RESUMEN
In exploration of congenital heart defects produced by TCE, Hepatocyte Nuclear Factor 4 alpha (HNF4a) transcriptional activity was identified as a central component. TCE exposure altered gene transcription in the chick heart in a non-monotonic pattern where only low dose exposure inhibited transcription by HNF4a. As the chick embryo is non-placental, we examine here HNF4a as a target of TCE in developing mouse embryos. Benfluorex and Bi6015, published agonist and antagonist, respectively, of HNF4a were compared to low dose TCE exposure. Pregnant mice were exposed to 10 ppb (76 nM) TCE, 5 µM Benfluorex, 5 µM Bi6015, or a combination of Bi6015 and TCE in drinking water. Litters (E12) were collected during a sensitive window in heart development. Embryonic hearts were collected, pooled for extraction of RNA and marker expression was examined by quantitative PCR. Multiple markers, previously identified as sensitive to TCE exposure in chicks or as published targets of HNF4a transcription were significantly affected by Benfluorex, Bi6015 and TCE. Activity of TCE and both HNF4a-specific reagents on transcription argues that HNF4a is a component of TCE cardiotoxicity and likely a proximal target of low dose exposure during development. The effectiveness of these reagents after delivery in maternal drinking water suggests that neither maternal metabolism, nor placental transport is protective of exposure.
Asunto(s)
Tricloroetileno/toxicidad , Animales , Femenino , Corazón/embriología , Factor Nuclear 4 del Hepatocito/genética , Ratones , EmbarazoRESUMEN
Exposure to trichloroethylene (TCE) is linked to formation of congenital heart defects in humans and animals. Prior interactome analysis identified the transcription factor, Hepatocyte Nuclear Factor 4 alpha (HNF4a), as a potential target of TCE exposure. As a role for HNF4a is unknown in the heart, we examined developing avian hearts for HNF4a expression and for sensitivity to TCE and the HNF4a agonist, Benfluorex. In vitro analysis using a HNF4a reporter construct showed both TCE and HFN4a to be antagonists of HNF4a-mediated transcription at the concentrations tested. HNF4a mRNA is expressed transiently in the embryonic heart during valve formation and cardiac development. Embryos were examined for altered gene expression in the presence of TCE or Benfluorex. TCE altered expression of selected mRNAs including HNF4a, TRAF6 and CYP2C45. There was a transition between inhibition and induction of marker gene expression in embryos as TCE concentration increased. Benfluorex was largely inhibitory to selected markers. Echocardiography of exposed embryos showed reduced cardiac function with both TCE and Benfluorex. Cardiac contraction was reduced by 29% and 23%, respectively at 10 ppb. The effects of TCE and Benfluorex on autocrine regulation of HNF4a, selected markers and cardiac function argue for a functional interaction of TCE and HNF4a. Further, the dose-sensitive shift between inhibition and induction of marker expression may explain the nonmonotonic-like dose response observed with TCE exposure in the heart.
