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1.
EMBO J ; 32(8): 1103-14, 2013 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-23524849

RESUMO

The cellular inhibitor of apoptosis (c-IAP) proteins are E3 ubiquitin ligases that are critical regulators of tumour necrosis factor (TNF) receptor (TNFR)-mediated signalling. Through their E3 ligase activity c-IAP proteins promote ubiquitination of receptor-interaction protein 1 (RIP1), NF-κB-inducing kinase (NIK) and themselves, and regulate the assembly of TNFR signalling complexes. Consequently, in the absence of c-IAP proteins, TNFR-mediated activation of NF-κB and MAPK pathways and the induction of gene expression are severely reduced. Here, we describe the identification of OTUB1 as a c-IAP-associated deubiquitinating enzyme that regulates c-IAP1 stability. OTUB1 disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo within the TWEAK receptor-signalling complex. Downregulation of OTUB1 promotes TWEAK- and IAP antagonist-stimulated caspase activation and cell death, and enhances c-IAP1 degradation. Furthermore, knockdown of OTUB1 reduces TWEAK-induced activation of canonical NF-κB and MAPK signalling pathways and modulates TWEAK-induced gene expression. Finally, suppression of OTUB1 expression in zebrafish destabilizes c-IAP (Birc2) protein levels and disrupts fish vasculature. These results suggest that OTUB1 regulates NF-κB and MAPK signalling pathways and TNF-dependent cell death by modulating c-IAP1 stability.


Assuntos
Cisteína Endopeptidases/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Animais , Vasos Sanguíneos/embriologia , Linhagem Celular , Enzimas Desubiquitinantes , Humanos , Hidrólise , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Peixe-Zebra/embriologia
3.
Blood ; 118(7): 1989-97, 2011 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-21700774

RESUMO

The Notch signaling pathway plays a fundamental role during blood vessel development. Notch signaling regulates blood vessel morphogenesis by promoting arterial endothelial differentiation and providing spatial and temporal control over "tip cell" phenotype during angiogenic sprouting. Components of the Notch signaling pathway have emerged as potential regulators of lymphatic development, joining the increasing examples of blood vessel regulators that are also involved in lymphatic development. However, in mammals a role for the Notch signaling pathway during lymphatic development remains to be demonstrated. In this report, we show that blockade of Notch1 and Dll4, with specific function-blocking antibodies, results in defective postnatal lymphatic development in mice. Mechanistically, Notch1-Dll4 blockade is associated with down-regulation of EphrinB2 expression, been shown to be critically involved in VEGFR3/VEGFC signaling, resulting in reduced lymphangiogenic sprouting. In addition, Notch1-Dll4 blockade leads to compromised expression of distinct lymphatic markers and to dilation of collecting lymphatic vessels with reduced and disorganized mural cell coverage. Finally, Dll4-blockade impairs wound closure and severely affects lymphangiogenesis during the wound healing in adult mouse skin. Thus, our study demonstrates for the first time in a mammalian system that Notch1-Dll4 signaling pathway regulates postnatal lymphatic development and pathologic lymphangiogenesis.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfangiogênese , Vasos Linfáticos/metabolismo , Proteínas de Membrana/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Linhagem Celular , Efrina-B2/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Vasos Linfáticos/citologia , Vasos Linfáticos/ultraestrutura , Camundongos
4.
Microvasc Res ; 89: 80-5, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23743248

