The Experts below are selected from a list of 123666 Experts worldwide ranked by ideXlab platform
Lucy Liaw - One of the best experts on this subject based on the ideXlab platform.
-
a receptor specific function for notch2 in mediating vascular smooth muscle cell growth arrest through cyclin dependent kinase inhibitor 1b
Circulation Research, 2013Co-Authors: Joshua M Boucher, Anne Harrington, Bahman Rostama, Volkhard Lindner, Lucy LiawAbstract:Rationale:Deregulated vascular smooth muscle cell (VSMC) proliferation contributes to multiple vascular pathologies, and Notch signaling regulates VSMC phenotype. Objective:Previous work focused on Notch1 and Notch3 in VSMC during vascular disease; however, the role of Notch2 is unknown. Because injured murine carotid arteries display increased Notch2 in VSMC as compared with uninjured arteries, we sought to understand the impact of Notch2 signaling in VSMCs. Methods and Results:In human primary VSMCs, Jagged-1 (Jag-1) significantly reduced proliferation through specific activation of Notch2. Increased levels of p27kip1 were observed downstream of Jag-1/Notch2 signaling and were required for cell cycle exit. Jag-1 activation of Notch resulted in increased phosphorylation on serine 10, decreased ubiquitination, and prolonged half-life of p27kip1. Jag-1/Notch2 signaling robustly decreased S-phase kinase–associated protein, an F-box protein that degrades p27kip1 during G1. Overexpression of S-phase kinase–as...
-
a receptor specific function for notch2 in mediating vascular smooth muscle cell growth arrest through p27kip1
Circulation Research, 2013Co-Authors: Joshua M Boucher, Anne Harrington, Bahman Rostama, Volkhard Lindner, Lucy LiawAbstract:Rationale: Deregulated vascular smooth muscle cell (VSMC) proliferation contributes to multiple vascular pathologies, and Notch signaling regulates VSMC phenotype. Objective: Previous work focused on Notch1 and Notch3 in VSMC during vascular disease; however, the role of Notch2 is unknown. Because injured murine carotid arteries display increased Notch2 in VSMC as compared to uninjured arteries, we sought to understand the impact of Notch2 signaling in VSMC. Methods and Results: In human primary VSMC, Jagged-1 (Jag-1) significantly reduced proliferation through specific activation of Notch2. Increased levels of p27 kip1 were observed downstream of Jag-1/Notch2 signaling, and required for cell cycle exit. Jag-1 activation of Notch resulted in increased phosphorylation on serine 10, decreased ubiquitination and prolonged half-life of p27 kip1 . Jag-1/Notch2 signaling robustly decreased S-phase kinase associated protein (Skp2), an F-box protein that degrades p27 kip1 during G1. Over expression of Skp2 prior to Notch activation by Jag-1 suppressed the induction of p27 kip1 . Additionally, increased Notch2 andp27 kip1 expression was co-localized to the non-proliferative zone of injured arteries as indicated by co-staining with proliferating cell nuclear antigen (PCNA), whereas Notch3 was expressed throughout normal and injured arteries, suggesting Notch2 may negatively regulate lesion formation. Conclusions: We propose a receptor specific function for Notch2 in regulating Jag-1-induced p27 kip1 expression and growth arrest in VSMC. During vascular remodeling, co-localization of Notch2 and p27 kip1 to the non-proliferating region supports a model where Notch2 activation may negatively regulate VSMC proliferation to lessen the severity of the lesion. Thus Notch2 is a potential target for control of VSMC hyperplasia.
-
Hairy-Related Transcription Factors Inhibit Notch-Induced Smooth Muscle α-Actin Expression by Interfering With Notch Intracellular Domain/CBF-1 Complex Interaction With the CBF-1–Binding Site
Circulation Research, 2008Co-Authors: Yuefeng Tang, Lucy LiawAbstract:Notch signaling regulates smooth muscle cell phenotype and is critical for vascular development. One Notch target is smooth muscle α-actin (SMA), a differentiated smooth muscle cell marker. The Notch intracellular domain (NotchICD) forms a complex with CBF-1 (C-promoter– binding factor-1) and directly induces SMA expression. Using primary human smooth muscle cells, we show that expression of the constitutive active ICD of human Notch1, Notch2, or Notch4 receptors increase SMA levels. NotchICD also induce expression of the transcriptional repressors HRT1 (Hairy-related transcription factor 1) and HRT2, in a CBF-1–dependent manner. However, unlike the activating effects of NotchICD, HRT1 or HRT2 represses basal SMA expression, and both are strong antagonists of NotchICD-induced SMA upregulation. This antagonism does not depend on histone deacetylase activity and occurs at the transcriptional level. Competitive coimmunoprecipitation experiments demonstrate that HRT does not disrupt the association of NotchICD and CBF-1, which form a complex in the presence or absence of HRTs. However, HRT suppresses NotchICD/CBF-1 binding to the SMA promoter, as measured by chromatin immunoprecipitation, and transactivation of an SMA promoter reporter spanning sequences −124/+32. SMA expression was regulated similarly following endogenous Notch activation in smooth muscle cells by coculture with endothelial cells, and this effect was also sensitive to HRT inhibition. Temporally defined HRT activity may constitute a negative feedback mechanism of Notch signaling. Our study presents a novel mechanism by which a balance between Notch signaling and HRT activity determines the expression of smooth muscle differentiation markers including SMA.
