RESUMEN
Caspase-1 or interleukin-1ß (IL-1ß) converting enzyme is a pro-inflammatory member of the caspase family. An IL-1ß-independent role for caspase-1 in cardiomyocyte cell death and heart failure has emerged but the mechanisms underlying these effects are incompletely understood. Here, we report that transcription factor GATA4, a key regulator of cardiomyocyte survival and adaptive stress response is an in vivo and in vitro substrate for caspase-1. Caspase-1 mediated cleavage of GATA4 generates a truncated protein that retains the ability to bind DNA but lacks transcriptional activation domains and acts as a dominant negative regulator of GATA4. We show that caspase-1 is rapidly activated in cardiomyocyte nuclei treated with the cell death inducing drug Doxorubicin. We also find that inhibition of caspase-1 alone is as effective as complete caspase inhibition at rescuing GATA4 degradation and myocyte cell death. Caspase-1 inhibition of GATA4 transcriptional activity is rescued by HSP70, which binds directly to GATA4 and masks the caspase recognition motif. The data identify a caspase-1 nuclear substrate and suggest a direct role for caspase-1 in transcriptional regulation. This mechanism may underlie the inflammation-independent action of caspase-1 in other organs.
Asunto(s)
Caspasa 1/metabolismo , Factor de Transcripción GATA4/metabolismo , Miocitos Cardíacos/enzimología , Animales , Caspasa 1/química , Caspasa 1/genética , Línea Celular , Núcleo Celular/enzimología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Supervivencia Celular , Células Cultivadas , Factor de Transcripción GATA4/genética , Proteínas del Choque Térmico HSP72/genética , Proteínas del Choque Térmico HSP72/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Activación TranscripcionalRESUMEN
BACKGROUND: Hairdressers are exposed to chemicals and work tasks that may cause respiratory symptoms. There is little awareness of occupational health among hairdressing salons in Palestine. AIMS: To characterize respiratory symptoms, lung function, and knowledge of exposure to hazards among female Palestinian hairdressers. METHODS: Cross-sectional study of female hairdressers and controls of female university students and staff. Working history and respiratory symptoms were collected using questionnaire. Lung function was measured. Working conditions were characterized in salons. RESULTS: A total of 170 hairdressers from 56 salons and 170 controls participated. Nineteen per cent of the hairdressers reported wheezing versus 11% in the control group. The mean forced vital capacity was 3.31 l compared with 3.42 l for controls. Adjusting for age and height, there was a forced expiratory volume in 1 s reduction of 0.093 l (95% confidence interval (CI) = 0.06-0.15) comparing hairdressers with controls. A small number of hairdressers used respiratory protective equipment, and satisfactory ventilation in salons were lacking. CONCLUSIONS: Female hairdressers had higher prevalence of reported asthma and respiratory symptoms than the controls, but these differences reduced markedly when adjusted for age, height, weight and years of education. They had lower lung function measurements than the control group. Increasing the awareness of occupational health hazards and improving the work conditions for the hairdressers in Palestine is needed. Possible bias could be present as hairdressers might have over reported symptoms or lung function measurements might be affected by differences in socioeconomic status between the two groups.
Asunto(s)
Asma/etiología , Peluquería , Industria de la Belleza , Sustancias Peligrosas/efectos adversos , Pulmón/fisiopatología , Enfermedades Profesionales/etiología , Exposición Profesional/efectos adversos , Adulto , Árabes , Asma/epidemiología , Asma/fisiopatología , Estudios de Casos y Controles , Estudios Transversales , Femenino , Volumen Espiratorio Forzado , Humanos , Medio Oriente/epidemiología , Enfermedades Profesionales/epidemiología , Enfermedades Profesionales/fisiopatología , Ocupaciones , Prevalencia , Ruidos Respiratorios , Enfermedades Respiratorias/epidemiología , Enfermedades Respiratorias/etiología , Enfermedades Respiratorias/fisiopatología , Encuestas y Cuestionarios , Capacidad VitalRESUMEN
From an analysis of tangent spherical drops in straining flow, Baldessari and Leal conclude that the drop-scale internal circulation, driven by the ambient flow, has a negligible influence on the drainage of the thin liquid film between drops under small-deformation conditions [F. Baldessari, L.G. Leal, J. Colloid Interface Sci. 289 (2005) 262]. However, their conclusion is incorrect as explained in this letter.
