hepatic steatosis. Metabolomic analysis reveals reduced methionine content in Cnr mutants. Methionine supplementation rescues developmental and metabolic defects in Cnr mutant livers, suggesting a causal relationship between EC signaling, methionine deficiency and impaired liver development. The effect of Cnr on methionine metabolism is regulated by sterol regulatory element-binding transcription factors (Srebfs), as their overexpression rescues Cnr mutant liver phenotypes in a methionine-dependent manner. Our work describes a novel developmental role for EC signaling, whereby Cnr-mediated regulation of Srebfs and methionine metabolism impacts liver development and function.

  1. 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.

  2. Activation and Regulation of Purinergic P2X Receptor Channels

    PubMed Central

    Coddou, Claudio; Yan, Zonghe; Obsil, Tomas; Huidobro-Toro, J. Pablo

    2011-01-01

    Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions. PMID:21737531

  3. 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

  4. Serotonin 2c receptors in pro-opiomelanocortin neurons regulate energy and glucose homeostasis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Energy and glucose homeostasis are regulated by central serotonin 2C receptors. These receptors are attractive pharmacological targets for the treatment of obesity; however, the identity of the serotonin 2C receptor-expressing neurons that mediate the effects of serotonin and serotonin 2C receptor a...

  5. Leptin receptors are developmentally regulated in rat pituitary and hypothalamus.

    PubMed

    Morash, Barbara A; Imran, Ali; Wilkinson, Diane; Ur, Ehud; Wilkinson, Michael

    2003-11-28

    We have previously reported that leptin is expressed in adult rat brain and pituitary gland, though the role of leptin in these sites has not been determined. Leptin mRNA is developmentally regulated in the brain and pituitary of male and female rats during early postnatal development, suggesting a role in the maturation of the brain-pituitary system. Here, we sought to extend our previous studies by evaluating (1) the ontogeny of leptin receptor mRNA levels in rat brain and pituitary and (2) pituitary leptin protein levels in neonatal and pre-pubertal rats. Pituitary leptin concentration was highest shortly after birth (postnatal day (PD) 4, 25 ng/mg protein) and fell significantly throughout postnatal development and into adulthood (PD 60, 3.5 ng/mg protein; P<0.005) coincident with a decline in pituitary leptin mRNA levels. Significant age-related effects on leptin receptor mRNA levels were also observed in the pituitary and the hypothalamus of male and female rats using semi-quantitative RT-PCR analysis. In the pituitary, the short form (OBRa) mRNA levels were highest in neonatal rats (PD 4) but declined throughout postnatal development (PD 4-22) paralleling the fall in pituitary leptin mRNA and protein levels. The long form (OBRb) mRNA levels were unaffected by age between PD 4 and 22. In contrast, hypothalamic, levels of OBRb mRNA were very low to undetectable shortly after birth (PD 4) and rose significantly between PD 4 and 14/22 while levels of OBRa mRNA were not significantly different between PD 4 and 22. Immunohistochemical detection of leptin receptor immunoreactivity (all forms) revealed the presence of OBR-like protein in pituitary and hypothalamus as early as PD 4. Cortical leptin receptor mRNA levels were similar throughout early postnatal development. No gender-related differences in leptin receptor mRNA levels were noted in brain or pituitary. In conclusion, these data, together with our previous work, indicate that the neonatal pituitary gland

  6. Chemokine receptor CXCR2: physiology regulator and neuroinflammation controller?

    PubMed Central

    Veenstra, Mike; Ransohoff, Richard M.

    2012-01-01

    The innate immune system is a crucial component of inflammatory reactions, while the central nervous system (CNS) is the most vulnerable site of the body to inflammatory tissue injury. Neuroinflammatory brain pathologies are disorders in which the CNS is threatened by its own immune system. Chemokine receptor CXCR2 and its ligands have been implicated in several neuroinflammatory brain pathologies, as well as in neutrophil recruitment and in the developmental positioning of neural cells. This review focuses on the basics of CXCR2, its regulating role in bone marrow neutrophil recruitment, oligodendrocyte progenitor cell positioning and neural repair mechanisms, as well as its diverse roles in neuroinflammatory brain pathologies. PMID:22445294

  7. 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

  8. Liver X Receptor (LXR) Regulates Human Adipocyte Lipolysis*

    PubMed Central

    Stenson, Britta M.; Rydén, Mikael; Venteclef, Nicolas; Dahlman, Ingrid; Pettersson, Annie M. L.; Mairal, Aline; Åström, Gaby; Blomqvist, Lennart; Wang, Victoria; Jocken, Johan W. E.; Clément, Karine; Langin, Dominique; Arner, Peter; Laurencikiene, Jurga

    2011-01-01

    The Liver X receptor (LXR) is an important regulator of carbohydrate and lipid metabolism in humans and mice. We have recently shown that activation of LXR regulates cellular fuel utilization in adipocytes. In contrast, the role of LXR in human adipocyte lipolysis, the major function of human white fat cells, is not clear. In the present study, we stimulated in vitro differentiated human and murine adipocytes with the LXR agonist GW3965 and observed an increase in basal lipolysis. Microarray analysis of human adipocyte mRNA following LXR activation revealed an altered gene expression of several lipolysis-regulating proteins, which was also confirmed by quantitative real-time PCR. We show that expression and intracellular localization of perilipin1 (PLIN1) and hormone-sensitive lipase (HSL) are affected by GW3965. Although LXR activation does not influence phosphorylation status of HSL, HSL activity is required for the lipolytic effect of GW3965. This effect is abolished by PLIN1 knockdown. In addition, we demonstrate that upon activation, LXR binds to the proximal regions of the PLIN1 and HSL promoters. By selective knock-down of either LXR isoform, we show that LXRα is the major isoform mediating the lipolysis-related effects of LXR. In conclusion, the present study demonstrates that activation of LXRα up-regulates basal human adipocyte lipolysis. This is at least partially mediated through LXR binding to the PLIN1 promoter and down-regulation of PLIN1 expression. PMID:21030586

  9. Liver X receptor (LXR) regulates human adipocyte lipolysis.

    PubMed

    Stenson, Britta M; Rydén, Mikael; Venteclef, Nicolas; Dahlman, Ingrid; Pettersson, Annie M L; Mairal, Aline; Aström, Gaby; Blomqvist, Lennart; Wang, Victoria; Jocken, Johan W E; Clément, Karine; Langin, Dominique; Arner, Peter; Laurencikiene, Jurga

    2011-01-01

    The Liver X receptor (LXR) is an important regulator of carbohydrate and lipid metabolism in humans and mice. We have recently shown that activation of LXR regulates cellular fuel utilization in adipocytes. In contrast, the role of LXR in human adipocyte lipolysis, the major function of human white fat cells, is not clear. In the present study, we stimulated in vitro differentiated human and murine adipocytes with the LXR agonist GW3965 and observed an increase in basal lipolysis. Microarray analysis of human adipocyte mRNA following LXR activation revealed an altered gene expression of several lipolysis-regulating proteins, which was also confirmed by quantitative real-time PCR. We show that expression and intracellular localization of perilipin1 (PLIN1) and hormone-sensitive lipase (HSL) are affected by GW3965. Although LXR activation does not influence phosphorylation status of HSL, HSL activity is required for the lipolytic effect of GW3965. This effect is abolished by PLIN1 knockdown. In addition, we demonstrate that upon activation, LXR binds to the proximal regions of the PLIN1 and HSL promoters. By selective knock-down of either LXR isoform, we show that LXRα is the major isoform mediating the lipolysis-related effects of LXR. In conclusion, the present study demonstrates that activation of LXRα up-regulates basal human adipocyte lipolysis. This is at least partially mediated through LXR binding to the PLIN1 promoter and down-regulation of PLIN1 expression. PMID:21030586

  10. Bile acid receptor agonist GW4064 regulates PPARγ coactivator-1α expression through estrogen receptor-related receptor α.

    PubMed

    Dwivedi, Shailendra Kumar Dhar; Singh, Nidhi; Kumari, Rashmi; Mishra, Jay Sharan; Tripathi, Sarita; Banerjee, Priyam; Shah, Priyanka; Kukshal, Vandana; Tyagi, Abdul Malik; Gaikwad, Anil Nilkanth; Chaturvedi, Rajnish Kumar; Mishra, Durga Prasad; Trivedi, Arun Kumar; Sanyal, Somali; Chattopadhyay, Naibedya; Ramachandran, Ravishankar; Siddiqi, Mohammad Imran; Bandyopadhyay, Arun; Arora, Ashish; Lundåsen, Thomas; Anakk, Sayee Priyadarshini; Moore, David D; Sanyal, Sabyasachi

    2011-06-01

    Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression. This induction in PGC-1α concomitantly enhances mitochondrial mass and expression of several PGC-1α target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor α (ERRα). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1α mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1α expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERRα-FXR-PGC-1α and small heterodimer partner offers new insights into the biological functions of ERRα and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiology.

  11. Pharmacological activation of lysophosphatidic acid receptors regulates erythropoiesis

    PubMed Central

    Lin, Kuan-Hung; Ho, Ya-Hsuan; Chiang, Jui-Chung; Li, Meng-Wei; Lin, Shi-Hung; Chen, Wei-Min; Chiang, Chi-Ling; Lin, Yu-Nung; Yang, Ya-Jan; Chen, Chiung-Nien; Lu, Jenher; Huang, Chang-Jen; Tigyi, Gabor; Yao, Chao-Ling; Lee, Hsinyu

    2016-01-01

    Lysophosphatidic acid (LPA), a growth factor-like phospholipid, regulates numerous physiological functions, including cell proliferation and differentiation. In a previous study, we have demonstrated that LPA activates erythropoiesis by activating the LPA 3 receptor subtype (LPA3) under erythropoietin (EPO) induction. In the present study, we applied a pharmacological approach to further elucidate the functions of LPA receptors during red blood cell (RBC) differentiation. In K562 human erythroleukemia cells, knockdown of LPA2 enhanced erythropoiesis, whereas knockdown of LPA3 inhibited RBC differentiation. In CD34+ human hematopoietic stem cells (hHSC) and K526 cells, the LPA3 agonist 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate (2S-OMPT) promoted erythropoiesis, whereas the LPA2 agonist dodecyl monophosphate (DMP) and the nonlipid specific agonist GRI977143 (GRI) suppressed this process. In zebrafish embryos, hemoglobin expression was significantly increased by 2S-OMPT treatment but was inhibited by GRI. Furthermore, GRI treatment decreased, whereas 2S-OMPT treatment increased RBC counts and amount of hemoglobin level in adult BALB/c mice. These results indicate that LPA2 and LPA3 play opposing roles during RBC differentiation. The pharmacological activation of LPA receptor subtypes represent a novel strategies for augmenting or inhibiting erythropoiesis. PMID:27244685

  12. Alcohol Related Changes in Regulation of NMDA Receptor Functions

    PubMed Central

    Nagy, József

    2008-01-01

    Long-term alcohol exposure may lead to development of alcohol dependence in consequence of altered neurotransmitter functions. Accumulating evidence suggests that the N-methyl-D-aspartate (NMDA) type of glutamate receptors is a particularly important site of ethanol’s action. Several studies showed that ethanol potently inhibits NMDA receptors (NMDARs) and prolonged ethanol exposition leads to a compensatory “up-regulation” of NMDAR mediated functions. Therefore, alterations in NMDAR function are supposed to contribute to the development of ethanol tolerance, dependence as well as to the acute and late signs of ethanol withdrawal. A number of publications report alterations in the expression and phosphorylation states of NMDAR subunits, in their interaction with scaffolding proteins or other receptors in consequence of chronic ethanol treatment. Our knowledge on the regulatory processes, which modulate NMDAR functions including factors altering transcription, protein expression and post-translational modifications of NMDAR subunits, as well as those influencing their interactions with different regulatory proteins or other downstream signaling elements are incessantly increasing. The aim of this review is to summarize the complex chain of events supposedly playing a role in the up-regulation of NMDAR functions in consequence of chronic ethanol exposure. PMID:19305787