Nataki C. Douglas - One of the best experts on this subject based on the ideXlab platform.
-
Dynamic maternal and fetal Notch activity and expression in placentation
Placenta, 2017Co-Authors: Heather Levin, Carrie J. Shawber, Virginia E. Papaioannou, Jan Kitajewski, Chantae S. Sullivan-pyke, Ronald J. Wapner, Nataki C. DouglasAbstract:Abstract Introduction Murine placentation requires trophoblast Notch2, while the Notch ligand, JAGGED1, is reduced in invasive trophoblasts from women with preeclampsia. However, the placental cells with active Notch signaling and expression of other Notch proteins and ligands in placentation have yet to be defined. We sought to identify endothelial cell and trophoblast subtypes with canonical Notch signaling in the decidua and placenta and correlate this to expression of Notch proteins and ligands. Methods Notch reporter transgenic mice were used to define canonical Notch activity and immunofluorescence staining performed to characterize expression of Notch1, 2, 3, 4 and ligands, Delta-like 4 (Dll4) and Jagged1 (Jag1) during early placentation and in the mature placenta. Results Notch signaling is active in maternal and fetal endothelial cells and trophoblasts during early placentation and in the mature placenta. Dll4, Jag1, Notch1, and Notch4 are expressed in maternal vasculature in the decidua. Dll4, Jag1 and Notch1 are expressed in fetal vasculature in the labyrinth. Dll4, Notch2 and Notch4 are co-expressed in the ectoplacental cone. Notch2 and Notch4 are expressed in parietal-trophoblast giant cells and junctional zone trophoblasts with active canonical Notch signaling and in labyrinthine syncytiotrophoblasts and sinusoidal-trophoblast giant cells. Discussion Canonical Notch activity and distinct expression patterns for Notch proteins and ligands was evident in endothelium and trophoblasts, suggesting Notch1, Notch2, Notch4, Dll4, and Jag1 have distinct and overlapping functions in placentation. Characterization of Notch signaling defects in existing mouse models of preeclampsia may shed light on the role of Notch in developing the preeclampsia phenotype.
-
Vascular Notch proteins and Notch signaling in the peri-implantation mouse uterus
Vascular Cell, 2015Co-Authors: Carrie J. Shawber, Lu Lin, Maria Gnarra, Mark V. Sauer, Virginia E. Papaioannou, Jan Kitajewski, Nataki C. DouglasAbstract:Angiogenesis is essential for uterine decidualization, the progesterone-mediated transformation of the uterus allowing embryo implantation and initiation of pregnancy. In the current study, we define the vasculature, expression of Notch proteins and Notch ligands, and Notch activity in both endothelial cells and vascular-associated mural cells of blood vessels in the pre-implantation endometrium and post-implantation decidua of the mouse uterus. We used immunofluorescence to determine the expression of Notch in endothelial cells and mural cells by co-staining for the endothelial cell marker, CD31, the pan-mural cell marker, platelet-derived growth factor receptor beta (PDGFR-β), the pericyte markers, neural/glial antigen 2 (NG2) and desmin, or the smooth muscle cell marker, alpha smooth muscle actin (SMA). A fluorescein isothiocyanate-labeled dextran tracer, was used to identify functional peri-implantation vasculature. CBF:H2B-Venus Notch reporter transgenic mice were used to determine Notch activity. Notch signaling is observed in endothelial cells and pericytes in the peri-implantation uterus. Prior to implantation, Notch1, Notch2 and Notch4 and Notch ligand, Delta-like 4 (Dll4) are expressed in capillary endothelial cells, while Notch3 is expressed in the pericytes. Jagged1 is expressed in both capillary endothelial cells and pericytes. After implantation, Notch1, Notch4 and Dll4 are expressed in endothelial cells of newly formed decidual capillaries. Jagged1 is expressed in endothelial cells of spiral arteries and a subset of decidual pericytes. Notch proteins are not expressed in lymphatic vessels or macrophages in the peri-implantation uterus. We show Notch activity and distinct expression patterns for Notch proteins and ligands, suggesting unique roles for Notch1, Notch4, Dll4, and Jag1 during decidual angiogenesis and early placentation. These data set the stage for loss-of-function and gain-of-function studies that will determine the cell-type specific requirements for Notch proteins in decidual angiogenesis and placentation.