RESUMO

RATIONALE: Loss of endothelial viability correlates with initiation and progress of vascular pathology. However, much remains to be learned about pathways required to maintain the balance between cell viability and apoptosis. Notch activation can enhance or inhibit apoptosis but its role in maintaining the endothelium needs further delineation. OBJECTIVE: This study aims to identify the mechanisms by which Notch activation regulates endothelial viability. METHODS AND RESULTS: Endothelial cells transduced with active Notch were treated with lipopolysaccharide (LPS) or homocysteine to induce endothelial apoptosis. Notch protected against LPS-induced cell death but exacerbated homocysteine-induced apoptosis. Inhibition of PI3K revealed that ligand-induced activation of endogenous Notch initiates parallel death and survival pathways and exhibits a differential effect on endothelial survival depending on the apoptotic stimulus. PI3K activity regulated the expression of Slug, which was required for survival in Notch-activated endothelial cells. Homocysteine, but not LPS, blocked both PI3K activity and Slug expression in Notch-activated cells, leading to increased endothelial apoptosis. CONCLUSIONS: Notch signaling leads to activation of parallel survival and apoptotic pathways in endothelial cells. The interaction of Notch with other signaling pathways plays an important contextual role in regulating endothelial viability.


Assuntos
Apoptose , Regulação da Expressão Gênica , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição/metabolismo , Sobrevivência Celular , Células Endoteliais/citologia , Inibidores Enzimáticos/química , Homocisteína/química , Humanos , Ligantes , Lipopolissacarídeos/química , Microcirculação , Microscopia de Fluorescência , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fatores de Tempo
5.
J Biol Chem ; 286(13): 11803-13, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21288908

RESUMO

Notch is a critical mediator of endothelial-to-mesenchymal transition (EndMT) during cardiac cushion development. Slug, a transcriptional repressor that is a Notch target, is an important Notch effector of EndMT in the cardiac cushion. Here, we report that the runt-related transcription factor RUNX3 is a novel direct Notch target in the endothelium. Ectopic expression of RUNX3 in endothelium induces Slug expression and EndMT independent of Notch activation. Interestingly, RUNX3 physically interacts with CSL, the Notch-interacting partner in the nucleus, and induces Slug in a CSL-dependent, but Notch-independent manner. Although RUNX3 may not be required for the initial induction of Slug and EndMT by Notch, because RUNX3 has a much longer half-life than Slug, it sustains the expression of Slug thereby maintaining the mesenchymal phenotype. CSL binds to the Runx3 promoter in the atrioventricular canal in vivo, and inhibition of Notch reduces RUNX3 expression in the cardiac cushion of embryonic hearts. Taken together, our results suggest that induction of RUNX3 may be a mechanism to maintain Notch-transformed mesenchymal cells during heart development.


Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Endotélio/embriologia , Transição Epitelial-Mesenquimal/fisiologia , Coração/embriologia , Mesoderma/metabolismo , Regiões Promotoras Genéticas/fisiologia , Receptores Notch/metabolismo , Linhagem Celular , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Meia-Vida , Humanos , Mesoderma/citologia , Organogênese/fisiologia , Receptores Notch/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
6.
Blood ; 115(8): 1654-61, 2010 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-19903896

RESUMO

In vertebrates, endothelial cells form 2 hierarchical tubular networks, the blood vessels and the lymphatic vessels. Despite the difference in their structure and function and genetic programs that dictate their morphogenesis, common signaling pathways have been recognized that regulate both vascular systems. ALK1 is a member of the transforming growth factor-beta type I family of receptors, and compelling genetic evidence suggests its essential role in regulating blood vascular development. Here we report that ALK1 signaling is intimately involved in lymphatic development. Lymphatic endothelial cells express key components of the ALK1 pathway and respond robustly to ALK1 ligand stimulation in vitro. Blockade of ALK1 signaling results in defective lymphatic development in multiple organs of neonatal mice. We find that ALK1 signaling regulates the differentiation of lymphatic endothelial cells to influence the lymphatic vascular development and remodeling. Furthermore, simultaneous inhibition of ALK1 pathway increases apoptosis in lymphatic vessels caused by blockade of VEGFR3 signaling. Thus, our study reveals a novel aspect of ALK1 signaling in regulating lymphatic development and suggests that targeting ALK1 pathway might provide additional control of lymphangiogenesis in human diseases.