  13. p75 neurotrophin receptor regulates energy balance in obesity

    PubMed Central

    Baeza-Raja, Bernat; Sachs, Benjamin D.; Li, Pingping; Christian, Frank; Vagena, Eirini; Davalos, Dimitrios; Le Moan, Natacha; Ryu, Jae Kyu; Sikorski, Shoana L.; Chan, Justin P.; Scadeng, Miriam; Taylor, Susan S.; Houslay, Miles D.; Baillie, George S.; Saltiel, Alan R.; Olefsky, Jerrold M.; Akassoglou, Katerina

    2015-01-01

    Summary Obesity and metabolic syndrome reflect the dysregulation of molecular pathways that control energy homeostasis. Here we show that upon high-fat diet (HFD), the p75 neurotrophin receptor (p75NTR) controls energy expenditure in obese mice. Despite no changes in food intake, p75NTR-null mice were protected from HFD-induced obesity and remained lean due to increased energy expenditure, without developing insulin resistance or liver steatosis. p75NTR directly interacts with the catalytic subunit of protein kinase A (PKA) and regulates cAMP signaling in adipocytes, leading to decreased lipolysis and thermogenesis. Adipocyte-specific depletion of p75NTR or transplantation of p75NTR-null white adipose tissue (WAT) into wild-type mice fed a HFD protected against weight gain and insulin resistance. Our results reveal that signaling from p75NTR to cAMP/PKA regulates energy balance and suggest that non-neuronal functions of neurotrophin receptor signaling could be a new target for treating obesity and the metabolic syndrome. PMID:26748707

  14. PACSIN1 regulates the dynamics of AMPA receptor trafficking.

    PubMed

    Widagdo, Jocelyn; Fang, Huaqiang; Jang, Se Eun; Anggono, Victor

    2016-01-01

    Dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses plays an important role in synaptic plasticity. We previously reported that the protein kinase C and casein kinase II substrate in neurons (PACSIN) forms a complex with AMPARs through its interaction with the protein interacting with C-kinase 1 (PICK1) to regulate NMDA receptor (NMDAR)-induced AMPAR endocytosis and cerebellar long-term depression. However, the molecular mechanism by which PACSIN regulates the dynamics of AMPAR trafficking remains unclear. Using a pH-sensitive green fluorescent protein, pHluorin, tagged to the extracellular domain of the GluA2 subunit of AMPARs, we demonstrate dual roles for PACSIN1 in controlling the internalization and recycling of GluA2 after NMDAR activation. Structure and function analysis reveals a requirement for the PACSIN1 F-BAR and SH3 domains in controlling these NMDAR-dependent processes. Interestingly, the variable region, which binds to PICK1, is not essential for NMDAR-dependent GluA2 internalization and is required only for the correct recycling of AMPARs. These results indicate that PACSIN is a versatile membrane deformation protein that links the endocytic and recycling machineries essential for dynamic AMPAR trafficking in neurons. PMID:27488904

  15. PACSIN1 regulates the dynamics of AMPA receptor trafficking

    PubMed Central

    Widagdo, Jocelyn; Fang, Huaqiang; Jang, Se Eun; Anggono, Victor

    2016-01-01

    Dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses plays an important role in synaptic plasticity. We previously reported that the protein kinase C and casein kinase II substrate in neurons (PACSIN) forms a complex with AMPARs through its interaction with the protein interacting with C-kinase 1 (PICK1) to regulate NMDA receptor (NMDAR)-induced AMPAR endocytosis and cerebellar long-term depression. However, the molecular mechanism by which PACSIN regulates the dynamics of AMPAR trafficking remains unclear. Using a pH-sensitive green fluorescent protein, pHluorin, tagged to the extracellular domain of the GluA2 subunit of AMPARs, we demonstrate dual roles for PACSIN1 in controlling the internalization and recycling of GluA2 after NMDAR activation. Structure and function analysis reveals a requirement for the PACSIN1 F-BAR and SH3 domains in controlling these NMDAR-dependent processes. Interestingly, the variable region, which binds to PICK1, is not essential for NMDAR-dependent GluA2 internalization and is required only for the correct recycling of AMPARs. These results indicate that PACSIN is a versatile membrane deformation protein that links the endocytic and recycling machineries essential for dynamic AMPAR trafficking in neurons. PMID:27488904

  16. Regulation of receptor protein-tyrosine phosphatase dimerization.

    PubMed

    van der Wijk, Thea; Blanchetot, Christophe; den Hertog, Jeroen

    2005-01-01

    Receptor protein-tyrosine phosphatases (RPTPs) are single membrane spanning proteins belonging to the family of PTPs that, together with the antagonistically acting protein-tyrosine kinases (PTKs), regulate the protein phosphotyrosine levels in cells. Protein-tyrosine phosphorylation is an important post-translational modification that has a major role in cell signaling by affecting protein-protein interactions and enzymatic activities. Increasing evidence indicates that RPTPs, like RPTKs, are regulated by dimerization. For RPTPalpha, we have shown that rotational coupling of the constitutive dimers in the cell membrane determines enzyme activity. Furthermore, oxidative stress, identified as an important second messenger during the past decade, is a regulator of rotational coupling of RPTPalpha dimers. In this review, we discuss the biochemical and cell biological techniques that we use to study the regulation of RPTPs by dimerization. These techniques include (co-) immunoprecipitation, RPTP activity assays, chemical and genetic cross-linking, detection of cell surface proteins by biotinylation, and analysis of RPTPalpha dimers, using conformation-sensitive antibody binding.

  17. Androgen receptor transcriptionally regulates μ-opioid receptor expression in rat trigeminal ganglia.

    PubMed

    Lee, Ki Seok; Zhang, Youping; Asgar, Jamila; Auh, Q-Schick; Chung, Man-Kyo; Ro, Jin Y

    2016-09-01

    The involvement of testosterone in pain, inflammation, and analgesia has been reported, but the role of androgen receptor (AR), a steroid receptor for testosterone, is not well understood. We have previously shown that peripheral inflammation upregulates μ-opioid receptor (MOR) in rat trigeminal ganglia (TG) in a testosterone-dependent manner. In this study, we hypothesized that testosterone regulates MOR expression via transcriptional activities of AR in TG. We first examined whether AR is co-expressed with MOR in TG neurons. Our immunohistochemical experiment revealed that AR staining is detected in neurons of all sizes in TG and that a subset of AR is expressed in MOR as well as in TRPV1-positive neurons. We identified the promoter region of the rat MOR gene contains putative AR binding sites. Using chromatin immunoprecipitation assay, we demonstrated that AR directly binds to these sites in TG extracts. We confirmed with luciferase reporter assay that AR activated the MOR promoter in response to androgens in a human neuroblastoma cell line (5H-5YSY). These data demonstrated that AR functions as a transcriptional regulator of the MOR gene activity. Finally, we showed that flutamide, a specific AR antagonist, prevents complete Freund's adjuvant (CFA)-induced upregulation of MOR mRNA in TG, and that flutamide dose-dependently blocks the efficacy of DAMGO, a specific MOR agonist, on CFA-induced mechanical hypersensitivity. Our results expand the knowledge regarding the role of androgens and their receptor in pain and analgesia and have important clinical implications, particularly for inflammatory pain patients with low or compromised plasma testosterone levels. PMID:27320211

  18. Self-renewal of CD133hi cells by IL6/Notch3 signalling regulates endocrine resistance in metastatic breast cancer

    PubMed Central

    Sansone, Pasquale; Ceccarelli, Claudio; Berishaj, Marjan; Chang, Qing; Rajasekhar, Vinagolu K.; Perna, Fabiana; Bowman, Robert L.; Vidone, Michele; Daly, Laura; Nnoli, Jennifer; Santini, Donatella; Taffurelli, Mario; Shih, Natalie N. C.; Feldman, Michael; Mao, Jun J.; Colameco , Christopher; Chen, Jinbo; DeMichele, Angela; Fabbri, Nicola; Healey, John H.; Cricca, Monica; Gasparre, Giuseppe; Lyden, David; Bonafé, Massimiliano; Bromberg, Jacqueline

    2016-01-01

    The mechanisms of metastatic progression from hormonal therapy (HT) are largely unknown in luminal breast cancer. Here we demonstrate the enrichment of CD133hi/ERlo cancer cells in clinical specimens following neoadjuvant endocrine therapy and in HT refractory metastatic disease. We develop experimental models of metastatic luminal breast cancer and demonstrate that HT can promote the generation of HT-resistant, self-renewing CD133hi/ERlo/IL6hi cancer stem cells (CSCs). HT initially abrogates oxidative phosphorylation (OXPHOS) generating self-renewal-deficient cancer cells, CD133hi/ERlo/OXPHOSlo. These cells exit metabolic dormancy via an IL6-driven feed-forward ERlo-IL6hi-Notchhi loop, activating OXPHOS, in the absence of ER activity. The inhibition of IL6R/IL6-Notch pathways switches the self-renewal of CD133hi CSCs, from an IL6/Notch-dependent one to an ER-dependent one, through the re-expression of ER. Thus, HT induces an OXPHOS metabolic editing of luminal breast cancers, paradoxically establishing HT-driven self-renewal of dormant CD133hi/ERlo cells mediating metastatic progression, which is sensitive to dual targeted therapy. PMID:26858125

  19. Photoperiodic regulation of androgen receptor and steroid receptor coactivator-1 in Siberian hamster brain.

    PubMed

    Tetel, Marc J; Ungar, Todd C; Hassan, Brett; Bittman, Eric L

    2004-11-24

    Seasonal changes in the neuroendocrine actions of gonadal steroid hormones are triggered by fluctuations in daylength. The mechanisms responsible for photoperiodic influences upon the feedback and behavioral effects of testosterone in Siberian hamsters are poorly understood. We hypothesized that daylength regulates the expression of androgen receptor (AR) and/or steroid receptor coactivator-1 (SRC-1) in specific forebrain regions. Hamsters were castrated and implanted with either oil-filled capsules or low doses of testosterone; half of the animals remained in 16L/8D and the rest were kept in 10L/14D for the ensuing 70 days. The number of AR-immunoreactive (AR-ir) cells was regulated by testosterone in medial amygdala and caudal arcuate, and by photoperiod in the medial preoptic nucleus and the posterodorsal medial amygdala. A significant interaction between photoperiod and androgen treatment was found in medial preoptic nucleus and posterodorsal medial amygdala. The molecular weight and distribution of SRC-1 were similar to reports in other rodent species, and short days reduced the number of SRC-1-ir cells in posteromedial bed nucleus of the stria terminalis (BNST) and posterodorsal medial amygdala. A significant interaction between androgen treatment and daylength in regulation of SRC-1-ir was found in anterior medial amygdala. The present results indicate that daylength-induced fluctuations in SRC-1 and AR expression may contribute to seasonally changing effects of testosterone.

  20. RAGE, receptor of advanced glycation endoproducts, negatively regulates chondrocytes differentiation.

    PubMed

    Kosaka, Tatsuya; Fukui, Rino; Matsui, Mio; Kurosaka, Yuko; Nishimura, Haruka; Tanabe, Motoki; Takakura, Yuuki; Iwai, Keisuke; Waki, Takuya; Fujita, Takashi

    2014-01-01

    RAGE, receptor for advanced glycation endoproducts (AGE), has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE) demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA) partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms.