Asunto(s)
Contaminantes Ambientales/toxicidad , Corazón/efectos de los fármacos , Factor Nuclear 4 del Hepatocito/genética , Transcripción Genética/efectos de los fármacos , Tricloroetileno/toxicidad , Animales , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Ecocardiografía , Fenfluramina/análogos & derivados , Fenfluramina/farmacología , Genes Reporteros , Corazón/diagnóstico por imagen , Corazón/embriología , Células Hep G2 , Factor Nuclear 4 del Hepatocito/agonistas , Humanos , Miocardio/metabolismoRESUMEN
Endothelia in the atrioventricular (AV) canal of the developing heart undergo a prototypical epithelial mesenchymal transition (EMT) to begin heart valve formation. Using an in vitro invasion assay, an extracellular matrix protein, Olfactomedin-1 (OLFM1), was found to increase mesenchymal cell numbers in AV canals from embryonic chick hearts. Treatment with both anti-OLFM1 antibody and siRNA targeting OLFM1 inhibits mesenchymal cell formation. OLFM1 does not alter cell proliferation, migration or apoptosis. Dispersion, but lack of invasion in the presence of inhibiting antibody, identifies a specific role for OLFM1 in cell invasion during EMT. This role is conserved in other epithelia, as OLFM1 similarly enhances invasion by MDCK epithelial cells in a transwell assay. Synergy is observed when TGFß2 and OLFM1 are added to MDCK cell cultures, indicating that OLFM-1 activity is cooperative with TGFß. Inhibition of both OLFM1 and TGFß in heart invasion assays shows a similar cooperative role during development. To explore OLFM1 activity during EMT, representative EMT markers were examined. Effects of OLFM1 protein and anti-OLFM1 on transcripts of cell-cell adhesion molecules and the transcription factors Snail-1, Snail-2, Twist1 and Sox-9 argue that OLFM1 does not initiate EMT. Rather, regulation of transcripts of Zeb1 and Zeb2, secreted proteases and mesenchymal cell markers by both OLFM1 and anti-OLFM1 is consistent with regulation of the cell invasion step of EMT. We conclude that OLFM1 is present and necessary during EMT in the embryonic chick heart. Its role in cell invasion and mesenchymal cell gene regulation suggests an invasion checkpoint in EMT where OLFM1 acts to promote cell invasion into the three-dimensional matrix.
Asunto(s)
Movimiento Celular , Transición Epitelial-Mesenquimal , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Corazón/embriología , Animales , Anticuerpos/farmacología , Biomarcadores/metabolismo , Muerte Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Perros , Transición Epitelial-Mesenquimal/efectos de los fármacos , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/inmunología , Glicoproteínas/genética , Glicoproteínas/inmunología , Corazón/efectos de los fármacos , Humanos , Células de Riñón Canino Madin Darby , Mesodermo/citología , Mesodermo/efectos de los fármacos , Mesodermo/embriología , Miocardio/citología , Miocardio/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Trichloroethylene (TCE) is an organic solvent and common environmental contaminant. TCE exposure is associated with heart defects in humans and animal models. Primary metabolism of TCE in adult rodent models is by specific hepatic cytochrome P450 enzymes (Lash et al. in Environ Health Perspect 108:177-200, 2000). As association of TCE exposure with cardiac defects is in exposed embryos prior to normal liver development, we investigated metabolism of TCE in the early embryo. Developing chick embryos were dosed in ovo with environmentally relevant doses of TCE (8 and 800 ppb) and RNA was extracted from cardiac and extra-cardiac tissue (whole embryo without heart). Real-time PCR showed upregulation of CYP2H1 transcripts in response to TCE exposure in the heart. No detectable cytochrome expression was found in extra-cardiac tissue. As seen previously, the dose response was non-monotonic and 8 ppb elicited stronger upregulation than 800 ppb. Immunostaining for CYP2C subfamily expression confirmed protein expression and showed localization in both myocardium and endothelium. TCE exposure increased protein expression in both tissues. These data demonstrate that the earliest embryonic expression of phase I detoxification enzymes is in the developing heart. Expression of these CYPs is likely to be relevant to the susceptibility of the developing heart to environmental teratogens.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Regulación Enzimológica de la Expresión Génica , Corazón/efectos de los fármacos , Corazón/embriología , Tricloroetileno/administración & dosificación , Animales , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Solventes/administración & dosificaciónRESUMEN
The cellular process of epithelial-mesenchymal cell transition (EMT) is a critical event in development that is reiterated in adult pathologies of metastasis and organ fibrosis. An initial understanding of the cellular and molecular events of this process emerged from an in vitro examination of heart valve development. Explants of the chick atrioventricular valve-forming region were placed on collagen gels and removed to show that EMT was regulated by a tissue interaction. Subsequent studies showed that specific TGFß isoforms and receptors were required and steps of activation and invasion could be distinguished. The assay was modified for mouse hearts and has been used to explore signal transduction and gene expression in both species. The principle advantages of the system are a defined temporal window, when EMT takes place and the ability to isolate cells at various stages of the EMT process. These advantages are largely unavailable in other developmental or adult models. As the mesenchymal cells produced by EMT in the heart are involved in defects found in congenital heart disease, there is also a direct relevance of cardiac EMT to human birth defects. This relationship has been explored in relation to environmental exposures and in a number of genetic models. This review provides both an overview of the findings developed from the assay and protocols to enable the use of the assay by other laboratories. The assay provides a versatile platform to explore roles of specific gene products, drugs, and environmental agents on a critical cellular process.