-
Vascular Notch proteins and Notch signaling in the peri-implantation mouse uterus
Vascular Cell, 2015Co-Authors: Carrie J. Shawber, Lu Lin, Maria Gnarra, Mark V. Sauer, Virginia E. Papaioannou, Jan Kitajewski, Nataki C. DouglasAbstract:Background Angiogenesis is essential for uterine decidualization, the progesterone-mediated transformation of the uterus allowing embryo implantation and initiation of pregnancy. In the current study, we define the vasculature, expression of Notch proteins and Notch ligands, and Notch activity in both endothelial cells and vascular-associated mural cells of blood vessels in the pre-implantation endometrium and post-implantation decidua of the mouse uterus. Methods We used immunofluorescence to determine the expression of Notch in endothelial cells and mural cells by co-staining for the endothelial cell marker, CD31, the pan-mural cell marker, platelet-derived growth factor receptor beta (PDGFR-β), the pericyte markers, neural/glial antigen 2 (NG2) and desmin, or the smooth muscle cell marker, alpha smooth muscle actin (SMA). A fluorescein isothiocyanate-labeled dextran tracer, was used to identify functional peri-implantation vasculature. CBF:H2B-Venus Notch reporter transgenic mice were used to determine Notch activity. Results Notch signaling is observed in endothelial cells and pericytes in the peri-implantation uterus. Prior to implantation, Notch1, Notch2 and Notch4 and Notch ligand, Delta-like 4 (Dll4) are expressed in capillary endothelial cells, while Notch3 is expressed in the pericytes. Jagged1 is expressed in both capillary endothelial cells and pericytes. After implantation, Notch1, Notch4 and Dll4 are expressed in endothelial cells of newly formed decidual capillaries. Jagged1 is expressed in endothelial cells of spiral arteries and a subset of decidual pericytes. Notch proteins are not expressed in lymphatic vessels or macrophages in the peri-implantation uterus. Conclusions We show Notch activity and distinct expression patterns for Notch proteins and ligands, suggesting unique roles for Notch1, Notch4, Dll4, and Jag1 during decidual angiogenesis and early placentation. These data set the stage for loss-of-function and gain-of-function studies that will determine the cell-type specific requirements for Notch proteins in decidual angiogenesis and placentation.
Shivendra V Singh - One of the best experts on this subject based on the ideXlab platform.
-
Notch2 activation is protective against anticancer effects of zerumbone in human breast cancer cells
Breast Cancer Research and Treatment, 2014Co-Authors: Anuradha Sehrawat, Kozue Sakao, Shivendra V SinghAbstract:We showed previously that zerumbone (ZER), a sesquiterpene isolated from subtropical ginger, inhibited in vitro (MCF-7 and MDA-MB-231cells) and in vivo (MDA-MB-231 cells) growth of human breast cancer cells in association with apoptosis induction. Here, we investigated the role of Notch receptors in anticancer effects of ZER (cell migration inhibition and apoptosis induction) using breast cancer cells. Western blotting was performed to determine protein expression changes. Effect of ZER on transcriptional activity of Notch was assessed by luciferase reporter assays. Transfection with small hairpin RNA or small interfering RNA was performed for knockdown of Notch2 or Presenilin-1 protein. Cell migration and apoptosis were quantitated by Boyden chamber assay and flow cytometry, respectively. Exposure of MDA-MB-231, MCF-7, and SUM159 cells to ZER resulted in increased cleavage of Notch2 in each cell line. On the other hand, levels of cleaved Notch1 and Notch4 proteins were decreased following ZER treatment. Increased cleavage of Notch2 in ZER-treated cells was accompanied by induction of Presenilin-1 protein and transcriptional activation of Notch. Inhibition of cell migration as well as apoptosis induction resulting from ZER exposure was significantly augmented by knockdown of Notch2 protein. ZER-mediated cleavage of Notch2 protein in MDA-MB-231 cells was markedly attenuated upon RNA interference of Presenilin-1. Knockdown of Presenilin-1 protein also resulted in escalation of ZER-induced apoptosis. The present study indicates that Notch2 activation by ZER inhibits its proapoptotic and anti-migratory response at least in breast cancer cells.
-
withaferin a causes activation of notch2 and notch4 in human breast cancer cells
Breast Cancer Research and Treatment, 2012Co-Authors: Joomin Lee, Anuradha Sehrawat, Shivendra V SinghAbstract:Ayurvedic medicine plants continue to draw attention for the discovery of novel anticancer agents. Withaferin A (WA) is one such small-molecule constituent of the ayurvedic medicine plant Withania somnifera with efficacy against cultured and xenografted human breast cancer cells. However, the mechanism underlying anticancer effect of WA is not fully understood. This study was undertaken to determine the role of Notch signaling in anticancer effects of WA using human breast cancer cells as a model. Notably, Notch signaling is often hyperactive in human breast cancers. Exposure of MDA-MB-231 and MCF-7 human breast cancer cells to pharmacological concentrations of WA resulted in cleavage (activation) of Notch2 as well as Notch4, which was accompanied by transcriptional activation of Notch as evidenced by RBP-Jk, HES-1A/B, and HEY-1 luciferase reporter assays. On the other hand, WA treatment caused a decrease in levels of both transmembrane and cleaved Notch1. The WA-mediated activation of Notch was associated with induction of γ-secretase complex components presenilin1 and/or nicastrin. Inhibition of MDA-MB-231 and MDA-MB-468 cell migration resulting from WA exposure was significantly augmented by knockdown of Notch2 as well as Notch4 protein. Activation of Notch2 was not observed in cells treated with withanone or withanolide A, which are structural analogs of WA. The results of this study indicate that WA treatment activates Notch2 and Notch4, which impede inhibitory effect of WA on breast cancer cell migration.
