RESUMEN
ES-cell-based cardiovascular repair requires an in-depth understanding of the molecular mechanisms underlying the differentiation of cardiovascular ES cells. A candidate cardiovascular-fate inducer is the bHLH transcription factor MesP1. As one of the earliest markers, it is expressed specifically in almost all cardiovascular precursors and is required for cardiac morphogenesis. Here we show that MesP1 is a key factor sufficient to induce the formation of ectopic heart tissue in vertebrates and increase cardiovasculogenesis by ES cells. Electrophysiological analysis showed all subtypes of cardiac ES-cell differentiation. MesP1 overexpression and knockdown experiments revealed a prominent function of MesP1 in a gene regulatory cascade, causing Dkk-1-mediated blockade of canonical Wnt-signalling. Independent evidence from ChIP and in vitro DNA-binding studies, expression analysis in wild-type and MesP knockout mice, and reporter assays confirm that Dkk-1 is a direct target of MesP1. Further analysis of the regulatory networks involving MesP1 will be required to preprogramme ES cells towards a cardiovascular fate for cell therapy and cardiovascular tissue engineering. This may also provide a tool to elicit cardiac transdifferentiation in native human adult stem cells.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/fisiología , Transducción de Señal , Proteínas Wnt/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Inmunoprecipitación de Cromatina , Células Madre Embrionarias/citología , Endotelio Vascular/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Xenopus laevis/metabolismoRESUMEN
Wnt signaling regulates cell fate decisions and cell proliferation during development and in adult tissues in both invertebrates and vertebrates. Here we describe the identification of Wnt genes, Wnt2a, 4, 5a, 5b, 6 and 11, expressed in mouse embryonic gut development. Each of these genes exhibits a characteristic and regional-specific expression pattern along the anterior-posterior axis of the digestive tube between embryonic day (E) 12.5 and 16.5 of embryonic development. The expression of Wnt5a is confined to the mesenchymal compartment, while expression of Wnt4 is found both in the intestinal epithelium and the mesenteric anlage. Wnt11 is expressed in the epithelium of esophagus and colon, but also in mesenchymal cells of the stomach. Wnt5b and Wnt6 exhibit restricted expression in the epithelium of the esophagus. A characteristic regionalized expression pattern is observed in the developing stomach. Wnt5a is expressed in the mesenchymal layer of the prospective gland region but becomes restricted to the tip of the gland region by E14.5. Wnt11 is highly expressed at the gastro-esophageal junctions, while Wnt4 is found in the epithelium lining the pyloric region of the stomach but not in the epithelium of the prospective gland region.
Asunto(s)
Proteínas del Huevo/biosíntesis , Glicoproteínas/biosíntesis , Intestinos/embriología , Proteínas , Proteínas Proto-Oncogénicas/biosíntesis , Animales , Hibridación in Situ , Ratones , Modelos Genéticos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estómago/embriología , Factores de Tiempo , Distribución Tisular , Proteínas Wnt , Proteína wnt2RESUMEN
During mammalian development, the Cdx1 homeobox gene exhibits an early period of expression when the embryonic body axis is established, and a later period where expression is restricted to the embryonic intestinal endoderm. Cdx1 expression is maintained throughout adulthood in the proliferative cell compartment of the continuously renewed intestinal epithelium, the crypts. In this study, we provide evidence in vitro and in vivo that Cdx1 is a direct transcriptional target of the Wnt/(beta)-catenin signaling pathway. Upon Wnt stimulation, expression of Cdx1 can be induced in mouse embryonic stem (ES) cells as well as in undifferentiated rat embryonic endoderm. Tcf4-deficient mouse embryos show abrogation of Cdx1 protein in the small intestinal epithelium, making Tcf4 the likely candidate to transduce Wnt signal in this part of gut. The promoter region of the Cdx1 gene contains several Tcf-binding motifs, and these bind Tcf/Lef1/(beta)-catenin complexes and mediate (beta)-catenin-dependent transactivation. The transcriptional regulation of the homeobox gene Cdx1 in the intestinal epithelium by Wnt/(beta)-catenin signaling underlines the importance of this signaling pathway in mammalian endoderm development.
Asunto(s)
Proteínas Aviares , Proteínas del Citoesqueleto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , Transactivadores , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra , Células 3T3 , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodermo , Genes Homeobox , Humanos , Mucosa Intestinal/metabolismo , Intestinos/embriología , Factor de Unión 1 al Potenciador Linfoide , Ratones , Ratas , Factores de Transcripción TCF , Proteína 2 Similar al Factor de Transcripción 7 , Factores de Transcripción/genética , Proteínas Wnt , beta CateninaRESUMEN
The down-regulation of E-cadherin is a common event in carcinogenesis. Phosphorylation/dephosphorylation is one posttranscriptional process which may regulate intercellular junctions. Here we show that in okadaic acid-treated keratinocytes, E-cadherin expression is shifted from the membrane to the cytoplasm, preventing cells from forming aggregates. These changes of E-cadherin localization and function are associated with a decrease in its phosphorylation state. The decrease in E-cadherin phosphorylation was essentially detected in okadaic acid-treated cell lysates isolated from 0.5% Triton-soluble fraction and not in the Triton-insoluble fraction linked to the cytoskeleton, suggesting a role of E-cadherin phosphorylation in cell-cell interactions. E-cadherin was markedly phosphorylated by CK2, either the purified recombinant enzyme or the endogenous enzyme. Using specific CK2 inhibitors such as heparin and 5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole, endogenous CK2 was confirmed as the main enzyme phosphorylating E-cadherin. The decrease in E-cadherin phosphorylation by endogenous CK2 was not restored by the addition of purified CK2, confirming that it is not due to a defect in CK2 expression or to its reduced activity, but rather to the incapacity of CK2 to phosphorylate E-cadherin. The co-immunoprecipitation and colocalization of E-cadherin and CK2 suggests that CK2 may play a critical role in the maintenance of epidermis cohesion.