  1. RAGE, Receptor of Advanced Glycation Endoproducts, Negatively Regulates Chondrocytes Differentiation

    PubMed Central

    Kurosaka, Yuko; Nishimura, Haruka; Tanabe, Motoki; Takakura, Yuuki; Iwai, Keisuke; Waki, Takuya; Fujita, Takashi

    2014-01-01

    RAGE, receptor for advanced glycation endoproducts (AGE), has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE) demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA) partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms. PMID:25275461

  2. Regulation of GABAA Receptor Dynamics by Interaction with Purinergic P2X2 Receptors*

    PubMed Central

    Shrivastava, Amulya Nidhi; Triller, Antoine; Sieghart, Werner; Sarto-Jackson, Isabella

    2011-01-01

    γ-Aminobutyric acid type A receptors (GABAARs) in the spinal cord are evolving as an important target for drug development against pain. Purinergic P2X2 receptors (P2X2Rs) are also expressed in spinal cord neurons and are known to cross-talk with GABAARs. Here, we investigated a possible “dynamic” interaction between GABAARs and P2X2Rs using co-immunoprecipitation and fluorescence resonance energy transfer (FRET) studies in human embryonic kidney (HEK) 293 cells along with co-localization and single particle tracking studies in spinal cord neurons. Our results suggest that a significant proportion of P2X2Rs forms a transient complex with GABAARs inside the cell, thus stabilizing these receptors and using them for co-trafficking to the cell surface, where P2X2Rs and GABAARs are primarily located extra-synaptically. Furthermore, agonist-induced activation of P2X2Rs results in a Ca2+-dependent as well as an apparently Ca2+-independent increase in the mobility and an enhanced degradation of GABAARs, whereas P2X2Rs are stabilized and form larger clusters. Antagonist-induced blocking of P2XRs results in co-stabilization of this receptor complex at the cell surface. These results suggest a novel mechanism where association of P2X2Rs and GABAARs could be used for specific targeting to neuronal membranes, thus providing an extrasynaptic receptor reserve that could regulate the excitability of neurons. We further conclude that blocking the excitatory activity of excessively released ATP under diseased state by P2XR antagonists could simultaneously enhance synaptic inhibition mediated by GABAARs. PMID:21343285

  3. Chemokine (C-X-C Motif) Receptor 4 and Atypical Chemokine Receptor 3 Regulate Vascular α1-Adrenergic Receptor Function

    PubMed Central

    Bach, Harold H; Wong, Yee M; Tripathi, Abhishek; Nevins, Amanda M; Gamelli, Richard L; Volkman, Brian F; Byron, Kenneth L; Majetschak, Matthias

    2014-01-01

    Chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor (ACKR) 3 ligands have been reported to modulate cardiovascular function in various disease models. The underlying mechanisms, however, remain unknown. Thus, it was the aim of the present study to determine how pharmacological modulation of CXCR4 and ACKR3 regulate cardiovascular function. In vivo administration of TC14012, a CXCR4 antagonist and ACKR3 agonist, caused cardiovascular collapse in normal animals. During the cardiovascular stress response to hemorrhagic shock, ubiquitin, a CXCR4 agonist, stabilized blood pressure, whereas coactivation of CXCR4 and ACKR3 with CXC chemokine ligand 12 (CXCL12), or blockade of CXCR4 with AMD3100 showed opposite effects. While CXCR4 and ACKR3 ligands did not affect myocardial function, they selectively altered vascular reactivity upon α1-adrenergic receptor (AR) activation in pressure myography experiments. CXCR4 activation with ubiquitin enhanced α1-AR-mediated vasoconstriction, whereas ACKR3 activation with various natural and synthetic ligands antagonized α1-AR-mediated vasoconstriction. The opposing effects of CXCR4 and ACKR3 activation by CXCL12 could be dissected pharmacologically. CXCR4 and ACKR3 ligands did not affect vasoconstriction upon activation of voltage-operated Ca2+ channels or endothelin receptors. Effects of CXCR4 and ACKR3 agonists on vascular α1-AR responsiveness were independent of the endothelium. These findings suggest that CXCR4 and ACKR3 modulate α1-AR reactivity in vascular smooth muscle and regulate hemodynamics in normal and pathological conditions. Our observations point toward CXCR4 and ACKR3 as new pharmacological targets to control vasoreactivity and blood pressure. PMID:25032954

  4. DSL-Notch signaling in the Drosophila brain in response to olfactory stimulation.

    PubMed

    Lieber, Toby; Kidd, Simon; Struhl, Gary

    2011-02-10

    Delta/Serrate/Lag2 (DSL) ligands and their Notch family receptors have profound and pervasive roles in development. They are also expressed in adult tissues, notably in mature neurons and glia in the brain, where their roles are unknown. Here, focusing on the sense of smell in adult Drosophila, we show that Notch is activated in select olfactory receptor neurons (ORNs) in an odorant-specific fashion. This response requires olfactory receptor activity and the Notch ligand Delta. We present evidence that Notch activation depends on synaptic transmission by the ORNs in which the receptors are active and is modulated by the activity of local interneurons in the antennal lobe. It is also subject to regulatory inputs from olfactory receptor activity in other ORNs. These findings identify a correlate of stimulus-dependent brain activity and potentially new forms of neural integration and plasticity.

  5. 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

  6. 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

  7. An exquisite cross-control mechanism among endothelial cell fate regulators directs the plasticity and heterogeneity of lymphatic endothelial cells

    PubMed Central

    Kang, Jinjoo; Yoo, Jaehyuk; Lee, Sunju; Tang, Wanli; Aguilar, Berenice; Ramu, Swapnika; Choi, Inho; Otu, Hasan H.; Shin, Jay W.; Dotto, G. Paolo; Koh, Chester J.; Detmar, Michael

    2010-01-01

    Arteriovenous-lymphatic endothelial cell fates are specified by the master regulators, namely, Notch, COUP-TFII, and Prox1. Whereas Notch is expressed in the arteries and COUP-TFII in the veins, the lymphatics express all 3 cell fate regulators. Previous studies show that lymphatic endothelial cell (LEC) fate is highly plastic and reversible, raising a new concept that all 3 endothelial cell fates may coreside in LECs and a subtle alteration can result in a reprogramming of LEC fate. We provide a molecular basis verifying this concept by identifying a cross-control mechanism among these cell fate regulators. We found that Notch signal down-regulates Prox1 and COUP-TFII through Hey1 and Hey2 and that activated Notch receptor suppresses the lymphatic phenotypes and induces the arterial cell fate. On the contrary, Prox1 and COUP-TFII attenuate vascular endothelial growth factor signaling, known to induce Notch, by repressing vascular endothelial growth factor receptor-2 and neuropilin-1. We show that previously reported podoplanin-based LEC heterogeneity is associated with differential expression of Notch1 in human cutaneous lymphatics. We propose that the expression of the 3 cell fate regulators is controlled by an exquisite feedback mechanism working in LECs and that LEC fate is a consequence of the Prox1-directed lymphatic equilibrium among the cell fate regulators. PMID:20351309

  8. 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

  9. DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur.

    PubMed

    Nichols, James T; Miyamoto, Alison; Olsen, Samantha L; D'Souza, Brendan; Yao, Christine; Weinmaster, Gerry

    2007-02-12

    Cleavage of Notch by furin is required to generate a mature, cell surface heterodimeric receptor that can be proteolytically activated to release its intracellular domain, which functions in signal transduction. Current models propose that ligand binding to heterodimeric Notch (hNotch) induces a disintegrin and metalloprotease (ADAM) proteolytic release of the Notch extracellular domain (NECD), which is subsequently shed and/or endocytosed by DSL ligand cells. We provide evidence for NECD release and internalization by DSL ligand cells, which, surprisingly, did not require ADAM activity. However, losses in either hNotch formation or ligand endocytosis significantly decreased NECD transfer to DSL ligand cells, as well as signaling in Notch cells. Because endocytosis-defective ligands bind hNotch, but do not dissociate it, additional forces beyond those produced through ligand binding must function to disrupt the intramolecular interactions that keep hNotch intact and inactive. Based on our findings, we propose that mechanical forces generated during DSL ligand endocytosis function to physically dissociate hNotch, and that dissociation is a necessary step in Notch activation.

  10. Estrogen Receptor α (ERα) and Estrogen Related Receptor α (ERRα) are both transcriptional regulators of the Runx2-I isoform.

    PubMed

    Kammerer, Martial; Gutzwiller, Sabine; Stauffer, Daniela; Delhon, Isabelle; Seltenmeyer, Yves; Fournier, Brigitte

    2013-04-30

    Runx2 is a master regulator of bone development and has also been described as an oncogene. Estrogen Receptor α (ERα) and Estrogen Related Receptor α (ERRα), both implicated in bone metabolism and breast cancer, have been shown to share common transcriptional targets. Here, we show that ERα is a positive regulator of Runx2-I transcription. Moreover, ERRα can act as a transcriptional activator of Runx2-I in presence of peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1α). In contrast, ERRα behaves as a negative regulator of Runx2-I transcription in presence of PGC-1β. ERα and ERRα cross-talk via a common estrogen receptor response element on the Runx2-I promoter. In addition, estrogen regulates PGC-1β that in turn is able to modulate both ERα and ERRα transcriptional activity.

  11. Liver X Receptor β and Peroxisome Proliferator-Activated Receptor δ regulate cholesterol transport in cholangiocytes

    PubMed Central

    Xia, Xuefeng; Jung, Dongju; Webb, Paul; Zhang, Aijun; Zhang, Bin; Li, Lifei; Ayers, Stephen D.; Gabbi, Chiara; Ueno, Yoshiyuki; Gustafsson, Jan-Åke; Alpini, Gianfranco; Moore, David D.; LeSage, Gene D.

    2012-01-01

    Nuclear receptors (NRs) play crucial roles in regulation of hepatic cholesterol synthesis, metabolism and conversion to bile acids, but their actions in cholangiocytes have not been examined. In this study, we investigated the roles of NRs in cholangiocyte physiology and cholesterol metabolism and flux. We examined the expression of NRs and other genes involved in cholesterol homeostasis in freshly isolated and cultured rodent cholangiocytes and found that these cells express a specific subset of NRs which includes Liver X Receptor β (LXRβ) and Peroxisome Proliferator-Activated Receptor δ (PPARδ). Activation of LXRβ and/or PPARδ in cholangiocytes induces ATP-binding cassette cholesterol transporter A1 (ABCA1) and increases cholesterol export at the basolateral compartment in polarized cultured cholangiocytes. In addition, PPARδ induces Niemann Pick C1 Like L1 (NPC1L1), which imports cholesterol into cholangiocytes and is expressed on the apical cholangiocyte membrane, via specific interaction with a PPRE within the NPC1L1 promoter. Based on these studies, we propose that (i) LXRβ and PPARδ coordinate NPC1L1/ABCA1 dependent vectorial cholesterol flux from bile through cholangiocytes and (ii) manipulation of these processes may influence bile composition with important applications in cholestatic liver disease and gallstone disease, serious health concerns for humans. PMID:22729460

  12. Vascular endothelial growth factor receptor 3 directly regulates murine neurogenesis

    PubMed Central

    Calvo, Charles-Félix; Fontaine, Romain H.; Soueid, Jihane; Tammela, Tuomas; Makinen, Taija; Alfaro-Cervello, Clara; Bonnaud, Fabien; Miguez, Andres; Benhaim, Lucile; Xu, Yunling; Barallobre, Maria-José; Moutkine, Imane; Lyytikkä, Johannes; Tatlisumak, Turgut; Pytowski, Bronislaw; Zalc, Bernard; Richardson, William; Kessaris, Nicoletta; Garcia-Verdugo, Jose Manuel; Alitalo, Kari; Eichmann, Anne; Thomas, Jean-Léon

    2011-01-01

    Neural stem cells (NSCs) are slowly dividing astrocytes that are intimately associated with capillary endothelial cells in the subventricular zone (SVZ) of the brain. Functionally, members of the vascular endothelial growth factor (VEGF) family can stimulate neurogenesis as well as angiogenesis, but it has been unclear whether they act directly via VEGF receptors (VEGFRs) expressed by neural cells, or indirectly via the release of growth factors from angiogenic capillaries. Here, we show that VEGFR-3, a receptor required for lymphangiogenesis, is expressed by NSCs and is directly required for neurogenesis. Vegfr3:YFP reporter mice show VEGFR-3 expression in multipotent NSCs, which are capable of self-renewal and are activated by the VEGFR-3 ligand VEGF-C in vitro. Overexpression of VEGF-C stimulates VEGFR-3-expressing NSCs and neurogenesis in the SVZ without affecting angiogenesis. Conversely, conditional deletion of Vegfr3 in neural cells, inducible deletion in subventricular astrocytes, and blocking of VEGFR-3 signaling with antibodies reduce SVZ neurogenesis. Therefore, VEGF-C/VEGFR-3 signaling acts directly on NSCs and regulates adult neurogenesis, opening potential approaches for treatment of neurodegenerative diseases. PMID:21498572

  13. Receptor Tyrosine Kinases: Molecular Switches Regulating CNS Axon Regeneration

    PubMed Central

    Vigneswara, Vasanthy; Kundi, Sarina; Ahmed, Zubair

    2012-01-01

    The poor or lack of injured adult central nervous system (CNS) axon regeneration results in devastating consequences and poor functional recovery. The interplay between the intrinsic and extrinsic factors contributes to robust inhibition of axon regeneration of injured CNS neurons. The insufficient or lack of trophic support for injured neurons is considered as one of the major obstacles contributing to their failure to survive and regrow their axons after injury. In the CNS, many of the signalling pathways associated with neuronal survival and axon regeneration are regulated by several classes of receptor tyrosine kinases (RTK) that respond to a variety of ligands. This paper highlights and summarises the most relevant recent findings pertinent to different classes of the RTK family of molecules, with a particular focus on elucidating their role in CNS axon regeneration. PMID:22848811

  14. Structures and regulation of non-X orphan nuclear receptors: A retinoid hypothesis.

    PubMed

    Zhi, Xiaoyong; Zhou, X Edward; Melcher, Karsten; Xu, H Eric

    2016-03-01

    Nuclear receptors are defined as a family of ligand regulated transcription factors [1-6]. While this definition reflects that ligand binding is a key property of nuclear receptors, it is still a heated subject of debate if all the nuclear receptors (48 human members) can bind ligands (ligands referred here to both physiological and synthetic ligands). Recent studies in nuclear receptor structure biology and pharmacology have undoubtedly increased our knowledge of nuclear receptor functions and their regulation. As a result, they point to new avenues for the discovery and development of nuclear receptor regulators, including nuclear receptor ligands. Here we review the recent literature on orphan nuclear receptor structural analysis and ligand identification, particularly on the orphan nuclear receptors that do not heterodimerize with retinoid X receptors, which we term as non-X orphan receptors. We also propose a speculative "retinoid hypothesis" for a subset of non-X orphan nuclear receptors, which we hope to help shed light on orphan nuclear receptor biology and drug discovery. This article is part of a Special Issue entitled 'Orphan Nuclear Receptors'.