-
Notch2 activation by benzyl isothiocyanate impedes its inhibitory effect on breast cancer cell migration
Breast Cancer Research and Treatment, 2012Co-Authors: Anuradha Sehrawat, Shivendra V SinghAbstract:Benzyl isothiocyanate (BITC) is a promising anticancer constituent of edible cruciferous vegetables with in vivo efficacy against chemically induced as well as oncogene-driven breast cancer in experimental rodents. However, the mechanism underlying anticancer effect of BITC is not fully understood. This study was undertaken to determine the role of Notch signaling in anticancer responses to BITC as this pathway is often hyperactive in human breast cancer. Exposure of MCF-7, MDA-MB-231, and SUM159 human breast cancer cells to pharmacologic concentrations of BITC (2.5 and 5 μM) resulted in cleavage (activation) of Notch1, Notch2, and Notch4, which was accompanied by induction of γ-secretase complex components Presenilin1 and/or Nicastrin. The BITC-mediated cleavage of Notch was associated with its transcriptional activation as revealed by RBP-Jk and Hes-1A/B luciferase reporter assays. Inhibition of cell migration or cell viability resulting from BITC exposure was not influenced by pharmacological suppression of Notch1 using a γ-secretase inhibitor or RNA interference of Notch1 as well as Notch4. On the other hand, the BITC-mediated inhibition of cell migration, but not cell viability, was significantly augmented by siRNA and shRNA knockdown of Notch2 protein. Furthermore, the BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with a significant increase in nuclear levels of cleaved Notch2 and Hes-1 proteins. In conclusion, the results of this study indicate that (a) BITC treatment activates Notch2 in cultured and xenografted human breast cancer cells, and (b) Notch2 activation impedes inhibitory effect of BITC on cell migration.
Carrie J. Shawber - One of the best experts on this subject based on the ideXlab platform.
-
Dynamic maternal and fetal Notch activity and expression in placentation
Placenta, 2017Co-Authors: Heather Levin, Carrie J. Shawber, Virginia E. Papaioannou, Jan Kitajewski, Chantae S. Sullivan-pyke, Ronald J. Wapner, Nataki C. DouglasAbstract:Abstract Introduction Murine placentation requires trophoblast Notch2, while the Notch ligand, JAGGED1, is reduced in invasive trophoblasts from women with preeclampsia. However, the placental cells with active Notch signaling and expression of other Notch proteins and ligands in placentation have yet to be defined. We sought to identify endothelial cell and trophoblast subtypes with canonical Notch signaling in the decidua and placenta and correlate this to expression of Notch proteins and ligands. Methods Notch reporter transgenic mice were used to define canonical Notch activity and immunofluorescence staining performed to characterize expression of Notch1, 2, 3, 4 and ligands, Delta-like 4 (Dll4) and Jagged1 (Jag1) during early placentation and in the mature placenta. Results Notch signaling is active in maternal and fetal endothelial cells and trophoblasts during early placentation and in the mature placenta. Dll4, Jag1, Notch1, and Notch4 are expressed in maternal vasculature in the decidua. Dll4, Jag1 and Notch1 are expressed in fetal vasculature in the labyrinth. Dll4, Notch2 and Notch4 are co-expressed in the ectoplacental cone. Notch2 and Notch4 are expressed in parietal-trophoblast giant cells and junctional zone trophoblasts with active canonical Notch signaling and in labyrinthine syncytiotrophoblasts and sinusoidal-trophoblast giant cells. Discussion Canonical Notch activity and distinct expression patterns for Notch proteins and ligands was evident in endothelium and trophoblasts, suggesting Notch1, Notch2, Notch4, Dll4, and Jag1 have distinct and overlapping functions in placentation. Characterization of Notch signaling defects in existing mouse models of preeclampsia may shed light on the role of Notch in developing the preeclampsia phenotype.