Asunto(s)
Cadherinas/metabolismo , Uniones Intercelulares/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Quinasa de la Caseína II , Adhesión Celular , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Uniones Intercelulares/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/fisiología , Ácido Ocadaico/farmacología , Fosforilación , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Beta-catenin, a member of the Armadillo repeat protein family, binds directly to the cytoplasmic domain of E-cadherin, linking it via alpha-catenin to the actin cytoskeleton. A 30-amino acid region within the cytoplasmic domain of E-cadherin, conserved among all classical cadherins, has been shown to be essential for beta-catenin binding. This region harbors several putative casein kinase II (CKII) and glycogen synthase kinase-3beta (GSK-3beta) phosphorylation sites and is highly phosphorylated. Here we report that in vitro this region is indeed phosphorylated by CKII and GSK-3beta, which results in an increased binding of beta-catenin to E-cadherin. Additionally, in mouse NIH3T3 fibroblasts expression of E-cadherin with mutations in putative CKII sites resulted in reduced cell-cell contacts. Thus, phosphorylation of the E-cadherin cytoplasmic domain by CKII and GSK-3beta appears to modulate the affinity between beta-catenin and E-cadherin, ultimately modifying the strength of cell-cell adhesion.
Asunto(s)
Cadherinas/metabolismo , Adhesión Celular , Proteínas del Citoesqueleto/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transactivadores , Células 3T3 , Animales , Secuencia de Bases , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Quinasa de la Caseína II , Citoplasma/enzimología , Citoplasma/metabolismo , Proteínas del Citoesqueleto/genética , Cartilla de ADN , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasas , Ratones , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Proteínas Recombinantes/metabolismo , beta CateninaRESUMEN
One of the hallmarks of polarized epithelial cells undergoing mitosis is their rounded morphology. This phenotype correlates with a reduced cell-substratum adhesion, apparently caused by a modulation of integrin function. However, it is still unclear whether the cadherin-mediated cell-cell adhesion is affected as well. To address this question, the cadherin complex was analyzed in different cell cycle stages of Madin-Darby canine kidney cells. By immunofluorescence, mitotic Madin-Darby canine kidney cells showed an increased staining of E-cadherin and the catenins (alpha-catenin, beta-catenin, plakoglobin, p120(ctn)) in the cytosol, suggesting a reorganization of the cadherin-catenin complex during mitosis. Biochemical analysis revealed that the overall amount of these components, as well as the proportion of the complex associated with the actin cytoskeleton, remained unchanged in mitotic cells. However, we found evidence for an internalization of E-cadherin during mitosis. In addition, the cadherin-catenin complex was analyzed for mitosis-specific changes in phosphorylation. We report a decrease in the tyrosine phosphorylation of beta-catenin, plakoglobin, and p120(ctn) during mitosis. Moreover, we observed a mitosis-specific Ser/Thr-phosphorylation of p120(ctn), as detected by the MPM-2 antibody. Hence, the cadherin/catenin complex is a target for different posttranslational modifications during mitosis, which may also have a profound impact on cadherin-mediated cell-cell adhesion.
Asunto(s)
Cadherinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Mitosis , Fosfoproteínas/metabolismo , Transactivadores , Proteína de la Poliposis Adenomatosa del Colon , Animales , Cadherinas/aislamiento & purificación , Cateninas , Moléculas de Adhesión Celular/aislamiento & purificación , Compartimento Celular , Proteínas del Citoesqueleto/aislamiento & purificación , Desmoplaquinas , Perros , Células Epiteliales/citología , Riñón/citología , Sustancias Macromoleculares , Mimosina/farmacología , Nocodazol/farmacología , Fosfoproteínas/aislamiento & purificación , Fosforilación , alfa Catenina , beta Catenina , gamma Catenina , Catenina deltaRESUMEN
A system for the documentation of radiographic diagnoses is described, based on the "documentation method for radiographic diagnosis" (Formmhold et al 1972) and on the diagnostic information system of the medical faculty of the University of Tübingen. The diagnostic indeces are dictated in relation to the findings and the diagnosis and fed on punched cards into a computer (IBM 1800). Various programmes provide patient-orientated diagnoses for clinical routine, as well as diagnoses-orientated lists of patients for demonstrations, teaching or research.