  15. Structures and regulation of non-X orphan nuclear receptors: A retinoid hypothesis.

    PubMed

    Zhi, Xiaoyong; Zhou, X Edward; Melcher, Karsten; Xu, H Eric

    2016-03-01

    Nuclear receptors are defined as a family of ligand regulated transcription factors [1-6]. While this definition reflects that ligand binding is a key property of nuclear receptors, it is still a heated subject of debate if all the nuclear receptors (48 human members) can bind ligands (ligands referred here to both physiological and synthetic ligands). Recent studies in nuclear receptor structure biology and pharmacology have undoubtedly increased our knowledge of nuclear receptor functions and their regulation. As a result, they point to new avenues for the discovery and development of nuclear receptor regulators, including nuclear receptor ligands. Here we review the recent literature on orphan nuclear receptor structural analysis and ligand identification, particularly on the orphan nuclear receptors that do not heterodimerize with retinoid X receptors, which we term as non-X orphan receptors. We also propose a speculative "retinoid hypothesis" for a subset of non-X orphan nuclear receptors, which we hope to help shed light on orphan nuclear receptor biology and drug discovery. This article is part of a Special Issue entitled 'Orphan Nuclear Receptors'. PMID:26159912

  16. Orphan Nuclear Receptor Estrogen-Related Receptor γ (ERRγ) Is Key Regulator of Hepatic Gluconeogenesis*

    PubMed Central

    Kim, Don-Kyu; Ryu, Dongryeol; Koh, Minseob; Lee, Min-Woo; Lim, Donghyun; Kim, Min-Jung; Kim, Yong-Hoon; Cho, Won-Jea; Lee, Chul-Ho; Park, Seung Bum; Koo, Seung-Hoi; Choi, Hueng-Sik

    2012-01-01

    Glucose homeostasis is tightly controlled by hormonal regulation of hepatic glucose production. Dysregulation of this system is often associated with insulin resistance and diabetes, resulting in hyperglycemia in mammals. Here, we show that the orphan nuclear receptor estrogen-related receptor γ (ERRγ) is a novel downstream mediator of glucagon action in hepatic gluconeogenesis and demonstrate a beneficial impact of the inverse agonist GSK5182. Hepatic ERRγ expression was increased by fasting-dependent activation of the cAMP-response element-binding protein-CRTC2 pathway. Overexpression of ERRγ induced Pck1 and G6PC gene expression and glucose production in primary hepatocytes, whereas abolition of ERRγ gene expression attenuated forskolin-mediated induction of gluconeogenic gene expression. Deletion and mutation analyses of the Pck1 promoter showed that ERRγ directly regulates the Pck1 gene transcription via ERR response elements of the Pck1 promoter as confirmed by ChIP assay and in vivo imaging analysis. We also demonstrate that GSK5182, an inverse agonist of ERRγ, specifically inhibits the transcriptional activity of ERRγ in a PGC-1α dependent manner. Finally, the ERRγ inverse agonist ameliorated hyperglycemia through inhibition of hepatic gluconeogenesis in db/db mice. Control of hepatic glucose production by an ERRγ-specific inverse agonist is a new potential therapeutic approach for the treatment of type 2 diabetes. PMID:22549789

  17. Inverse expression states of the BRN2 and MITF transcription factors in melanoma spheres and tumour xenografts regulate the NOTCH pathway

    PubMed Central

    Thurber, A.E.; Douglas, G.; Sturm, E.C.; Zabierowski, S.E.; Smit, D.J.; Ramakrishnan, S.N.; Hacker, E.; Leonard, J.H.; Herlyn, M.; Sturm, R.A.

    2013-01-01

    The use of adherent monolayer cultures have produced many insights into melanoma cell growth and differentiation, but often novel therapeutics demonstrated to act on these cells are not active in vivo. It is imperative that new methods of growing melanoma cells that reflect growth in vivo are investigated. To this end, a range of human melanoma cell lines passaged as adherent cultures or induced to form melanoma spheres (melanospheres) in stem cell media have been studied to compare cellular characteristics and protein expression. Melanoma spheres and tumours grown from cell lines as mouse xenografts had increased heterogeneity when compared to adherent cells and 3D-spheroids in agar (aggregates). Furthermore, cells within the melanoma spheres and mouse xenografts each displayed a high level of reciprocal BRN2 or MITF expression, which matched more closely the pattern seen in human melanoma tumours in situ, rather than the propensity for co-expression of these important melanocytic transcription factors seen in adherent cells and 3D-spheroids. Notably, when the levels of the BRN2 and MITF proteins were each independently repressed using siRNA treatment of adherent melanoma cells, members of the NOTCH pathway responded by decreasing or increasing expression respectively. This links BRN2 as an activator and conversely MITF as a repressor of the NOTCH pathway in melanoma cells. Loss of the BRN2-MITF axis in antisense ablated cell lines decreased melanoma sphere forming capability, cell adhesion during 3D-spheroid formation, and invasion through a collagen matrix. Combined, this evidence suggests that the melanoma sphere culture system induces subpopulations of cells that may more accurately portray the in vivo disease, than growth as adherent melanoma cells. PMID:21358674

  18. 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

  19. Loss of Notch1-dependent p21Waf1/Cip1 expression influences the Notch1 outcome in tumorigenesis

    PubMed Central

    Cialfi, Samantha; Palermo, Rocco; Manca, Sonia; De Blasio, Carlo; Vargas Romero, Paula; Checquolo, Saula; Bellavia, Diana; Uccelletti, Daniela; Saliola, Michele; D'Alessandro, Angelo; Zolla, Lello; Gulino, Alberto; Screpanti, Isabella; Talora, Claudio

    2014-01-01

    Notch signaling plays a complex role in carcinogenesis, and its signaling pathway has both tumor-suppressor and oncogenic components. In this study we investigated the effects of reactive oxygen species (ROS) on Notch1 signaling outcome in keratinocyte biology. We demonstrate that Notch1 function contributes to the arsenic-induced keratinocyte transformation. We found that acute exposure to arsenite increases oxidative stress and inhibits proliferation of keratinocyte cells by upregulation of p21waf1/Cip1. The necessity of p21waf1/Cip1 for arsenite-induced cell death was demonstrated by targeted downregulation of p21waf1/Cip1 by using RNA interference. We further demonstrated that on acute exposure to arsenite, p21waf1/Cip1 is upregulated and Notch1 downmodulated, whereas on chronic exposure to arsenite, malignant progression of arsenite-treated keratinocytes cells was accompanied by regained expression and activity of Notch1. Notch1 activity in arsenite-transformed keratinocytes inhibits arsenite-induced upregulation of p21waf1/Cip1 by sustaining c-myc expression. We further demonstrated that c-myc collaborates with Nrf2, a key regulator for the maintenance of redox homeostasis, to promote metabolic activities that support cell proliferation and cytoprotection. Therefore, Notch1-mediated repression of p21waf1/Cip1 expression results in the inhibition of cell death and keratinocytes transformation. Our results not only demonstrate that sustained Notch1 expression is at least one key event implicated in the arsenite human skin carcinogenic effect, but also may provide mechanistic insights into the molecular aspects that determine whether Notch signaling will be either oncogenic or tumor suppressive. PMID:24801890

  20. notch3 is essential for oligodendrocyte development and vascular integrity in zebrafish

    PubMed Central

    Zaucker, Andreas; Mercurio, Sara; Sternheim, Nitzan; Talbot, William S.; Marlow, Florence L.

    2013-01-01

    abnormalities observed in heterozygous larvae and adults. Our analysis of zebrafish notch3 mutants indicates that Notch3 regulates OPC development and mbp gene expression in larvae, and maintains vascular integrity in adults. PMID:23720232

  1. Regulation of somatostatins and their receptors in fish.

    PubMed

    Nelson, Laura E; Sheridan, Mark A

    2005-05-15

    The multifunctional nature of the somatostatin (SS) family of peptides results from a multifaceted signaling system consisting of many forms of SS peptides that bind to a variety of receptor (SSTR) subtypes. Research in fish has contributed important information about the components, function, evolution, and regulation of this system. Somatostatins or mRNAs encoding SSs have been isolated from over 20 species of fish. Peptides and deduced peptides differ in their amino acid chain length and/or composition, and most species of fish possess more than one form of SS. The structural heterogeneity of SSs results from differential processing of the hormone precursor, preprosomatostatin (PPSS), and from the existence of multiple genes that give rise to multiple PPSSs. The PPSS genes appear to have arisen through a series of gene duplication events over the course of vertebrate evolution. The numerous PPSSs of fish are differentially expressed, both in terms of the distribution among tissues and in terms of the relative abundance within a tissue. Accumulated evidence suggests that nutritional state, season/stage of sexual maturation, and many hormones [insulin (INS), glucagon, growth hormone (GH), insulin-like growth factor-I (IGF-I), and 17beta-estradiol (E2)] regulate the synthesis and release of particular SSs. Fish and mammals possess multiple SSTRs; four different SSTRs have been described in fish and several of these occur as isoforms. SSTRs are also wide spread and are differentially expressed, both in terms of distribution of tissues as well as in terms of relative abundance within tissues. The pattern of distribution of SSTRs may underlie tissue-specific responses of SSs. The synthesis of SSTR mRNA and SS-binding capacity are regulated by nutritional state and numerous hormones (INS, GH, IGF-I, and E2). Accumulated evidence suggests the possibility of both tissue- and subtype-specific mechanisms of regulation. In many instances, there appears to be coordinate

  2. Progesterone receptor membrane component-1 regulates hepcidin biosynthesis

    PubMed Central

    Li, Xiang; Rhee, David K.; Malhotra, Rajeev; Mayeur, Claire; Hurst, Liam A.; Ager, Emily; Shelton, Georgia; Kramer, Yael; McCulloh, David; Keefe, David; Bloch, Kenneth D.; Bloch, Donald B.; Peterson, Randall T.

    2015-01-01

    Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism. PMID:26657863

  3. Androgen receptor regulates SRC expression through microRNA-203.

    PubMed

    Siu, Man Kit; Chen, Wei-Yu; Tsai, Hong-Yuan; Yeh, Hsiu-Lien; Yin, Juan Juan; Liu, Shih-Yang; Liu, Yen-Nien

    2016-05-01

    The SRC kinase has pivotal roles in multiple developmental processes and in tumor progression. An inverse relationship has been observed between androgen receptor (AR) activity and SRC signaling in advanced prostate cancer (PCa); however, the modulation of AR/SRC crosstalk that leads to metastatic PCa is unclear. Here, we showed that patients with high SRC levels displayed correspondingly low canonical AR gene signatures. Our results demonstrated that activated AR induced miR-203 and reduced SRC levels in PCa model systems. miR-203 directly binds to the 3' UTR of SRC and regulates the stability of SRC mRNA upon AR activation. Moreover, we found that progressive PCa cell migration and growth were associated with a decrease in AR-regulated miR-203 and an increase in SRC. Relationships among AR, miR-203, and SRC were also confirmed in clinical datasets and specimens. We suggest that the induction of SRC results in increased PCa metastasis that is linked to the dysregulation of the AR signaling pathway through the inactivation of miR-203.