-
Vascular Notch proteins and Notch signaling in the peri-implantation mouse uterus
Vascular Cell, 2015Co-Authors: Carrie J. Shawber, Lu Lin, Maria Gnarra, Mark V. Sauer, Virginia E. Papaioannou, Jan Kitajewski, Nataki C. DouglasAbstract:Angiogenesis is essential for uterine decidualization, the progesterone-mediated transformation of the uterus allowing embryo implantation and initiation of pregnancy. In the current study, we define the vasculature, expression of Notch proteins and Notch ligands, and Notch activity in both endothelial cells and vascular-associated mural cells of blood vessels in the pre-implantation endometrium and post-implantation decidua of the mouse uterus. We used immunofluorescence to determine the expression of Notch in endothelial cells and mural cells by co-staining for the endothelial cell marker, CD31, the pan-mural cell marker, platelet-derived growth factor receptor beta (PDGFR-β), the pericyte markers, neural/glial antigen 2 (NG2) and desmin, or the smooth muscle cell marker, alpha smooth muscle actin (SMA). A fluorescein isothiocyanate-labeled dextran tracer, was used to identify functional peri-implantation vasculature. CBF:H2B-Venus Notch reporter transgenic mice were used to determine Notch activity. Notch signaling is observed in endothelial cells and pericytes in the peri-implantation uterus. Prior to implantation, Notch1, Notch2 and Notch4 and Notch ligand, Delta-like 4 (Dll4) are expressed in capillary endothelial cells, while Notch3 is expressed in the pericytes. Jagged1 is expressed in both capillary endothelial cells and pericytes. After implantation, Notch1, Notch4 and Dll4 are expressed in endothelial cells of newly formed decidual capillaries. Jagged1 is expressed in endothelial cells of spiral arteries and a subset of decidual pericytes. Notch proteins are not expressed in lymphatic vessels or macrophages in the peri-implantation uterus. We show Notch activity and distinct expression patterns for Notch proteins and ligands, suggesting unique roles for Notch1, Notch4, Dll4, and Jag1 during decidual angiogenesis and early placentation. These data set the stage for loss-of-function and gain-of-function studies that will determine the cell-type specific requirements for Notch proteins in decidual angiogenesis and placentation.
-
Vascular Notch proteins and Notch signaling in the peri-implantation mouse uterus
Vascular Cell, 2015Co-Authors: Carrie J. Shawber, Lu Lin, Maria Gnarra, Mark V. Sauer, Virginia E. Papaioannou, Jan Kitajewski, Nataki C. DouglasAbstract:Background Angiogenesis is essential for uterine decidualization, the progesterone-mediated transformation of the uterus allowing embryo implantation and initiation of pregnancy. In the current study, we define the vasculature, expression of Notch proteins and Notch ligands, and Notch activity in both endothelial cells and vascular-associated mural cells of blood vessels in the pre-implantation endometrium and post-implantation decidua of the mouse uterus. Methods We used immunofluorescence to determine the expression of Notch in endothelial cells and mural cells by co-staining for the endothelial cell marker, CD31, the pan-mural cell marker, platelet-derived growth factor receptor beta (PDGFR-β), the pericyte markers, neural/glial antigen 2 (NG2) and desmin, or the smooth muscle cell marker, alpha smooth muscle actin (SMA). A fluorescein isothiocyanate-labeled dextran tracer, was used to identify functional peri-implantation vasculature. CBF:H2B-Venus Notch reporter transgenic mice were used to determine Notch activity. Results Notch signaling is observed in endothelial cells and pericytes in the peri-implantation uterus. Prior to implantation, Notch1, Notch2 and Notch4 and Notch ligand, Delta-like 4 (Dll4) are expressed in capillary endothelial cells, while Notch3 is expressed in the pericytes. Jagged1 is expressed in both capillary endothelial cells and pericytes. After implantation, Notch1, Notch4 and Dll4 are expressed in endothelial cells of newly formed decidual capillaries. Jagged1 is expressed in endothelial cells of spiral arteries and a subset of decidual pericytes. Notch proteins are not expressed in lymphatic vessels or macrophages in the peri-implantation uterus. Conclusions We show Notch activity and distinct expression patterns for Notch proteins and ligands, suggesting unique roles for Notch1, Notch4, Dll4, and Jag1 during decidual angiogenesis and early placentation. These data set the stage for loss-of-function and gain-of-function studies that will determine the cell-type specific requirements for Notch proteins in decidual angiogenesis and placentation.
-
notch alters vegf responsiveness in human and murine endothelial cells by direct regulation of vegfr 3 expression
Journal of Clinical Investigation, 2007Co-Authors: Carrie J. Shawber, Yasuhiro Funahashi, Esther Francisco, Marina Vorontchikhina, Yukari Ido Kitamura, Stephanie A Stowell, Valeriya Borisenko, Nikki Feirt, Simona Podgrabinska, Kazuko ShiraishiAbstract:The Notch family of cell surface receptors and its ligands are highly conserved proteins that regulate cell fate determination, including those involved in mammalian vascular development. We report that Notch induces VEGFR-3 expression in vitro in human endothelial cells and in vivo in mice. In vitro, Notch in complex with the DNA-binding protein CBF-1/suppressor of hairless/Lag1 (CSL) bound the VEGFR-3 promoter and transactivated VEGFR-3 specifically in endothelial cells. Through induction of VEGFR-3, Notch increased endothelial cell responsiveness to VEGF-C, promoting endothelial cell survival and morphological changes. In vivo, VEGFR-3 was upregulated in endothelial cells with active Notch signaling. Mice heterozygous for null alleles of both Notch1 and VEGFR-3 had significantly reduced viability and displayed midgestational vascular patterning defects analogous to Notch1 nullizygous embryos. We found that Notch1 and Notch4 were expressed in normal and tumor lymphatic endothelial cells and that Notch1 was activated in lymphatic endothelium of invasive mammary micropapillary carcinomas. These results demonstrate that Notch1 and VEGFR-3 interact genetically, that Notch directly induces VEGFR-3 in blood endothelial cells to regulate vascular development, and that Notch may function in tumor lymphangiogenesis.
