RESUMEN
ß-catenin is the key component of the canonical Wnt pathway and plays a crucial role in a multitude of developmental and homeostatic processes. The different tasks of ß-catenin are orchestrated by its subcellular localization and participation in multiprotein complexes. To gain a better understanding of ß-catenin's role in living cells we have generated a new set of single domain antibodies, referred to as nanobodies, derived from heavy chain antibodies of camelids. We selected nanobodies recognizing the N-terminal, core or C-terminal domain of ß-catenin and applied these new high-affinity binders as capture molecules in sandwich immunoassays and co-immunoprecipitations of endogenous ß-catenin complexes. In addition, we engineered intracellularly functional anti-ß-catenin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. For the first time, we were able to visualize the subcellular localization and nuclear translocation of endogenous ß-catenin in living cells using these chromobodies. Moreover, the chromobody signal allowed us to trace the accumulation of diffusible, hypo-phosphorylated ß-catenin in response to compound treatment in real time using High Content Imaging. The anti-ß-catenin nanobodies and chromobodies characterized in this study are versatile tools that enable a novel and unique approach to monitor the dynamics of subcellular ß-catenin in biochemical and cell biological assays.
Asunto(s)
Camélidos del Nuevo Mundo/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , beta Catenina/química , beta Catenina/metabolismo , Animales , Sitios de Unión , Línea Celular , Núcleo Celular/metabolismo , Cromatografía de Afinidad , Citoplasma/metabolismo , Técnica del Anticuerpo Fluorescente/métodos , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Transporte de ProteínasRESUMEN
ES cells can self-renew while preserving pluripotency and are able to differentiate into many cell types. In both processes, different signal transduction pathways are implicated, including the Wnt/beta-catenin pathway, which we here further analyzed. We found that a loss of beta-catenin in ES cells leads to altered expression of stem cell marker genes. TCF/beta-catenin reporter gene assays indicate that undifferentiated murine ES cells are capable of reacting to LiCl and Wnt3a but not Wnt5a stimulation, but have low endogenous TCF/beta-catenin activity. Oct-3/4, nanog and Wnt11 were able to repress TCF/beta-catenin transcriptional activity. During differentiation, activation of the Wnt/beta-catenin pathway influences formation of mesoderm and cardiomyocytes in a time and dose dependent manner.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , beta Catenina/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Células Madre Embrionarias/efectos de los fármacos , Proteínas Fetales/genética , Cloruro de Litio/farmacología , Ratones , Ratones Noqueados , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Proteínas de Dominio T Box/genética , Factores de Transcripción TCF/metabolismo , Factores de Transcripción/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteínas Wnt/farmacología , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/deficiencia , beta Catenina/genéticaRESUMEN
Advances in embryology, genetics, and regenerative medicine regularly attract attention from scientists, scholars, journalists, and policymakers, yet implications of these advances may be broader than commonly supposed. Laboratories culturing human embryos, editing human genes, and creating human-animal chimeras have been working along lines that are now becoming intertwined. Embryogenic methods are weaving traditional in vivo and in vitro distinctions into a new "in vivitro" (in life in glass) fabric. These and other methods known to be in use or thought to be in development promise soon to bring society to startling choices and discomfiting predicaments, all in a global effort to supply reliably rejuvenating stem cells, to grow immunologically non-provocative replacement organs, and to prevent, treat, cure, or even someday eradicate diseases having genetic or epigenetic mechanisms. With humanity's human-engineering era now begun, procedural prohibitions, funding restrictions, institutional controls, and transparency rules are proving ineffective, and business incentives are migrating into the most basic life-sciences inquiries, wherein lie huge biomedical potentials and bioethical risks. Rights, health, and heritage are coming into play with bioethical presumptions and formal protections urgently needing reassessment.