  4. Progesterone receptor membrane component-1 regulates hepcidin biosynthesis.

    PubMed

    Li, Xiang; Rhee, David K; Malhotra, Rajeev; Mayeur, Claire; Hurst, Liam A; Ager, Emily; Shelton, Georgia; Kramer, Yael; McCulloh, David; Keefe, David; Bloch, Kenneth D; Bloch, Donald B; Peterson, Randall T

    2016-01-01

    Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism. PMID:26657863

  5. Androgen receptor regulates SRC expression through microRNA-203

    PubMed Central

    Tsai, Hong-Yuan; Yeh, Hsiu-Lien; Yin, Juan Juan; Liu, Shih-Yang; Liu, Yen-Nien

    2016-01-01

    The SRC kinase has pivotal roles in multiple developmental processes and in tumor progression. An inverse relationship has been observed between androgen receptor (AR) activity and SRC signaling in advanced prostate cancer (PCa); however, the modulation of AR/SRC crosstalk that leads to metastatic PCa is unclear. Here, we showed that patients with high SRC levels displayed correspondingly low canonical AR gene signatures. Our results demonstrated that activated AR induced miR-203 and reduced SRC levels in PCa model systems. miR-203 directly binds to the 3′ UTR of SRC and regulates the stability of SRC mRNA upon AR activation. Moreover, we found that progressive PCa cell migration and growth were associated with a decrease in AR-regulated miR-203 and an increase in SRC. Relationships among AR, miR-203, and SRC were also confirmed in clinical datasets and specimens. We suggest that the induction of SRC results in increased PCa metastasis that is linked to the dysregulation of the AR signaling pathway through the inactivation of miR-203. PMID:27028864

  6. Transient receptor potential channels and regulation of lung endothelial permeability

    PubMed Central

    2013-01-01

    Abstract This review highlights our current knowledge regarding expression of transient receptor potential (TRP) cation channels in lung endothelium and evidence for their involvement in regulation of lung endothelial permeability. Six mammalian TRP families have been identified and organized on the basis of sequence homology: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPML (mucolipin), TRPP (polycystin), and TRPA (ankyrin). To date, only TRPC1/4, TRPC6, TRPV4, and TRPM2 have been extensively studied in lung endothelium. Calcium influx through each of these channels has been documented to increase lung endothelial permeability, although their channel-gating mechanisms, downstream signaling mechanisms, and impact on endothelial structure and barrier integrity differ. While other members of the TRPC, TRPV, and TRPM families may be expressed in lung endothelium, we have little or no evidence linking these to regulation of lung endothelial permeability. Further, neither the expression nor functional role(s) of any TRPML, TRPP, and TRPA family members has been studied in lung endothelium. In addition to this assessment organized by TRP channel family, we also discuss TRP channels and lung endothelial permeability from the perspective of lung endothelial heterogeneity, using outcomes of studies focused on TRPC1/4 and TRPV4 channels. The diversity within the TRP channel family and the relative paucity of information regarding roles of a number of these channels in lung endothelium make this field ripe for continued investigation. PMID:25006396

  7. CaMKII regulation of cardiac ryanodine receptors and inositol triphosphate receptors.

    PubMed

    Camors, Emmanuel; Valdivia, Héctor H

    2014-01-01

    Ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3Rs) are structurally related intracellular calcium release channels that participate in multiple primary or secondary amplified Ca(2+) signals, triggering muscle contraction and oscillatory Ca(2+) waves, or activating transcription factors. In the heart, RyRs play an indisputable role in the process of excitation-contraction coupling as the main pathway for Ca(2+) release from sarcoplasmic reticulum (SR), and a less prominent role in the process of excitation-transcription coupling. Conversely, InsP3Rs are believed to contribute in subtle ways, only, to contraction of the heart, and in more important ways to regulation of transcription factors. Because uncontrolled activity of either RyRs or InsP3Rs may elicit life-threatening arrhythmogenic and/or remodeling Ca(2+) signals, regulation of their activity is of paramount importance for normal cardiac function. Due to their structural similarity, many regulatory factors, accessory proteins, and post-translational processes are equivalent for RyRs and InsP3Rs. Here we discuss regulation of RyRs and InsP3Rs by CaMKII phosphorylation, but touch on other kinases whenever appropriate. CaMKII is emerging as a powerful modulator of RyR and InsP3R activity but interestingly, some of the complexities and controversies surrounding phosphorylation of RyRs also apply to InsP3Rs, and a clear-cut effect of CaMKII on either channel eludes investigators for now. Nevertheless, some effects of CaMKII on global cellular activity, such as SR Ca(2+) leak or force-frequency potentiation, appear clear now, and this constrains the limits of the controversies and permits a more tractable approach to elucidate the effects of phosphorylation at the single channel level.

  8. The novel plasminogen receptor, plasminogen receptor(KT) (Plg-R(KT)), regulates catecholamine release.

    PubMed

    Bai, Hongdong; Baik, Nagyung; Kiosses, William B; Krajewski, Stan; Miles, Lindsey A; Parmer, Robert J

    2011-09-23

    Neurotransmitter release by catecholaminergic cells is negatively regulated by prohormone cleavage products formed from plasmin-mediated proteolysis. Here, we investigated the expression and subcellular localization of Plg-R(KT), a novel plasminogen receptor, and its role in catecholaminergic cell plasminogen activation and regulation of catecholamine release. Prominent staining with anti-Plg-R(KT) mAb was observed in adrenal medullary chromaffin cells in murine and human tissue. In Western blotting, Plg-R(KT) was highly expressed in bovine adrenomedullary chromaffin cells, human pheochromocytoma tissue, PC12 pheochromocytoma cells, and murine hippocampus. Expression of Plg-R(KT) fused in-frame to GFP resulted in targeting of the GFP signal to the cell membrane. Phase partitioning, co-immunoprecipitation with urokinase-type plasminogen activator receptor (uPAR), and FACS analysis with antibody directed against the C terminus of Plg-R(KT) were consistent with Plg-R(KT) being an integral plasma membrane protein on the surface of catecholaminergic cells. Cells stably overexpressing Plg-R(KT) exhibited substantial enhancement of plasminogen activation, and antibody blockade of non-transfected PC12 cells suppressed plasminogen activation. In functional secretion assays, nicotine-evoked [(3)H]norepinephrine release from cells overexpressing Plg-R(KT) was markedly decreased (by 51 ± 2%, p < 0.001) when compared with control transfected cells, and antibody blockade increased [(3)H]norepinephrine release from non-transfected PC12 cells. In summary, Plg-R(KT) is present on the surface of catecholaminergic cells and functions to stimulate plasminogen activation and modulate catecholamine release. Plg-R(KT) thus represents a new mechanism and novel control point for regulating the interface between plasminogen activation and neurosecretory cell function.

  9. Perilipin, a critical regulator of fat storage and breakdown, is a target gene of estrogen receptor-related receptor {alpha}

    SciTech Connect

    Akter, Mst. Hasina; Yamaguchi, Tomohiro; Hirose, Fumiko; Osumi, Takashi

    2008-04-11

    Perilipin is a protein localized on lipid droplet surfaces in adipocytes and steroidogenic cells, playing a central role in regulated lipolysis. Expression of the perilipin gene is markedly induced during adipogenesis. We found that transcription from the perilipin gene promoter is activated by an orphan nuclear receptor, estrogen receptor-related receptor (ERR){alpha}. A response element to this receptor was identified in the promoter region by a gene reporter assay, the electrophoretic-gel mobility-shift assay and the chromatin immunoprecipitation assay. Peroxisome proliferator-activated receptor {gamma} coactivator (PGC)-1{alpha} enhanced, whereas small heterodimer partner (SHP) repressed, the transactivating function of ERR{alpha} on the promoter. Thus, the perilipin gene expression is regulated by a transcriptional network controlling energy metabolism, substantiating the functional importance of perilipin in the maintenance of body energy balance.

  10. Down-regulation of the chemokine receptor CCR5 by activation of chemotactic formyl peptide receptor in human monocytes.

    PubMed

    Shen, W; Li, B; Wetzel, M A; Rogers, T J; Henderson, E E; Su, S B; Gong, W; Le, Y; Sargeant, R; Dimitrov, D S; Oppenheim, J J; Wang, J M

    2000-10-15

    Interactions between cell surface receptors are important regulatory elements in the complex host responses to infections. In this study, it is shown that a classic chemotactic factor, the bacterial chemotactic peptide N-formyl-methionyl-leucylphenyl-alanine (fMLF), rapidly induced a protein-kinase-C-mediated serine phosphorylation and down-regulation of the chemokine receptor CCR5, which serves as a major human immunodeficiency virus (HIV)-1 coreceptor. The fMLF binding to its receptor, formyl peptide receptor (FPR), resulted in significant attenuation of cell responses to CCR5 ligands and in inhibition of HIV-1-envelope-glycoprotein-mediated fusion and infection of cells expressing CD4, CCR5, and FPR. The finding that the expression and function of CCR5 can be regulated by peptides that use an unrelated receptor may provide a novel approach to the design of anti-inflamatory and antiretroviral agents. (Blood. 2000;96:2887-2894)

  11. Liver X Receptors Regulate the Transcriptional Activity of the Glucocorticoid Receptor: Implications for the Carbohydrate Metabolism

    PubMed Central

    Nader, Nancy; Ng, Sinnie Sin Man; Wang, Yonghong; Abel, Brent S.; Chrousos, George P.; Kino, Tomoshige

    2012-01-01

    GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase), a key enzyme of gluconeogenesis, and suppress the immune system through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. Since the actions of these receptors overlap with each other, we evaluated the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXRα/RXRα binding to GREs. Endogenous LXRα/RXRα bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of

  12. Nanoparticle optical notch filters

    NASA Astrophysics Data System (ADS)

    Kasinadhuni, Pradeep Kumar

    Developing novel light blocking products involves the design of a nanoparticle optical notch filter, working on the principle of localized surface plasmon resonance (LSPR). These light blocking products can be used in many applications. One such application is to naturally reduce migraine headaches and light sensitivity. Melanopsin ganglion cells present in the retina of the human eye, connect to the suprachiasmatic nucleus (SCN-the body's clock) in the brain, where they participate in the entrainment of the circadian rhythms. As the Melanopsin ganglion cells are involved in triggering the migraine headaches in photophobic patients, it is necessary to block the part of visible spectrum that activates these cells. It is observed from the action potential spectrum of the ganglion cells that they absorb light ranging from 450-500nm (blue-green part) of the visible spectrum with a λmax (peak sensitivity) of around 480nm (blue line). Currently prescribed for migraine patients is the FL-41 coating, which blocks a broad range of wavelengths, including wavelengths associated with melanopsin absorption. The nanoparticle optical notch filter is designed to block light only at 480nm, hence offering an effective prescription for the treatment of migraine headaches.

  13. Down-regulation of insulin receptors is related to insulin internalization

    SciTech Connect

    Geiger, D.; Carpentier, J.L.; Gorden, P.; Orci, L. )

    1989-11-01

    In the present study, we have tested the influence of inhibition of endocytosis by hypertonic medium on the regulation of cell surface insulin receptors. We show that active internalization of {sup 125}I-insulin is markedly inhibited by hypertonic media and that, in parallel, cell surface invaginations are significantly diminished. These two events are accompanied by a marked inhibition of cell surface insulin receptor down-regulation. These data provide further strong evidence that receptor-mediated endocytosis is the major mechanism by which insulin receptors are regulated at the surface of target cells.

  14. NFAT regulates calcium-sensing receptor-mediated TNF production

    SciTech Connect

    abdullah, huda ismail; Pedraza, Paulina L.; Hao, Shoujin; Rodland, Karin D.; McGiff, John C.; Ferreri, Nicholas R.