-
Jagged: A mammalian ligand that activates notch1
Cell, 1995Co-Authors: Claire E. Lindsell, Carrie J. Shawber, Jim Boulter, Gerry WeinmasterAbstract:Here we report the isolation of a rat cDNA clone, Jagged, which we show encodes a ligand for vertebrate Notch. Our conclusion is based on three observations. First, sequence analysis reveals substantial homology between Jagged and invertebrate ligands for the LIN-12/Notch proteins. Second, in situ hybridization of rat embryos identifies both distinct and overlapping patterns of gene expression for Jagged with those for Notch1, Notch2, and Notch3. Finally, the biological activity of Jagged was tested using a cell culture assay in which Jagged activates rat Notch1 expressed in myoblasts and prevents muscle cell differentiation. Our data support the hypothesis that Notch-ligand interactions function in maintaining mammalian cells in an undifferentiated state.
Jan Kitajewski - One of the best experts on this subject based on the ideXlab platform.
-
Dynamic maternal and fetal Notch activity and expression in placentation
Placenta, 2017Co-Authors: Heather Levin, Carrie J. Shawber, Virginia E. Papaioannou, Jan Kitajewski, Chantae S. Sullivan-pyke, Ronald J. Wapner, Nataki C. DouglasAbstract:Abstract Introduction Murine placentation requires trophoblast Notch2, while the Notch ligand, JAGGED1, is reduced in invasive trophoblasts from women with preeclampsia. However, the placental cells with active Notch signaling and expression of other Notch proteins and ligands in placentation have yet to be defined. We sought to identify endothelial cell and trophoblast subtypes with canonical Notch signaling in the decidua and placenta and correlate this to expression of Notch proteins and ligands. Methods Notch reporter transgenic mice were used to define canonical Notch activity and immunofluorescence staining performed to characterize expression of Notch1, 2, 3, 4 and ligands, Delta-like 4 (Dll4) and Jagged1 (Jag1) during early placentation and in the mature placenta. Results Notch signaling is active in maternal and fetal endothelial cells and trophoblasts during early placentation and in the mature placenta. Dll4, Jag1, Notch1, and Notch4 are expressed in maternal vasculature in the decidua. Dll4, Jag1 and Notch1 are expressed in fetal vasculature in the labyrinth. Dll4, Notch2 and Notch4 are co-expressed in the ectoplacental cone. Notch2 and Notch4 are expressed in parietal-trophoblast giant cells and junctional zone trophoblasts with active canonical Notch signaling and in labyrinthine syncytiotrophoblasts and sinusoidal-trophoblast giant cells. Discussion Canonical Notch activity and distinct expression patterns for Notch proteins and ligands was evident in endothelium and trophoblasts, suggesting Notch1, Notch2, Notch4, Dll4, and Jag1 have distinct and overlapping functions in placentation. Characterization of Notch signaling defects in existing mouse models of preeclampsia may shed light on the role of Notch in developing the preeclampsia phenotype.
-
Vascular Notch proteins and Notch signaling in the peri-implantation mouse uterus
Vascular Cell, 2015Co-Authors: Carrie J. Shawber, Lu Lin, Maria Gnarra, Mark V. Sauer, Virginia E. Papaioannou, Jan Kitajewski, Nataki C. DouglasAbstract:Angiogenesis is essential for uterine decidualization, the progesterone-mediated transformation of the uterus allowing embryo implantation and initiation of pregnancy. In the current study, we define the vasculature, expression of Notch proteins and Notch ligands, and Notch activity in both endothelial cells and vascular-associated mural cells of blood vessels in the pre-implantation endometrium and post-implantation decidua of the mouse uterus. We used immunofluorescence to determine the expression of Notch in endothelial cells and mural cells by co-staining for the endothelial cell marker, CD31, the pan-mural cell marker, platelet-derived growth factor receptor beta (PDGFR-β), the pericyte markers, neural/glial antigen 2 (NG2) and desmin, or the smooth muscle cell marker, alpha smooth muscle actin (SMA). A fluorescein isothiocyanate-labeled dextran tracer, was used to identify functional peri-implantation vasculature. CBF:H2B-Venus Notch reporter transgenic mice were used to determine Notch activity. Notch signaling is observed in endothelial cells and pericytes in the peri-implantation uterus. Prior to implantation, Notch1, Notch2 and Notch4 and Notch ligand, Delta-like 4 (Dll4) are expressed in capillary endothelial cells, while Notch3 is expressed in the pericytes. Jagged1 is expressed in both capillary endothelial cells and pericytes. After implantation, Notch1, Notch4 and Dll4 are expressed in endothelial cells of newly formed decidual capillaries. Jagged1 is expressed in endothelial cells of spiral arteries and a subset of decidual pericytes. Notch proteins are not expressed in lymphatic vessels or macrophages in the peri-implantation uterus. We show Notch activity and distinct expression patterns for Notch proteins and ligands, suggesting unique roles for Notch1, Notch4, Dll4, and Jag1 during decidual angiogenesis and early placentation. These data set the stage for loss-of-function and gain-of-function studies that will determine the cell-type specific requirements for Notch proteins in decidual angiogenesis and placentation.