Asunto(s)
Embriología/métodos , Terapia Genética/métodos , Medicina Regenerativa/métodos , Sistemas CRISPR-Cas , Embriología/ética , Embriología/legislación & jurisprudencia , Desarrollo Embrionario/fisiología , Edición Génica/ética , Edición Génica/métodos , Terapia Genética/ética , Terapia Genética/legislación & jurisprudencia , Humanos , Medicina Regenerativa/ética , Medicina Regenerativa/legislación & jurisprudencia , Investigación con Células Madre/ética , Investigación con Células Madre/legislación & jurisprudenciaRESUMEN
Advances in embryology, genetics, and regenerative medicine regularly attract attention from scientists, scholars, journalists, and policymakers, yet implications of these advances may be broader than commonly supposed. Laboratories culturing human embryos, editing human genes, and creating human-animal chimeras have been working along lines that are now becoming intertwined. Embryogenic methods are weaving traditional in vivo and in vitro distinctions into a new "in vivitro" (in life in glass) fabric. These and other methods known to be in use or thought to be in development promise soon to bring society to startling choices and discomfiting predicaments, all in a global effort to supply reliably rejuvenating stem cells, to grow immunologically non-provocative replacement organs, and to prevent, treat, cure, or even someday eradicate diseases having genetic or epigenetic mechanisms. With humanity's human-engineering era now begun, procedural prohibitions, funding restrictions, institutional controls, and transparency rules are proving ineffective, and business incentives are migrating into the most basic life-sciences inquiries, wherein lie huge biomedical potentials and bioethical risks. Rights, health, and heritage are coming into play with bioethical presumptions and formal protections urgently needing reassessment.
Asunto(s)
Embriología , Ingeniería Genética/métodos , Genética , Medicina Regenerativa , Bioética , Sistemas CRISPR-Cas/fisiología , Quimera/metabolismo , Edición Génica/ética , Edición Génica/métodos , Ingeniería Genética/ética , Humanos , Técnicas In VitroRESUMEN
The fundamental role of p38 mitogen-activated protein kinases (MAPKs) in inflammation underlines their importance as therapeutic targets for various inflammatory medical conditions, including infectious, vascular, neurobiological and autoimmune disease. Although decades of research have yielded several p38 inhibitors, most clinical trials have failed, due to lack of selectivity and efficacy in vivo. This underlines the continuous need to screen for novel structures and chemotypes of p38 inhibitors. Here we report an optimized MK2-EGFP translocation assay in a semi-automated image based High Content Analysis (HCA) system to screen a combinatorial library of 3362 proprietary compounds with extensive variations of chemotypes. By determining the levels of redistribution of MK2-EGFP upon activation of the Rac/p38 pathway in combination with compound treatment, new candidates were identified, which modulate p38 activity in living cells. Based on integrated analysis of TNFα release from human whole blood, biochemical kinase activity assays and JNK3 selectivity testing, we show that this cell based assay reveals a high overlap and predictability for cellular efficacy, selectivity and potency of tested compounds. As a result we disclose a new comprehensive short-list of subtype inhibitors which are functional in the low nanomolar range and might provide the basis for further lead-optimization. In accordance to previous reports, we demonstrate that the MK2-EGFP translocation assay is a suitable primary screening approach for p38-MAPK drug development and provide an attractive labor- and cost saving alternative to other cell based methods including determination of cytokine release from hPBMCs or whole blood.
Asunto(s)
Bioensayo/métodos , Proteínas Fluorescentes Verdes/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Inhibidores Enzimáticos/farmacología , Proteínas Fluorescentes Verdes/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas/genética , Transporte de Proteínas/fisiologíaRESUMEN
Telomerase activity controls telomere length and plays a pivotal role in stem cells, aging, and cancer. Here, we report a molecular link between Wnt/ß-catenin signaling and the expression of the telomerase subunit Tert. ß-Catenin-deficient mouse embryonic stem (ES) cells have short telomeres; conversely, ES cell expressing an activated form of ß-catenin (ß-cat(ΔEx3/+)) have long telomeres. We show that ß-catenin regulates Tert expression through the interaction with Klf4, a core component of the pluripotency transcriptional network. ß-Catenin binds to the Tert promoter in a mouse intestinal tumor model and in human carcinoma cells. We uncover a previously unknown link between the stem cell and oncogenic potential whereby ß-catenin regulates Tert expression, and thereby telomere length, which could be critical in human regenerative therapy and cancer.