Assuntos
Receptores de Activinas Tipo II/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Diferenciação Celular/fisiologia , Células Endoteliais/metabolismo , Linfangiogênese/fisiologia , Vasos Linfáticos/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/fisiologia , Células Cultivadas , Humanos , Ligantes , Camundongos , Transdução de Sinais , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
7.
Cell Rep ; 36(1): 109309, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34233193

RESUMO

αvß8 integrin, a key activator of transforming growth factor ß (TGF-ß), inhibits anti-tumor immunity. We show that a potent blocking monoclonal antibody against αvß8 (ADWA-11) causes growth suppression or complete regression in syngeneic models of squamous cell carcinoma, mammary cancer, colon cancer, and prostate cancer, especially when combined with other immunomodulators or radiotherapy. αvß8 is expressed at the highest levels in CD4+CD25+ T cells in tumors, and specific deletion of ß8 from T cells is as effective as ADWA-11 in suppressing tumor growth. ADWA-11 increases expression of a suite of genes in tumor-infiltrating CD8+ T cells normally inhibited by TGF-ß and involved in tumor cell killing, including granzyme B and interferon-γ. The in vitro cytotoxic effect of tumor CD8 T cells is inhibited by CD4+CD25+ cells, and this suppressive effect is blocked by ADWA-11. These findings solidify αvß8 integrin as a promising target for cancer immunotherapy.


Assuntos
Imunidade , Imunoterapia , Integrinas/metabolismo , Modelos Biológicos , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T/imunologia , Animais , Anticorpos Antineoplásicos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Granzimas/metabolismo , Interferon gama/metabolismo , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Transdução de Sinais , Proteína Smad3/metabolismo , Análise de Sobrevida , Linfócitos T Citotóxicos/imunologia , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
8.
Physiol Genomics ; 40(3): 150-7, 2010 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-19952280

RESUMO

Valve formation during embryonic heart development involves a complex interplay of regional specification, cell transformations, and remodeling events. While many studies have addressed the role of specific genes during this process, a global understanding of the genetic basis for the regional specification and development of the heart valves is incomplete. We have undertaken genome-wide transcriptional profiling of the developing heart valves in the mouse. Four Serial Analysis of Gene Expression libraries were generated and analyzed from the mouse atrio-ventricular canal (AVC) at embryonic days 9.5-12.5, covering the stages from initiation of endothelial to mesenchymal transition (EMT) through to the beginning of endocardial cushion remodeling. We identified 14 distinct temporal patterns of gene expression during AVC development. These were associated with specific functions and signaling pathway members. We defined the temporal distribution of mesenchyme genes during the EMT process and of specific Notch and transforming growth factor-beta targets. This work provides the first comprehensive temporal dataset during the formation of heart valves. These results identify molecular signatures that distinguish different phases of early heart valve formation allowing gene expression and function to be further investigated.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genoma , Genômica , Valvas Cardíacas/embriologia , Animais , Diferenciação Celular , Embrião de Mamíferos/metabolismo , Endotélio/metabolismo , Valvas Cardíacas/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores Notch/genética , Receptores Notch/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
9.
Circ Res ; 102(10): 1169-81, 2008 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-18497317

RESUMO

The Notch signaling pathway has been demonstrated to play a critical role during mammalian cardiac development based on recent findings from gene-targeted mice. In addition, mutations in the Notch signaling pathway have been associated with human congenital heart defects such as Alagille syndrome, bicuspid aortic valve disease, calcification of the heart valves, and ventricular septal defects. Recently, it was demonstrated that Notch activation in the endocardium regulates ventricular myocardial development and that the Notch downstream target genes Hey1 and Hey2 are required for the establishment of the atrioventricular canal myocardial boundary. The Notch pathway has previously been implicated in regulating endothelial-to-mesenchymal transition during development of the heart valves, and recent reports further dissect the role of individual Notch downstream target genes during this process. In addition, a role for the Notch pathway during cardiac neural crest cell development has been identified, which provides a potential mechanism for the findings seen in Alagille syndrome. This review focuses on recently reported findings that elucidate mechanisms regulated by the Notch pathway during ventricular, atrioventricular canal, and outflow tract development.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Coração/fisiologia , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos
10.
Circ Res ; 98(12): 1468-70, 2006 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-16741155