-
Vascular Notch proteins and Notch signaling in the peri-implantation mouse uterus
Vascular Cell, 2015Co-Authors: Carrie J. Shawber, Lu Lin, Maria Gnarra, Mark V. Sauer, Virginia E. Papaioannou, Jan Kitajewski, Nataki C. DouglasAbstract:Background Angiogenesis is essential for uterine decidualization, the progesterone-mediated transformation of the uterus allowing embryo implantation and initiation of pregnancy. In the current study, we define the vasculature, expression of Notch proteins and Notch ligands, and Notch activity in both endothelial cells and vascular-associated mural cells of blood vessels in the pre-implantation endometrium and post-implantation decidua of the mouse uterus. Methods We used immunofluorescence to determine the expression of Notch in endothelial cells and mural cells by co-staining for the endothelial cell marker, CD31, the pan-mural cell marker, platelet-derived growth factor receptor beta (PDGFR-β), the pericyte markers, neural/glial antigen 2 (NG2) and desmin, or the smooth muscle cell marker, alpha smooth muscle actin (SMA). A fluorescein isothiocyanate-labeled dextran tracer, was used to identify functional peri-implantation vasculature. CBF:H2B-Venus Notch reporter transgenic mice were used to determine Notch activity. Results Notch signaling is observed in endothelial cells and pericytes in the peri-implantation uterus. Prior to implantation, Notch1, Notch2 and Notch4 and Notch ligand, Delta-like 4 (Dll4) are expressed in capillary endothelial cells, while Notch3 is expressed in the pericytes. Jagged1 is expressed in both capillary endothelial cells and pericytes. After implantation, Notch1, Notch4 and Dll4 are expressed in endothelial cells of newly formed decidual capillaries. Jagged1 is expressed in endothelial cells of spiral arteries and a subset of decidual pericytes. Notch proteins are not expressed in lymphatic vessels or macrophages in the peri-implantation uterus. Conclusions We show Notch activity and distinct expression patterns for Notch proteins and ligands, suggesting unique roles for Notch1, Notch4, Dll4, and Jag1 during decidual angiogenesis and early placentation. These data set the stage for loss-of-function and gain-of-function studies that will determine the cell-type specific requirements for Notch proteins in decidual angiogenesis and placentation.
-
A switch in Notch gene expression parallels stem cell to endothelial transition in infantile hemangioma
Angiogenesis, 2010Co-Authors: June K. Wu, Omotinuwe Adepoju, Dinuka Silva, Keith Baribault, Elisa Boscolo, Joyce Bischoff, Jan KitajewskiAbstract:Background Infantile hemangioma (IH) is the most common benign tumor of infancy, yet its pathogenesis is poorly understood. Notch family members are known to play a role in vascular development during embryogenesis and postnatal tumor angiogenesis, yet the role of Notch signaling in the pathogenesis of IH has not been investigated. This study aims to survey Notch expression in IH. Materials and methods RNA from resected hemangioma tissue and hemangioma-derived stem cells (HemSCs) and endothelial cells (HemECs) was used for gene expression analyses by real-time PCR. Results were confirmed with immunofluorescence for protein expression in tissue. Results Real-time PCR showed that Notch family gene expression in IH is distinct from placenta and skin. Notch3 is expressed in HemSCs, but not in HemECs, indicating Notch3 is downregulated as HemSCs differentiate into HemECs. Moreover, expression of endothelial-associated Notch proteins, Notch1, Notch4, and Jagged-1 are increased in involuting hemangiomas and HemECs, suggesting that as hemangioma progresses toward involution, it acquires more differentiated endothelium. A subset of cells stained double positive for Notch3 and CD31, pointing to a potential intermediate between the HemSC cellular differentiation into HemEC. Conclusion HemSCs have distinct Notch expression patterns from differentiated HemECs and from normal human endothelial cells. Notch3 is expressed in HemSCs, while Notch1, Notch4, and Jagged-1 have higher expression levels in HemECs. Notch3 was localized to the interstitial cells outside of the nascent vascular channels in proliferating IH tissue sections, but became more apparent in the perivascular cells in involuting IH. In summary, the pattern of Notch gene expression mirrors the progression from immature cells to endothelial-lined vascular channels (i.e., endothelial differentiation) that characterizes the growth and involution of IH.