Asunto(s)
Células Madre Adultas/metabolismo , Células Madre Embrionarias/metabolismo , Neoplasias/metabolismo , Telomerasa/genética , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Neoplasias/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Telomerasa/metabolismo , Telómero/metabolismo , Telómero/ultraestructura , Homeostasis del Telómero , Sitio de Iniciación de la Transcripción , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMEN
MicroRNAs (miRs) and the canonical Wnt pathway are known to be dysregulated in human cancers and play key roles during cancer initiation and progression. To identify miRs that can modulate the activity of the Wnt pathway we performed a cell-based overexpression screen of 470 miRs in human HEK293 cells. We identified 38 candidate miRs that either activate or repress the canonical Wnt pathway. A literature survey of all verified candidate miRs revealed that the Wnt-repressing miRs tend to be anti-oncomiRs and down-regulated in cancers while Wnt-activating miRs tend to be oncomiRs and upregulated during tumorigenesis. Epistasis-based functional validation of three candidate miRs, miR-1, miR-25 and miR-613, confirmed their inhibitory role in repressing the Wnt pathway and suggest that while miR-25 may function at the level of â-catenin (ß-cat), miR-1 and miR-613 act upstream of ß-cat. Both miR-25 and miR-1 inhibit cell proliferation and viability during selection of human colon cancer cell lines that exhibit dysregulated Wnt signaling. Finally, transduction of miR-1 expressing lentiviruses into primary mammary organoids derived from Conductin-lacZ mice significantly reduced the expression of the Wnt-sensitive ß-gal reporter. In summary, these findings suggest the potential use of Wnt-modulating miRs as diagnostic and therapeutic tools in Wnt-dependent diseases, such as cancer.
Asunto(s)
MicroARNs/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Animales , Línea Celular Tumoral , Proliferación Celular , Evaluación Preclínica de Medicamentos/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Neoplasias/genética , Neoplasias/metabolismo , Vía de Señalización Wnt/genéticaRESUMEN
The vertebrate heart comprises a variety of cell types, the majority of which are cardiomyocytes, smooth muscle and endothelial cells. Their origin is still an intriguing research topic and the question is whether these cells derive from a common or from multiple distinct progenitor cell(s). Three recent publications not only suggest the existence of a single progenitor cell that can give rise to cardiovascular lineages but additionally uncovered, at least in part, the molecular identity of such a multipotent precursor cell. These findings constitute major progress in the quest for stem-cell therapies for cardiac diseases.
Asunto(s)
Miocardio/metabolismo , Animales , Biomarcadores , Diferenciación Celular , Miocardio/citología , Transducción de Señal , Células Madre/citología , Células Madre/metabolismoRESUMEN
Wnt proteins form a family of secreted signaling proteins that play a key role in various developmental events such as cell differentiation, cell migration, cell polarity and cell proliferation. It is currently thought that Wnt proteins activate at least three different signaling pathways by binding to seven transmembrane receptors of the Frizzled family and the co-receptor LRP6. Despite our growing knowledge of intracellular components that mediate a Wnt signal, the molecular events at the membrane have remained rather unclear. Now several publications(1-4) indicate that Frizzled receptors are G-protein coupled and kinases were identified that phosphorylate the co-receptor LRP6. These data deepen our understanding of Wnt-mediated signal transduction and provide more insight into how specificity may be achieved.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/metabolismo , Fosfotransferasas/metabolismo , Proteínas Wnt/metabolismo , Animales , Proteínas Relacionadas con Receptor de LDL/metabolismo , Unión Proteica , Transducción de SeñalRESUMEN
The evolutionary origin of the anterior-posterior and the dorsoventral body axes of Bilateria is a long-standing question. It is unclear how the main body axis of Cnidaria, the sister group to the Bilateria, is related to the two body axes of Bilateria. The conserved antagonism between two secreted factors, BMP2/4 (Dpp in Drosophila) and its antagonist Chordin (Short gastrulation in Drosophila) is a crucial component in the establishment of the dorsoventral body axis of Bilateria and could therefore provide important insight into the evolutionary origin of bilaterian axes. Here, we cloned and characterized two BMP ligands, dpp and GDF5-like as well as two secreted antagonists, chordin and gremlin, from the basal cnidarian Nematostella vectensis. Injection experiments in zebrafish show that the ventralizing activity of NvDpp mRNA is counteracted by NvGremlin and NvChordin, suggesting that Gremlin and Chordin proteins can function as endogenous antagonists of NvDpp. Expression analysis during embryonic and larval development of Nematostella reveals asymmetric expression of all four genes along both the oral-aboral body axis and along an axis perpendicular to this one, the directive axis. Unexpectedly, NvDpp and NvChordin show complex and overlapping expression on the same side of the embryo, whereas NvGDF5-like and NvGremlin are both expressed on the opposite side. Yet, the two pairs of ligands and antagonists only partially overlap, suggesting complex gradients of BMP activity along the directive axis but also along the oral-aboral axis. We conclude that a molecular interaction between BMP-like molecules and their secreted antagonists was already employed in the common ancestor of Cnidaria and Bilateria to create axial asymmetries, but that there is no simple relationship between the oral-aboral body axis of Nematostella and one particular body axis of Bilateria.