RESUMO

Intercellular signaling mediated by Notch receptors is essential for proper cardiovascular development and homeostasis. Notch regulates cell fate decisions that affect proliferation, survival, and differentiation of endothelial and smooth muscle cells. It has been reported that Jagged1-Notch interactions may participate in endocardial cushion formation by inducing endothelial-to-mesenchymal transformation. Here, we show that Notch directly regulates expression of the mesenchymal and smooth muscle cell marker smooth muscle alpha-actin (SMA) in endothelial and vascular smooth muscle cells via activation of its major effector, CSL. Notch/CSL activation induces SMA expression during endothelial-to-mesenchymal transformation, and Notch activation is required for expression of SMA in vascular smooth muscle cells. CSL directly binds a conserved cis element in the SMA promoter, and this consensus sequence is required for Notch-mediated SMA induction. This is the first evidence of the requirement for Notch activation in the regulation of SMA expression.


Assuntos
Actinas/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/fisiologia , Músculo Liso/metabolismo , Receptores Notch/fisiologia , Actinas/genética , Células Cultivadas , Sequência Consenso , Células Endoteliais/metabolismo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Miócitos de Músculo Liso/metabolismo , Regiões Promotoras Genéticas
11.
Circ Res ; 97(2): 102-4, 2005 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-15976316

RESUMO

Perturbation of the Notch signaling pathway has been implicated in the pathogenesis of human cardiovascular diseases, and animal models have confirmed the requirement of Notch during cardiovascular development. We recently demonstrated that Notch activation delays S-phase entry and contributes to endothelial contact inhibition. Minichromosome maintenance (MCM) proteins, components of the prereplicative complex (pre-RC), are essential for DNA replication. Here, we report that Notch-mediated cell cycle arrest is associated with downregulation of MCM2 and MCM6 in endothelial cells and human fibroblasts. Downregulation of MCM proteins is also observed on activation of C promoter binding factor (CBF1) and is mediated by inhibition of Rb phosphorylation, as demonstrated using a constitutively active Rb mutant. Although the effects of the Notch pathway are cell-type specific and context-dependent, in cell types where Notch has an antiproliferative effect, downregulation of MCM proteins may be a common mechanism to inhibit DNA replication.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Ciclo Celular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Superfície Celular/fisiologia , Fatores de Transcrição/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina , Componente 2 do Complexo de Manutenção de Minicromossomo , Componente 6 do Complexo de Manutenção de Minicromossomo , Proteínas Nucleares/fisiologia , Fosforilação , Receptor Notch1 , Receptor Notch4 , Receptores Notch , Proteína do Retinoblastoma/metabolismo
12.
Circ Res ; 94(7): 910-7, 2004 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-14988227

RESUMO

Various studies have identified a critical role for Notch signaling in cardiovascular development. In this and other systems, Notch receptors and ligands are expressed in regions that undergo epithelial-to-mesenchymal transformation. However, there is no direct evidence that Notch activation can induce mesenchymal transdifferentiation. In this study we show that Notch activation in endothelial cells results in morphological, phenotypic, and functional changes consistent with mesenchymal transformation. These changes include downregulation of endothelial markers (vascular endothelial [VE]-cadherin, Tie1, Tie2, platelet-endothelial cell adhesion molecule-1, and endothelial NO synthase), upregulation of mesenchymal markers (alpha-smooth muscle actin, fibronectin, and platelet-derived growth factor receptors), and migration toward platelet-derived growth factor-BB. Notch-induced endothelial-to-mesenchymal transformation does not seem to require external regulation and is restricted to cells expressing activated Notch. Jagged1 stimulation of endothelial cells induces a similar mesenchymal transformation, and Jagged1, Notch1, and Notch4 are expressed in the ventricular outflow tract during stages of endocardial cushion formation. This is the first evidence that Jagged1-Notch interactions induce endothelial-to-mesenchymal transformation, and our findings suggest that Notch signaling may be required for proper endocardial cushion differentiation and/or vascular smooth muscle cell development.