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca2+ (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca2+ were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  15. Regulation of μ and δ opioid receptor functions: involvement of cyclin-dependent kinase 5

    PubMed Central

    Beaudry, H; Mercier-Blais, A-A; Delaygue, C; Lavoie, C; Parent, J-L; Neugebauer, W; Gendron, L

    2015-01-01

    Background and Purpose Phosphorylation of δ opioid receptors (DOP receptors) by cyclin-dependent kinase 5 (CDK5) was shown to regulate the trafficking of this receptor. Therefore, we aimed to determine the role of CDK5 in regulating DOP receptors in rats treated with morphine or with complete Freund's adjuvant (CFA). As μ (MOP) and DOP receptors are known to be co-regulated, we also sought to determine if CDK5-mediated regulation of DOP receptors also affects MOP receptor functions. Experimental Approach The role of CDK5 in regulating opioid receptors in CFA- and morphine-treated rats was studied using roscovitine as a CDK inhibitor and a cell-penetrant peptide mimicking the second intracellular loop of DOP receptors (C11-DOPri2). Opioid receptor functions were assessed in vivo in a series of behavioural experiments and correlated by measuring ERK1/2 activity in dorsal root ganglia homogenates. Key Results Chronic roscovitine treatment reduced the antinociceptive and antihyperalgesic effects of deltorphin II (Dlt II) in morphine- and CFA-treated rats respectively. Repeated administrations of C11-DOPri2 also robustly decreased Dlt II-induced analgesia. Interestingly, DAMGO-induced analgesia was significantly increased by roscovitine and C11-DOPri2. Concomitantly, in roscovitine-treated rats the Dlt II-induced ERK1/2 activation was decreased, whereas the DAMGO-induced ERK1/2 activation was increased. An acute roscovitine treatment had no effect on Dlt II- or DAMGO-induced analgesia. Conclusions and Implications Together, our results demonstrate that CDK5 is a key player in the regulation of DOP receptors in morphine- and CFA-treated rats and that the regulation of DOP receptors by CDK5 is sufficient to modulate MOP receptor functions through an indirect process. PMID:25598508

  16. Developmental regulation of human truncated nerve growth factor receptor

    SciTech Connect

    DiStefano, P.S.; Clagett-Dame, M.; Chelsea, D.M.; Loy, R. )

    1991-01-01

    Monoclonal antibodies (designated XIF1 and IIIG5) recognizing distinct epitopes of the human truncated nerve growth factor receptor (NGF-Rt) were used in a two-site radiometric immunosorbent assay to monitor levels of NGF-Rt in human urine as a function of age. Urine samples were collected from 70 neurologically normal subjects ranging in age from 1 month to 68 years. By using this sensitive two-site radiometric immunosorbent assay, NGF-Rt levels were found to be highest in urine from 1-month old subjects. By 2.5 months, NGF-Rt values were half of those seen at 1 month and decreased more gradually between 0.5 and 15 years. Between 15 and 68 years, urine NGF-Rt levels were relatively constant at 5% of 1-month values. No evidence for diurnal variation of adult NGF-Rt was apparent. Pregnant women in their third trimester showed significantly elevated urine NGF-Rt values compared with age-matched normals. Affinity labeling of NGF-Rt with 125I-NGF followed by immunoprecipitation with ME20.4-IgG and gel autoradiography indicated that neonatal urine contained high amounts of truncated receptor (Mr = 50 kd); decreasingly lower amounts of NGF-Rt were observed on gel autoradiograms with development, indicating that the two-site radiometric immunosorbent assay correlated well with the affinity labeling technique for measuring NGF-Rt. NGF-Rt in urines from 1-month-old and 36-year-old subjects showed no differences in affinities for NGF or for the monoclonal antibody IIIG5. These data show that NGF-Rt is developmentally regulated in human urine, and are discussed in relation to the development and maturation of the peripheral nervous system.

  17. Transport regulation of two-dimensional receptor-ligand association.

    PubMed

    Ju, Lining; Qian, Jin; Zhu, Cheng

    2015-04-01

    The impact of flow disturbances on platelet adhesion is complex and incompletely understood. At the molecular scale, platelet glycoprotein Ibα (GPIbα) must associate with the von Willebrand factor A1 domain (VWF-A1) with a rapid on-rate under high hemodynamic forces, as occurs in arterial thrombosis, where various transport mechanisms are at work. Here, we theoretically modeled the coupled transport-reaction process of the two-dimensional (2D) receptor-ligand association kinetics in a biomembrane force probe to explicitly account for the effects of molecular length, confinement stiffness, medium viscosity, surface curvature, and separation distance. We experimentally verified the theoretical approach by visualizing association and dissociation of individual VWF-A1-GPIbα bonds in a real-time thermal fluctuation assay. The apparent on-rate, reciprocal of the average time intervals between sequential bonds, decreased with the increasing gap distance between A1- and GPIbα-bearing surfaces with an 80-nm threshold (beyond which bond formation became prohibitive) identified as the combined contour length of the receptor and ligand molecules. The biomembrane force probe spring constant and diffusivity of the protein-bearing beads also significantly influenced the apparent on-rate, in accordance with the proposed transport mechanisms. The global agreement between the experimental data and the model predictions supports the hypothesis that receptor-ligand association behaves distinctly in the transport- and reaction-limited scenarios. To our knowledge, our results represent the first detailed quantification of physical regulation of the 2D on-rate that allows platelets to sense and respond to local changes in their hemodynamic environment. In addition, they provide an approach for determining the intrinsic kinetic parameters that employs simultaneous experimental measurements and theoretical modeling of bond association in a single assay. The 2D intrinsic forward rate

  18. Transport Regulation of Two-Dimensional Receptor-Ligand Association

    PubMed Central

    Ju, Lining; Qian, Jin; Zhu, Cheng

    2015-01-01

    The impact of flow disturbances on platelet adhesion is complex and incompletely understood. At the molecular scale, platelet glycoprotein Ibα (GPIbα) must associate with the von Willebrand factor A1 domain (VWF-A1) with a rapid on-rate under high hemodynamic forces, as occurs in arterial thrombosis, where various transport mechanisms are at work. Here, we theoretically modeled the coupled transport-reaction process of the two-dimensional (2D) receptor-ligand association kinetics in a biomembrane force probe to explicitly account for the effects of molecular length, confinement stiffness, medium viscosity, surface curvature, and separation distance. We experimentally verified the theoretical approach by visualizing association and dissociation of individual VWF-A1-GPIbα bonds in a real-time thermal fluctuation assay. The apparent on-rate, reciprocal of the average time intervals between sequential bonds, decreased with the increasing gap distance between A1- and GPIbα-bearing surfaces with an 80-nm threshold (beyond which bond formation became prohibitive) identified as the combined contour length of the receptor and ligand molecules. The biomembrane force probe spring constant and diffusivity of the protein-bearing beads also significantly influenced the apparent on-rate, in accordance with the proposed transport mechanisms. The global agreement between the experimental data and the model predictions supports the hypothesis that receptor-ligand association behaves distinctly in the transport- and reaction-limited scenarios. To our knowledge, our results represent the first detailed quantification of physical regulation of the 2D on-rate that allows platelets to sense and respond to local changes in their hemodynamic environment. In addition, they provide an approach for determining the intrinsic kinetic parameters that employs simultaneous experimental measurements and theoretical modeling of bond association in a single assay. The 2D intrinsic forward rate

  19. O-glucose trisaccharide is present at high but variable stoichiometry at multiple sites on mouse Notch1.

    PubMed

    Rana, Nadia A; Nita-Lazar, Aleksandra; Takeuchi, Hideyuki; Kakuda, Shinako; Luther, Kelvin B; Haltiwanger, Robert S

    2011-09-01

    Notch activity is regulated by both O-fucosylation and O-glucosylation, and Notch receptors contain multiple predicted sites for both. Here we examine the occupancy of the predicted O-glucose sites on mouse Notch1 (mN1) using the consensus sequence C(1)XSXPC(2). We show that all of the predicted sites are modified, although the efficiency of modifying O-glucose sites is site- and cell type-dependent. For instance, although most sites are modified at high stoichiometries, the site at EGF 27 is only partially glucosylated, and the occupancy of the site at EGF 4 varies with cell type. O-Glucose is also found at a novel, non-traditional consensus site at EGF 9. Based on this finding, we propose a revision of the consensus sequence for O-glucosylation to allow alanine N-terminal to cysteine 2: C(1)XSX(A/P)C(2). We also show through biochemical and mass spectral analyses that serine is the only hydroxyamino acid that is modified with O-glucose on EGF repeats. The O-glucose at all sites is efficiently elongated to the trisaccharide Xyl-Xyl-Glc. To establish the functional importance of individual O-glucose sites in mN1, we used a cell-based signaling assay. Elimination of most individual sites shows little or no effect on mN1 activation, suggesting that the major effects of O-glucose are mediated by modification of multiple sites. Interestingly, elimination of the site in EGF 28, found in the Abruptex region of Notch, does significantly reduce activity. These results demonstrate that, like O-fucose, the O-glucose modifications of EGF repeats occur extensively on mN1, and they play important roles in Notch function.

  20. M2 muscarinic receptor activation regulates Schwann cell differentiation and myelin organization.

    PubMed

    Uggenti, Carolina; De Stefano, M Egle; Costantino, Michele; Loreti, Simona; Pisano, Annalinda; Avallone, Bice; Talora, Claudio; Magnaghi, Valerio; Tata, Ada Maria

    2014-07-01

    Glial cells express acetylcholine receptors. In particular, rat Schwann cells express different muscarinic receptor subtypes, the most abundant of which is the M2 subtype. M2 receptor activation causes a reversible arrest of the cell cycle. This negative effect on Schwann cell proliferation suggests that these cells may possibly progress into a differentiating program. In this study we analyzed the in vitro modulation, by the M2 agonist arecaidine, of transcription factors and specific signaling pathways involved in Schwann cell differentiation. The arecaidine-induced M2 receptor activation significantly upregulates transcription factors involved in the promyelinating phase (e.g., Sox10 and Krox20) and downregulates proteins involved in the maintenance of the undifferentiated state (e.g., c-jun, Notch-1, and Jagged-1). Furthermore, arecaidine stimulation significantly increases the expression of myelin proteins, which is accompanied by evident changes in cell morphology, as indicated by electron microscopy analysis, and by substantial cellular re-distribution of actin and cell adhesion molecules. Moreover, ultrastructural and morphometric analyses on sciatic nerves of M2/M4 knockout mice show numerous degenerating axons and clear alterations in myelin organization compared with wild-type mice. Therefore, our data demonstrate that acetylcholine mediates axon-glia cross talk, favoring Schwann cell progression into a differentiated myelinating phenotype and contributing to compact myelin organization.

  1. 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

  2. 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.

  3. 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

  4. Regulation of renal peripheral benzodiazepine receptors by anion transport inhibitors

    SciTech Connect

    Basile, A.S.; Lueddens, W.M.; Skolnick, P.

    1988-01-01

    The in vitro and in vivo regulation of (/sup 3/H)Ro 5-4864 binding to peripheral benzodiazepine receptors (PBR) by ion transport/exchange inhibitors was studied in the kidney. The potencies of 9-anthroic acid, furosemide, bumetanide, hydrochlorothiazide and SITS as inhibitors of (/sup 3/H)Ro 5-4864 binding to renal membranes were consistent with their actions as anion transport inhibitors (Ki approx. = 30 - 130 ..mu..M). In contrast, spironolactone, amiloride, acetazolamide, and ouabain were less potent (Ki=100-1000 ..mu..M). Administration of furosemide to rats for five days resulted in a profound diuresis accompanied by a significant increase in PBR density (43%) that was apparent by the fifth day of treatment. Administration of hydrochlorothiazide or Ro 5-4864 for five days also caused diuresis and increased renal PBR density. Both the diuresis and increased density of PBR produced by Ro 5-4864 were blocked by coadministration of PK 11195, which alone had no effect on either PBR density or urine volume. The equilibrium binding constants of (/sup 3/H)Ro 5-4864 to cardiac membranes were unaffected by administration of any of these drugs. These findings suggest that renal PBR may be selectively modulated in vivo and in vitro by administration of ion transport/exchange inhibitors. 36 references, 4 tables.