-
dll4 a novel notch ligand expressed in arterial endothelium
Genes & Development, 2000Co-Authors: John R Shutter, Jan Kitajewski, Chris Kintner, Sheila Scully, William G Richards, Gisele A Deblandre, Kevin L StarkAbstract:Cell-to-cell communication is required for many biological processes such as differentiation, proliferation, and homeostasis. One system utilized by a wide range of eukaryotes is the Notch-signaling pathway. The Notch gene family consists of structurally conserved cell surface receptors that are activated by one or more membrane-bound ligands of the emerging Delta/Serrate/Lag-2 (DSL) gene family (Fleming 1998). Previous studies with invertebrates have shown that the Notch-signaling pathway allows a subset of cells within an equipotent precursor population to acquire a specific cell fate in a spatially and temporally restricted manner. In this process, known as lateral inhibition or specification, cells destined for one fate inhibit neighboring cells from adopting the same fate (Artavanis-Tsakonas et al. 1999). In Drosophila, for example, equipotent cells in the embryonic ectoderm give rise to both neural and epidermal cells. As cells assume a neural fate, expression of neurogenic genes results in increased expression of Delta on the cell surface, leading to an inhibition of these same genes in neighboring cells via activation of their Notch receptors. Notch-receptor activation results in the transactivation of the Enhancer of Split complex [E(spl)-C] which, in turn, suppresses the expression of downstream genes, including Delta, necessary for neurogenesis (Artavanis-Tsakonas et al. 1999). Accordingly, mutant flies lacking Notch have excessive numbers of neuronal cells, whereas activating mutations of Notch lead to an inhibition of neuron production (Struhl et al. 1993). Although Delta–Notch interactions in vertebrates are less well understood, the identification of several family members suggests that these genes play an important role in mammalian development. Four mammalian Notch receptors have been identified, Notch1/TAN-1 (Ellisen et al. 1991; del Amo et al. 1993), Notch2 (Weinmaster et al. 1992), Notch3 (Lardelli et al. 1994), and Notch4/int-3 (Uyttendaele et al. 1996). Dysregulated Notch signaling has been shown to result in several developmental abnormalities and diseases. Chromosomal translocations resulting in the truncation of the human Notch1 homolog, TAN1, have been found in certain T-cell acute lymphoblastic leukemias (T-ALL) (Ellisen et al. 1991). A frequent site for insertional activation by the mouse mammary tumor virus (MMTV) in malignant mammary carcinomas is the Notch4 locus. The insertion of MMTV results in an aberrantly expressed intracellular form of Notch4 originally identified as Int-3 (Gallahan and Callahan 1987). Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is a disorder that leads to ischaemic strokes and dementia in adults, and has been traced recently to missense mutations of the Notch3 receptor (Joutel et al. 1997). Notch ligands are divided into two subclasses, the Delta family and the Serrate family. All Notch ligands share some structural features (Fleming 1998) including epidermal growth factor (EGF)-like repeats, a characteristic DSL domain necessary for Notch binding (Muskavitch 1994) and a transmembrane region. However, an extracellular cysteine-rich domain and insertions that interrupt some EGF-like repeats are common only to the Serrate family. It is these structural differences that categorize a Notch ligand as a Delta or Serrate family member. Mammalian ligands reported include two members of the Serrate family, Jagged1/JAG1 (Lindsell et al. 1995; Oda et al. 1997a) and Jagged2 (Shawber et al. 1996), and two members of the Delta family, murine Dll1 (Bettenhausen et al. 1995) and murine Dll3 (Dunwoodie et al. 1997). Ligand mutations can also result in the disruption of the Notch-signaling pathway, leading to developmental abnormalities. Mutations of human JAG1 have been attributed to the development of Alagille syndrome (AGS), an autosomal dominant disorder characterized by developmental abnormalities of the heart, skeleton, muscle, liver, and eyes (Li et al. 1997; Oda et al. 1997b). Mice homozygous for the pudgy (pu) mutation exhibit severe deformities of the ribs and vertebrae, and this mutation has been mapped to the Dll3 locus (Kusumi et al. 1998). Dll1-deficient mice generated by homologous recombination exhibit neonatal lethality (Hrabe de Angelis et al. 1997), and disruption of murine Jagged2 revealed an essential role for this gene in limb, craniofacial, and thymic development (Jiang et al. 1998). The pleiotropic effects of dysregulated Notch signaling validate this pathway as an important regulator of development, and the existence of multiple ligands and receptors suggests a more specialized role for these genes in mammals than in lower eukaryotes. Here we report the molecular cloning and characterization of Dll4, the third mammalian member of the Delta family of Notch ligands. The ability to activate Notch receptors and the specific expression pattern of Dll4 implicate a role for this gene in the regulation of vascular biology.