Assuntos
Endotélio Vascular/metabolismo , Coração Fetal/metabolismo , Septos Cardíacos/embriologia , Mesoderma/citologia , Músculo Liso Vascular/citologia , Proteínas/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Superfície Celular/fisiologia , Fatores de Transcrição , Actinas/biossíntese , Actinas/genética , Animais , Antígenos CD , Becaplermina , Caderinas/metabolismo , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Linhagem Celular , Endocárdio/citologia , Endotélio Vascular/citologia , Coração Fetal/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteína Jagged-1 , Proteínas de Membrana , Camundongos , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Biossíntese de Proteínas , Proteínas/genética , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-sis , Receptor Notch1 , Receptor Notch4 , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores Notch , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Serrate-Jagged , Ovinos , Transdução de Sinais/fisiologia , Transdução Genética
13.
Neurology ; 81(10): 865-71, 2013 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-23925765

RESUMO

OBJECTIVE: To find biomarkers identifying patients at risk for the development of progressive multifocal leukoencephalopathy (PML) during natalizumab treatment. METHODS: Patients were recruited from 10 European and US cohorts. Of 289 patients with multiple sclerosis (MS), 224 had been treated with natalizumab (18-80 months), 21 received other immune-modulatory treatments, and 28 were untreated. We had access to samples from 16 natalizumab PML patients. Eight of these patients had given blood before the diagnosis of PML. We also analyzed non-natalizumab-treated patients who developed PML (n = 10) and age- and sex-matched healthy donors (n = 31). All flow cytometric assessments were done on previously cryopreserved, viable peripheral blood mononuclear cells. RESULTS: The percentage of l-selectin-expressing CD4+ T cells was significantly lower in patients treated long-term with natalizumab (40.2%) when compared with patients not receiving natalizumab treatment (47.2%; p = 0.016) or healthy controls (61.0%; p < 0.0001). An unusually low percentage (9-fold lower; 4.6%) was highly correlated with the risk of developing PML in the patient group with available pre-PML samples when compared with non-PML natalizumab-treated patients (p ≤ 0.0001). Samples were gathered between 4 and 26 months before PML diagnosis. CONCLUSIONS: The cell-based assessment of the percentage of l-selectin-expressing CD4 T cells could provide an urgently needed biomarker for individual PML risk assessment.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Selectina L/sangue , Leucoencefalopatia Multifocal Progressiva/sangue , Esclerose Múltipla/sangue , Anticorpos Monoclonais Humanizados/efeitos adversos , Biomarcadores/sangue , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Estudos de Coortes , Humanos , Leucoencefalopatia Multifocal Progressiva/diagnóstico , Leucoencefalopatia Multifocal Progressiva/patologia , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/patologia , Natalizumab , Fatores de Risco , Resultado do Tratamento
14.
Dev Cell ; 21(2): 288-300, 2011 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-21839921

RESUMO

The heart is the most common site of congenital defects, and valvuloseptal defects are the most common of the cardiac anomalies seen in the newborn. The process of endothelial-to-mesenchymal transition (EndMT) in the cardiac cushions is a required step during early valve development, and Notch signaling is required for this process. Here we show that Notch activation induces the transcription of both subunits of the soluble guanylyl cyclase (sGC) heterodimer, GUCY1A3 and GUCY1B3, which form the nitric oxide receptor. In parallel, Notch also promotes nitric oxide (NO) production by inducing Activin A, thereby activating a PI3-kinase/Akt pathway to phosphorylate eNOS. We thus show that the activation of sGC by NO through a Notch-dependent autocrine loop is necessary to drive early EndMT in the developing atrioventricular canal (AVC).