  5. Perineuronal net, CSPG receptor and their regulation of neural plasticity.

    PubMed

    Miao, Qing-Long; Ye, Qian; Zhang, Xiao-Hui

    2014-08-25

    Perineuronal nets (PNNs) are reticular structures resulting from the aggregation of extracellular matrix (ECM) molecules around the cell body and proximal neurite of specific population of neurons in the central nervous system (CNS). Since the first description of PNNs by Camillo Golgi in 1883, the molecular composition, developmental formation and potential functions of these specialized extracellular matrix structures have only been intensively studied over the last few decades. The main components of PNNs are hyaluronan (HA), chondroitin sulfate proteoglycans (CSPGs) of the lectican family, link proteins and tenascin-R. PNNs appear late in neural development, inversely correlating with the level of neural plasticity. PNNs have long been hypothesized to play a role in stabilizing the extracellular milieu, which secures the characteristic features of enveloped neurons and protects them from the influence of malicious agents. Aberrant PNN signaling can lead to CNS dysfunctions like epilepsy, stroke and Alzheimer's disease. On the other hand, PNNs create a barrier which constrains the neural plasticity and counteracts the regeneration after nerve injury. Digestion of PNNs with chondroitinase ABC accelerates functional recovery from the spinal cord injury and restores activity-dependent mechanisms for modifying neuronal connections in the adult animals, indicating that PNN is an important regulator of neural plasticity. Here, we review recent progress in the studies on the formation of PNNs during early development and the identification of CSPG receptor - an essential molecular component of PNN signaling, along with a discussion on their unique regulatory roles in neural plasticity.

  6. The aryl hydrocarbon receptor (AHR) transcription factor regulates megakaryocytic polyploidization

    PubMed Central

    Lindsey, Stephan; T. Papoutsakis, Eleftherios

    2012-01-01

    Summary We propose that the aryl hydrocarbon receptor (AHR) is a novel transcriptional regulator of megakaryopoietic polyploidization. Functional evidence was obtained that AHR impacts in vivo megakaryocytic differentiation and maturation; compared to wild-type mice, AHR-null mice had lower platelet counts, fewer numbers of newly synthesized platelets, increased bleeding times and lower-ploidy megakaryocytes (Mks). AHR mRNA increased 3·6-fold during ex vivo megakaryocytic differentiation, but reduced or remained constant during parallel isogenic granulocytic or erythroid differentiation. We interrogated the role of AHR in megakaryopoiesis using a validated Mk model of megakaryopoiesis, the human megakaryoblastic leukaemia CHRF cell line. Upon CHRF Mk differentiation, AHR mRNA and protein levels increased, AHR protein shifted from the cytoplasm to the nucleus and AHR binding to its consensus DNA binding sequence increased. Protein and mRNA levels of the AHR transcriptional target HES1 also increased. Mk differentiation of CHRF cells where AHR or HES1 was knocked-down using RNAi resulted in lower ploidy distributions and cells that were incapable of reaching ploidy classes ≥16n. AHR knockdown also resulted in increased DNA synthesis of lower ploidy cells, without impacting apoptosis. Together, these data support a role for AHR in Mk polyploidization and in vivo platelet function, and warrant further detailed investigations. PMID:21226706

  7. Regulation of T-cell receptor signalling by membrane microdomains

    PubMed Central

    Razzaq, Tahir M; Ozegbe, Patricia; Jury, Elizabeth C; Sembi, Phupinder; Blackwell, Nathan M; Kabouridis, Panagiotis S

    2004-01-01

    There is now considerable evidence suggesting that the plasma membrane of mammalian cells is compartmentalized by functional lipid raft microdomains. These structures are assemblies of specialized lipids and proteins and have been implicated in diverse biological functions. Analysis of their protein content using proteomics and other methods revealed enrichment of signalling proteins, suggesting a role for these domains in intracellular signalling. In T lymphocytes, structure/function experiments and complementary pharmacological studies have shown that raft microdomains control the localization and function of proteins which are components of signalling pathways regulated by the T-cell antigen receptor (TCR). Based on these studies, a model for TCR phosphorylation in lipid rafts is presented. However, despite substantial progress in the field, critical questions remain. For example, it is unclear if membrane rafts represent a homogeneous population and if their structure is modified upon TCR stimulation. In the future, proteomics and the parallel development of complementary analytical methods will undoubtedly contribute in further delineating the role of lipid rafts in signal transduction mechanisms. PMID:15554919

  8. A neural basis for melanocortin-4 receptor regulated appetite

    PubMed Central

    Garfield, Alastair S.; Li, Chia; Madara, Joseph C.; Shah, Bhavik P.; Webber, Emily; Steger, Jennifer S.; Campbell, John N.; Gavrilova, Oksana; Lee, Charlotte E.; Olson, David P.; Elmquist, Joel K.; Tannous, Bakhos A.; Krashes, Michael J.; Lowell, Bradford B.

    2015-01-01

    Pro-opiomelanocortin (POMC)- and agouti-related peptide (AgRP)-expressing neurons are oppositely regulated by caloric depletion and co-ordinately stimulate and inhibit homeostatic satiety, respectively. This bimodality is principally underscored by the antagonistic actions of these ligands at downstream melanocortin-4 receptors (MC4R) within the paraventricular nucleus of the hypothalamus. Although this population is critical to energy balance the underlying neural circuitry remains unknown. Enabled by mice expressing Cre-recombinase in MC4R neurons, we demonstrate bidirectional control of feeding following real-time activation and inhibition of PVHMC4R neurons and further identify these cells as a functional exponent of ARCAgRP neuron-driven hunger. Moreover, we reveal this function to be mediated by a PVHMC4R→lateral parabrachial nucleus (LPBN) pathway. Activation of this circuit encodes positive valence, but only in calorically depleted mice. Thus, the satiating and appetitive nature of PVHMC4R→LPBN neurons supports the principles of drive reduction and highlights this circuit as a promising target for anti-obesity drug development. PMID:25915476

  9. Reduction of NOTCH1 expression pertains to maturation abnormalities of keratinocytes in squamous neoplasms.

    PubMed

    Sakamoto, Kei; Fujii, Takuma; Kawachi, Hiroshi; Miki, Yoshio; Omura, Ken; Morita, Kei-ichi; Kayamori, Kou; Katsube, Ken-ichi; Yamaguchi, Akira

    2012-05-01

    Notch is a transmembrane receptor functioning in the determination of cell fate. Abnormal Notch signaling promotes tumor development, showing either oncogenic or tumor suppressive activity. The uncertainty about the exact role of Notch signaling, partially, stems from inconsistencies in descriptions of Notch expression in human cancers. Here, we clarified basal-cell dominant expression of NOTCH1 in squamous epithelium. NOTCH1 was downregulated in squamous neoplasms of oral mucosa, esophagus and uterine cervix, compared with the normal basal cells, although the expression tended to be retained in cervical lesions. NOTCH1 downregulation was observed even in precancers, and there was little difference between cancers and high-grade precancerous lesions, suggesting its minor contribution to cancer-specific events such as invasion. In culture experiments, reduction of NOTCH1 expression resulted in downregulation of keratin 13 and keratin 15, and upregulation of keratin 17, and NOTCH1 knockdown cells formed a dysplastic stratified epithelium mimicking a precancerous lesion. The NOTCH1 downregulation and the concomitant alterations of those keratin expressions were confirmed in the squamous neoplasms both by immunohistochemical and cDNA microarray analyses. Our data indicate that reduction of NOTCH1 expression directs the basal cells to cease terminal differentiation and to form an immature epithelium, thereby playing a major role in the histopathogenesis of epithelial dysplasia. Furthermore, downregulation of NOTCH1 expression seems to be an inherent mechanism for switching the epithelium from a normal and mature state to an activated and immature state, suggesting its essential role in maintaining the epithelial integrity.

  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. Down-regulation of phospholipase C-beta1 following chronic muscarinic receptor activation.

    PubMed

    Sorensen, S D; Linseman, D A; Fisher, S K

    1998-04-01

    To determine whether prolonged activation of a phospholipase C-coupled receptor can lead to a down-regulation of its effector enzyme, SH-SY5Y neuroblastoma cells were incubated for 24 h with the muscarinic receptor agonist, oxotremorine-M. Under these conditions, significant reductions (46-53%) in muscarinic cholinergic receptor density, G(alphaq/11) and phospholipase C-beta1 (but not the beta3-or gamma1 isoforms) were observed. These results suggest that a selective down-regulation of phospholipase C-beta1 may play a role in adaptation to chronic muscarinic receptor activation. PMID:9617763

  12. Abnormal recruitment of extracellular matrix proteins by excess Notch3 ECD: a new pathomechanism in CADASIL.

    PubMed

    Monet-Leprêtre, Marie; Haddad, Iman; Baron-Menguy, Céline; Fouillot-Panchal, Maï; Riani, Meriem; Domenga-Denier, Valérie; Dussaule, Claire; Cognat, Emmanuel; Vinh, Joelle; Joutel, Anne

    2013-06-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the NOTCH3 receptor (Notch3(ECD)) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3(ECD)-containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel pathology. Herein, we hypothesized that excess Notch3(ECD) could recruit and sequester functionally important proteins within small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immunohistochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and artery samples demonstrated that mutant Notch3(ECD) accumulates in disulphide cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3(ECD)-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the formation of Notch3(ECD)-TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of both insoluble cross

  13. The dicrotic notch analyzed by a numerical model.

    PubMed

    Politi, María Teresa; Ghigo, Arthur; Fernández, Juan Manuel; Khelifa, Ismaïl; Gaudric, Julien; Fullana, José María; Lagrée, Pierre-Yves

    2016-05-01

    Divergent concepts on the origin of the dicrotic notch are widespread in medical literature and education. Since most medical textbooks explain the origin of the dicrotic notch as caused by the aortic valve closure itself, this is commonly transmitted in medical physiology courses. We present clinical data and numerical simulations to demonstrate that reflected pressure waves could participate as one of the causes of the dicrotic notch. Our experimental data from continuous arterial pressure measurements from adult patients undergoing vascular surgery suggest that isolated changes in peripheral vascular resistance using an intravenous bolus of phenylephrine (a selective alpha 1-receptor agonist and thus a potent vasoconstrictor) modify the dicrotic notch. We then explore the mechanisms behind this phenomenon by using a numerical model based on integrated axisymmetric Navier-Stokes equations to compute the hemodynamic flow. Our model illustrates clearly how modifications in peripheral artery resistance may result in changes in the amplitude of the dicrotic notch by modifying reflected pressure waves. We believe that this could be a useful tool in teaching medical physiology courses. PMID:27016670

  14. E3 Ubiquitin Ligases Pellinos as Regulators of Pattern Recognition Receptor Signaling and Immune responses

    PubMed Central

    Medvedev, Andrei E.; Murphy, Michael; Zhou, Hao; Li, Xiaoxia

    2015-01-01

    SUMMARY Pellinos are a family of E3 ubiquitin ligases discovered for their role in catalyzing K63-linked polyubiquitination of Pelle, an IL-1 receptor-associated kinase homologue in the Drosophila Toll pathway. Subsequent studies have revealed the central and non-redundant roles of mammalian Pellino-1, Pellino-2 and Pelino-3 in signaling pathways emanating from IL-1 receptors, Toll-like receptors, NOD-like receptors, T- and B-cell receptors. While Pellinos ability to interact with many signaling intermediates suggested their scaffolding roles, recent findings in mice expressing ligase-inactive Pellinos demonstrated the importance of Pellino ubiquitin ligase activity. Cell-specific functions of Pellinos have emerged, e.g., Pellino-1 being a negative regulator in T-lymphocytes and a positive regulator in myeloid cells, and details of molecular regulation of receptor signaling by various members of the Pellino family have been revealed. In this review, we have summarized current information about Pellino-mediated regulation of signaling by pattern recognition receptors, T-cell and B-cell receptors and TNF receptors, and discuss Pellino’s role in sepsis and infectious diseases, as well as in autoimmune, inflammatory and allergic disorders. We also provide our perspective on the potential of targeting Pellinos with peptide- or small molecule-based drug compounds as a new therapeutic approach for septic shock and autoimmune pathologies. PMID:26085210

  15. E3 ubiquitin ligases Pellinos as regulators of pattern recognition receptor signaling and immune responses.

    PubMed

    Medvedev, Andrei E; Murphy, Michael; Zhou, Hao; Li, Xiaoxia

    2015-07-01

    Pellinos are a family of E3 ubiquitin ligases discovered for their role in catalyzing K63-linked polyubiquitination of Pelle, an interleukin-1 (IL-1) receptor-associated kinase homolog in the Drosophila Toll pathway. Subsequent studies have revealed the central and non-redundant roles of mammalian Pellino-1, Pellino-2, and Pelino-3 in signaling pathways emanating from IL-1 receptors, Toll-like receptors, NOD-like receptors, T- and B-cell receptors. While Pellinos ability to interact with many signaling intermediates suggested their scaffolding roles, recent findings in mice expressing ligase-inactive Pellinos demonstrated the importance of Pellino ubiquitin ligase activity. Cell-specific functions of Pellinos have emerged, e.g. Pellino-1 being a negative regulator in T lymphocytes and a positive regulator in myeloid cells, and details of molecular regulation of receptor signaling by various members of the Pellino family have been revealed. In this review, we summarize current information about Pellino-mediated regulation of signaling by pattern recognition receptors, T-cell and B-cell receptors and tumor necrosis factor receptors, and discuss Pellinos roles in sepsis and infectious diseases, as well as in autoimmune, inflammatory, and allergic disorders. We also provide our perspective on the potential of targeting Pellinos with peptide- or small molecule-based drug compounds as a new therapeutic approach for septic shock and autoimmune pathologies.