Asunto(s)
Colágeno , Transición Epitelial-Mesenquimal/fisiología , Corazón/embriología , Modelos Biológicos , Miocardio/citología , Animales , Diferenciación Celular , Embrión de Pollo , Células Epiteliales/citología , Regulación del Desarrollo de la Expresión Génica , Humanos , Mesodermo/citología , Ratones , Miocardio/metabolismo , Transducción de SeñalRESUMEN
Arsenic is a naturally occurring metalloid and environmental contaminant. Arsenic exposure in drinking water is reported to cause cancer of the liver, kidneys, lung, bladder, and skin as well as birth defects, including neural tube, facial, and vasculogenic defects. The early embryonic period most sensitive to arsenic includes a variety of cellular processes. One key cellular process is epithelial-mesenchymal transition (EMT) where epithelial sheets develop into three-dimensional structures. An embryonic prototype of EMT is found in the atrioventricular (AV) canal of the developing heart, where endothelia differentiate to form heart valves. Effects of arsenic on this cellular process were examined by collagen gel invasion assay (EMT assay) using explanted AV canals from chicken embryo hearts. AV canals treated with 12.5-500 ppb arsenic showed a loss of mesenchyme at 12.5 ppb, and mesenchyme formation was completely inhibited at 500 ppb. Altered gene expression in arsenic-treated explants was investigated by microarray analysis. Genes whose expression was altered consistently at exposure levels of 10, 25, and 100 ppb were identified, and results showed that 25 ppb in vitro was particularly effective. Three hundred and eighty two genes were significantly altered at this exposure level. Cytoscape analysis of the microarray data using the chicken interactome identified four clusters of altered genes based on published relationships and pathways. This analysis identified cytoskeleton and cell adhesion-related genes whose disruption is consistent with an altered ability to undergo EMT. These studies show that EMT is sensitive to arsenic and that an interactome-based approach can be useful in identifying targets.
Asunto(s)
Arsénico/toxicidad , Colágeno , Células Epiteliales/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Mesodermo/efectos de los fármacos , Animales , Secuencia de Bases , Embrión de Pollo , Cartilla de ADN , Etiquetado Corte-Fin in Situ , Mesodermo/citologíaRESUMEN
Increasing evidence indicates that maintenance of neuronal homeostasis involves the activation of the cell cycle machinery in postmitotic neurons. Our recent findings suggest that cell cycle activation is essential for DNA damage-induced neuronal apoptosis. However, whether the cell division cycle also participates in DNA repair and survival of postmitotic, terminally differentiated neurons is unknown. Here, we tested the hypothesis that G(1) phase components contribute to the repair of DNA and are involved in the DNA damage response of postmitotic neurons. In cortical terminally differentiated neurons, treatment with subtoxic concentrations of hydrogen peroxide (H(2)O(2)) caused repairable DNA double strand breaks (DSBs) and the activation of G(1) components of the cell cycle machinery. Importantly, DNA repair was attenuated if cyclin-dependent kinases CDK4 and CDK6, essential elements of G(0) --> G(1) transition, were suppressed. Our data suggest that G(1) cell cycle components are involved in DNA repair and survival of postmitotic neurons.