Assuntos
Coxins Endocárdicos/citologia , Endotélio/fisiologia , Guanilato Ciclase/metabolismo , Mesoderma/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Notch/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/deficiência , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Interferência de RNA/fisiologia , Transdução de Sinais , Guanilil Ciclase Solúvel
15.
J Biol Chem ; 284(29): 19452-62, 2009 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-19473993

RESUMO

Notch and transforming growth factor beta (TGFbeta) play critical roles in endothelial-to-mesenchymal transition (EndMT), a process that is essential for heart development. Previously, we have shown that Notch and TGFbeta signaling synergistically induce Snail expression in endothelial cells, which is required for EndMT in cardiac cushion morphogenesis. Here, we report that Notch activation modulates TGFbeta signaling pathways in a receptor-activated Smad (R-Smad)-specific manner. Notch activation inhibits TGFbeta/Smad1 and TGFbeta/Smad2 signaling pathways by decreasing the expression of Smad1 and Smad2 and their target genes. In contrast, Notch increases SMAD3 mRNA expression and protein half-life and regulates the expression of TGFbeta/Smad3 target genes in a gene-specific manner. Inhibition of Notch in the cardiac cushion of mouse embryonic hearts reduces Smad3 expression. Notch and TGFbeta synergistically up-regulate a subset of genes by recruiting Smad3 to both Smad and CSL binding sites and cooperatively inducing histone H4 acetylation. This is the first evidence that Notch activation affects R-Smad expression and that cooperative induction of histone acetylation at specific promoters underlies the selective synergy between Notch and TGFbeta signaling pathways.


Assuntos
Células Endoteliais/efeitos dos fármacos , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação/genética , Proteínas de Ligação ao Cálcio , Linhagem Celular , Imunoprecipitação da Cromatina , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores Notch/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção
16.
J Cell Biol ; 182(2): 315-25, 2008 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-18663143

RESUMO

Snail family proteins are key regulators of epithelial-mesenchymal transition, but their role in endothelial-to-mesenchymal transition (EMT) is less well studied. We show that Slug, a Snail family member, is expressed by a subset of endothelial cells as well as mesenchymal cells of the atrioventricular canal and outflow tract during cardiac cushion morphogenesis. Slug deficiency results in impaired cellularization of the cardiac cushion at embryonic day (E)-9.5 but is compensated by increased Snail expression at E10.5, which restores cardiac cushion EMT. We further demonstrate that Slug, but not Snail, is directly up-regulated by Notch in endothelial cells and that Slug expression is required for Notch-mediated repression of the vascular endothelial cadherin promoter and for promoting migration of transformed endothelial cells. In contrast, transforming growth factor beta (TGF-beta) induces Snail but not Slug. Interestingly, activation of Notch in the context of TGF-beta stimulation results in synergistic up-regulation of Snail in endothelial cells. Collectively, our data suggest that combined expression of Slug and Snail is required for EMT in cardiac cushion morphogenesis.


Assuntos
Coxins Endocárdicos/embriologia , Coração/embriologia , Organogênese/genética , Receptor Notch1/metabolismo , Fatores de Transcrição/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Movimento Celular/genética , Coxins Endocárdicos/citologia , Coxins Endocárdicos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Mesoderma/citologia , Mesoderma/metabolismo , Regiões Promotoras Genéticas/genética , Receptor Notch1/genética , Fatores de Transcrição da Família Snail , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima/genética
17.
Am J Physiol Cell Physiol ; 293(1): C1-11, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17376817

RESUMO

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.