  16. The F-BAR Protein PACSIN2 Regulates Epidermal Growth Factor Receptor Internalization

    PubMed Central

    de Kreuk, Bart-Jan; Anthony, Eloise C.; Geerts, Dirk; Hordijk, Peter L.

    2012-01-01

    Signaling via growth factor receptors, including the epidermal growth factor (EGF) receptor, is key to various cellular processes, such as proliferation, cell survival, and cell migration. In a variety of human diseases such as cancer, aberrant expression and activation of growth factor receptors can lead to disturbed signaling. Intracellular trafficking is crucial for proper signaling of growth factor receptors. As a result, the level of cell surface expression of growth factor receptors is an important determinant for the outcome of downstream signaling. BAR domain-containing proteins represent an important family of proteins that regulate membrane dynamics. In this study, we identify a novel role for the F-BAR protein PACSIN2 in the regulation of EGF receptor signaling. We show that internalized EGF as well as the (activated) EGF receptor translocated to PACSIN2-positive endosomes. Furthermore, loss of PACSIN2 increased plasma membrane expression of the EGF receptor in resting cells and increased EGF-induced phosphorylation of the EGF receptor. As a consequence, EGF-induced activation of Erk and Akt as well as cell proliferation were enhanced in PACSIN2-depleted cells. In conclusion, this study identifies a novel role for the F-BAR-domain protein PACSIN2 in regulating EGF receptor surface levels and EGF-induced downstream signaling. PMID:23129763

  17. GluN2B-Containing NMDA Receptors Regulate AMPA Receptor Traffic through Anchoring of the Synaptic Proteasome.

    PubMed

    Ferreira, Joana S; Schmidt, Jeannette; Rio, Pedro; Águas, Rodolfo; Rooyakkers, Amanda; Li, Ka Wan; Smit, August B; Craig, Ann Marie; Carvalho, Ana Luisa

    2015-06-01

    NMDA receptors play a central role in shaping the strength of synaptic connections throughout development and in mediating synaptic plasticity mechanisms that underlie some forms of learning and memory formation in the CNS. In the hippocampus and the neocortex, GluN1 is combined primarily with GluN2A and GluN2B, which are differentially expressed during development and confer distinct molecular and physiological properties to NMDA receptors. The contribution of each subunit to the synaptic traffic of NMDA receptors and therefore to their role during development and in synaptic plasticity is still controversial. We report a critical role for the GluN2B subunit in regulating NMDA receptor synaptic targeting. In the absence of GluN2B, the synaptic levels of AMPA receptors are increased and accompanied by decreased constitutive endocytosis of GluA1-AMPA receptor. We used quantitative proteomic analysis to identify changes in the composition of postsynaptic densities from GluN2B(-/-) mouse primary neuronal cultures and found altered levels of several ubiquitin proteasome system components, in particular decreased levels of proteasome subunits. Enhancing the proteasome activity with a novel proteasome activator restored the synaptic levels of AMPA receptors in GluN2B(-/-) neurons and their endocytosis, revealing that GluN2B-mediated anchoring of the synaptic proteasome is responsible for fine tuning AMPA receptor synaptic levels under basal conditions.

  18. Calcium-Sensing Receptor Gene: Regulation of Expression.

    PubMed

    Hendy, Geoffrey N; Canaff, Lucie

    2016-01-01

    The human calcium-sensing receptor gene (CASR) has 8 exons, and localizes to chromosome 3q. Exons 1A and 1B encode alternative 5'-untranslated regions (UTRs) that splice to exon 2 encoding the AUG initiation codon. Exons 2-7 encode the CaSR protein of 1078 amino acids. Promoter P1 has TATA and CCAAT boxes upstream of exon 1A, and promoter P2 has Sp1/3 motifs at the start site of exon 1B. Exon 1A transcripts from the P1 promoter are reduced in parathyroid tumors and colon carcinomas. Studies of colon carcinomas and neuroblastomas have emphasized the importance of epigenetic changes-promoter methylation of the GC-rich P2 promoter, histone acetylation-as well as involvement of microRNAs in bringing about CASR gene silencing and reduced CaSR expression. Functional cis-elements in the CASR promoters responsive to 1,25-dihydroxyvitamin D [1,25(OH)2D], proinflammatory cytokines, and the transcription factor glial cells missing-2 (GCM2) have been characterized. Reduced levels of CaSR and reduced responsiveness to active vitamin D in parathyroid neoplasia and colon carcinoma may blunt the "tumor suppressor" activity of the CaSR. The hypocalcemia of critically ill patients with burn injury or sepsis is associated with CASR gene upregulation by TNF-alpha and IL-1beta via kappaB elements, and by IL-6 via Stat1/3 and Sp1/3 elements in the CASR gene promoters, respectively. The CASR is transactivated by GCM2-the expression of which is essential for parathyroid gland development. Hyperactive forms of GCM2 may contribute to later parathyroid hyperactivity or tumorigenesis. The expression of the CaSR-the calciostat-is regulated physiologically and pathophysiologically at the gene level. PMID:27679579

  19. Calcium-Sensing Receptor Gene: Regulation of Expression

    PubMed Central

    Hendy, Geoffrey N.; Canaff, Lucie

    2016-01-01

    The human calcium-sensing receptor gene (CASR) has 8 exons, and localizes to chromosome 3q. Exons 1A and 1B encode alternative 5′-untranslated regions (UTRs) that splice to exon 2 encoding the AUG initiation codon. Exons 2–7 encode the CaSR protein of 1078 amino acids. Promoter P1 has TATA and CCAAT boxes upstream of exon 1A, and promoter P2 has Sp1/3 motifs at the start site of exon 1B. Exon 1A transcripts from the P1 promoter are reduced in parathyroid tumors and colon carcinomas. Studies of colon carcinomas and neuroblastomas have emphasized the importance of epigenetic changes—promoter methylation of the GC-rich P2 promoter, histone acetylation—as well as involvement of microRNAs in bringing about CASR gene silencing and reduced CaSR expression. Functional cis-elements in the CASR promoters responsive to 1,25-dihydroxyvitamin D [1,25(OH)2D], proinflammatory cytokines, and the transcription factor glial cells missing-2 (GCM2) have been characterized. Reduced levels of CaSR and reduced responsiveness to active vitamin D in parathyroid neoplasia and colon carcinoma may blunt the “tumor suppressor” activity of the CaSR. The hypocalcemia of critically ill patients with burn injury or sepsis is associated with CASR gene upregulation by TNF-alpha and IL-1beta via kappaB elements, and by IL-6 via Stat1/3 and Sp1/3 elements in the CASR gene promoters, respectively. The CASR is transactivated by GCM2—the expression of which is essential for parathyroid gland development. Hyperactive forms of GCM2 may contribute to later parathyroid hyperactivity or tumorigenesis. The expression of the CaSR—the calciostat—is regulated physiologically and pathophysiologically at the gene level. PMID:27679579

  20. Calcium-Sensing Receptor Gene: Regulation of Expression

    PubMed Central

    Hendy, Geoffrey N.; Canaff, Lucie

    2016-01-01

    The human calcium-sensing receptor gene (CASR) has 8 exons, and localizes to chromosome 3q. Exons 1A and 1B encode alternative 5′-untranslated regions (UTRs) that splice to exon 2 encoding the AUG initiation codon. Exons 2–7 encode the CaSR protein of 1078 amino acids. Promoter P1 has TATA and CCAAT boxes upstream of exon 1A, and promoter P2 has Sp1/3 motifs at the start site of exon 1B. Exon 1A transcripts from the P1 promoter are reduced in parathyroid tumors and colon carcinomas. Studies of colon carcinomas and neuroblastomas have emphasized the importance of epigenetic changes—promoter methylation of the GC-rich P2 promoter, histone acetylation—as well as involvement of microRNAs in bringing about CASR gene silencing and reduced CaSR expression. Functional cis-elements in the CASR promoters responsive to 1,25-dihydroxyvitamin D [1,25(OH)2D], proinflammatory cytokines, and the transcription factor glial cells missing-2 (GCM2) have been characterized. Reduced levels of CaSR and reduced responsiveness to active vitamin D in parathyroid neoplasia and colon carcinoma may blunt the “tumor suppressor” activity of the CaSR. The hypocalcemia of critically ill patients with burn injury or sepsis is associated with CASR gene upregulation by TNF-alpha and IL-1beta via kappaB elements, and by IL-6 via Stat1/3 and Sp1/3 elements in the CASR gene promoters, respectively. The CASR is transactivated by GCM2—the expression of which is essential for parathyroid gland development. Hyperactive forms of GCM2 may contribute to later parathyroid hyperactivity or tumorigenesis. The expression of the CaSR—the calciostat—is regulated physiologically and pathophysiologically at the gene level.

  1. Cholestenoic acids regulate motor neuron survival via liver X receptors

    PubMed Central

    Theofilopoulos, Spyridon; Griffiths, William J.; Crick, Peter J.; Yang, Shanzheng; Meljon, Anna; Ogundare, Michael; Kitambi, Satish Srinivas; Lockhart, Andrew; Tuschl, Karin; Clayton, Peter T.; Morris, Andrew A.; Martinez, Adelaida; Reddy, M. Ashwin; Martinuzzi, Andrea; Bassi, Maria T.; Honda, Akira; Mizuochi, Tatsuki; Kimura, Akihiko; Nittono, Hiroshi; De Michele, Giuseppe; Carbone, Rosa; Criscuolo, Chiara; Yau, Joyce L.; Seckl, Jonathan R.; Schüle, Rebecca; Schöls, Ludger; Sailer, Andreas W.; Kuhle, Jens; Fraidakis, Matthew J.; Gustafsson, Jan-Åke; Steffensen, Knut R.; Björkhem, Ingemar; Ernfors, Patrik; Sjövall, Jan; Arenas, Ernest; Wang, Yuqin

    2014-01-01

    Cholestenoic acids are formed as intermediates in metabolism of cholesterol to bile acids, and the biosynthetic enzymes that generate cholestenoic acids are expressed in the mammalian CNS. Here, we evaluated the cholestenoic acid profile of mammalian cerebrospinal fluid (CSF) and determined that specific cholestenoic acids activate the liver X receptors (LXRs), enhance islet-1 expression in zebrafish, and increase the number of oculomotor neurons in the developing mouse in vitro and in vivo. While 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA) promoted motor neuron survival in an LXR-dependent manner, 3β-hydroxy-7-oxocholest-5-en-26-oic acid (3βH,7O-CA) promoted maturation of precursors into islet-1+ cells. Unlike 3β,7α-diHCA and 3βH,7O-CA, 3β-hydroxycholest-5-en-26-oic acid (3β-HCA) caused motor neuron cell loss in mice. Mutations in CYP7B1 or CYP27A1, which encode enzymes involved in cholestenoic acid metabolism, result in different neurolo