Assuntos
Anormalidades Cardiovasculares/metabolismo , Sistema Cardiovascular/embriologia , Sistema Cardiovascular/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Artérias/embriologia , Artérias/metabolismo , Padronização Corporal , Anormalidades Cardiovasculares/genética , Coração/embriologia , Humanos , Ligantes , Modelos Cardiovasculares , Modelos Moleculares , Conformação Molecular , Mutação , Miocárdio/metabolismo , Receptores Notch/química , Receptores Notch/genética , Veias/embriologia , Veias/metabolismo
18.
J Exp Med ; 204(12): 2935-48, 2007 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-17984306

RESUMO

Aberrant expression of Jagged1 and Notch1 are associated with poor outcome in breast cancer. However, the reason that Jagged1 and/or Notch overexpression portends a poor prognosis is unknown. We identify Slug, a transcriptional repressor, as a novel Notch target and show that elevated levels of Slug correlate with increased expression of Jagged1 in various human cancers. Slug was essential for Notch-mediated repression of E-cadherin, which resulted in beta-catenin activation and resistance to anoikis. Inhibition of ligand-induced Notch signaling in xenografted Slug-positive/E-cadherin-negative breast tumors promoted apoptosis and inhibited tumor growth and metastasis. This response was associated with down-regulated Slug expression, reexpression of E-cadherin, and suppression of active beta-catenin. Our findings suggest that ligand-induced Notch activation, through the induction of Slug, promotes tumor growth and metastasis characterized by epithelial-to-mesenchymal transition and inhibition of anoikis.


Assuntos
Caderinas/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/fisiologia , Células Epiteliais/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas de Membrana/fisiologia , Mesoderma/fisiologia , Receptor Notch1/fisiologia , Fatores de Transcrição/fisiologia , Sítios de Ligação , Mama/citologia , Neoplasias da Mama/genética , Caderinas/genética , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Proteínas de Membrana/genética , Mesoderma/citologia , Receptor Notch1/genética , Proteínas Serrate-Jagged , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética
19.
J Immunol ; 175(5): 2890-9, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16116175

RESUMO

During embryogenesis, vascular and hemopoietic cells originate from a common precursor, the hemangioblast. Recent evidence suggests the existence of endothelial precursors in adult bone marrow cells, but it is unclear whether those precursors have a role in tumor neovascularization. In this report, we demonstrate that murine bone marrow contains endothelial progenitors, which arise from a cell with self-renewing capacity, and can integrate into tumor microvasculature, albeit at a very low frequency. A transgenic double-reporter strategy allowed us to demonstrate definitively that tumor bone marrow-derived endothelial cells arise by transdifferentiation of marrow progenitors rather than by cell fusion. Single cell transplants showed that a common precursor contributes to both the hemopoietic and endothelial lineages, thus demonstrating the presence of an adult hemangioblast. Furthermore, we demonstrate that increased vascular endothelial growth factor (VEGF)-A secretion by tumor cells, as well as activation of VEGF receptor-2 in bone marrow cells does not alter the mobilization and incorporation of marrow-derived endothelial progenitors into tumor vasculature. Finally, in human umbilical cord blood cells, we show that endothelial precursors make up only approximately 1 in 10(7) mononuclear cells but are highly enriched in the CD133+ cell population. By ruling out cell fusion, we clearly demonstrate the existence of an adult hemangioblast, but the differentiation of marrow stem cells toward the endothelial lineage is an extremely rare event. Furthermore, we show that VEGF-A stimulation of hemopoietic cells does not significantly alter this process.


Assuntos
Células da Medula Óssea/citologia , Células Endoteliais/citologia , Células-Tronco Hematopoéticas/citologia , Neoplasias Experimentais/irrigação sanguínea , Antígeno AC133 , Animais , Antígenos CD/análise , Diferenciação Celular , Linhagem da Célula , Sangue Fetal/citologia , Glicoproteínas/análise , Camundongos , Células NIH 3T3 , Peptídeos/análise , Fator A de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia
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