RESUMEN
We analyze axisymmetric near-contact motion of two drops under the action of an external force or imposed flow. It is shown that hydrodynamic stresses in the near-contact region that are associated with the outer (drop-scale) flow can qualitatively affect the drainage of the thin fluid film separating the drops. If this far-field stress acts radially inward, film drainage is arrested at long times; exponential film drainage occurs if this stress acts outward. An asymptotic analysis of the stationary long-time film profile is presented for small-deformation conditions, and the critical strength of van der Waals attraction for film rupture is calculated. The effect of an insoluble surfactant is also considered. Hindered and enhanced drop coalescence are not predicted by the current theories, because the influence of the outer flow on film drainage is ignored.
RESUMEN
To identify the mechanisms that couple hemodynamic stress to alterations in cardiac gene expression, DNA constructs containing the rat B-type natriuretic peptide (BNP) promoter were injected into the myocardium of rats, which underwent bilateral nephrectomy or were sham-operated. Ventricular BNP mRNA levels were induced about 4-fold; and the BNP reporter construct containing the proximal 2200 bp, 5-fold, in response to 1-d nephrectomy. Deletion of sequences between bp -2200 and -114 did not affect basal or inducible activity of the BNP promoter. An activator protein-1-like site and two tandem GATA elements are located within this 114-bp sequence. Both deletion and mutation of the AP-1-like motif decreased basal activity but did not abolish the response to nephrectomy. In contrast, mutation or deletion of -90 bp GATA-sites abrogated the response to hemodynamic stress. The importance of these GATA elements to BNP promoter activation was further confirmed by the corresponding 38-bp oligonucleotide conferring hemodynamic stress responsiveness to a minimal BNP promoter. In gel mobility shift assays, nephrectomy increased left ventricular BNP GATA4 binding activity significantly. In conclusion, GATA elements are necessary and sufficient to confer transcriptional activation of BNP gene in response to hemodynamic stress.
Asunto(s)
Factor Natriurético Atrial/genética , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/fisiología , Hemodinámica/fisiología , Estrés Fisiológico/fisiopatología , Factores de Transcripción/fisiología , Secuencias de Aminoácidos/fisiología , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción GATA4 , Factor de Transcripción GATA6 , Corazón/fisiología , Ventrículos Cardíacos , Hipertensión/fisiopatología , Masculino , Miocardio/metabolismo , Péptido Natriurético Encefálico , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Regulación hacia ArribaRESUMEN
Rho-like GTPases play a pivotal role in the orchestration of changes in the actin cytoskeleton in response to receptor stimulation, and have been implicated in transcriptional activation, cell growth regulation, and oncogenic transformation. Recently, a role for RhoA in the regulation of cardiac contractility and hypertrophic cardiomyocyte growth has been suggested but the mechanisms underlying RhoA function in the heart remain undefined. We now report that transcription factor GATA-4, a key regulator of cardiac genes, is a nuclear mediator of RhoA signaling and is involved in the control of sarcomere assembly in cardiomyocytes. Both RhoA and GATA-4 are essential for sarcomeric reorganization in response to hypertrophic growth stimuli and overexpression of either protein is sufficient to induce sarcomeric reorganization. Consistent with convergence of RhoA and GATA signaling, RhoA potentiates the transcriptional activity of GATA-4 via a p38 MAPK-dependent pathway that phosphorylates GATA-4 activation domains and GATA binding sites mediate RhoA activation of target cardiac promoters. Moreover, a dominant-negative GATA-4 protein abolishes RhoA-induced sarcomere reorganization. The identification of transcription factor GATA-4 as a RhoA mediator in sarcomere reorganization and cardiac gene regulation provides a link between RhoA effects on transcription and cell remodeling.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Miocardio/metabolismo , Factores de Transcripción/metabolismo , Proteína de Unión al GTP rhoA/genética , Adenoviridae/genética , Northern Blotting , Western Blotting , Células Cultivadas , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Endotelina-1/farmacología , Elementos de Facilitación Genéticos , Técnica del Anticuerpo Fluorescente , Factor de Transcripción GATA4 , Regulación de la Expresión Génica , Humanos , Hibridación in Situ , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfoproteínas Fosfatasas/farmacología , Regiones Promotoras Genéticas , ARN/metabolismo , Sarcómeros/fisiología , Factores de Transcripción/genética , Proteínas Quinasas p38 Activadas por Mitógenos , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The identification and molecular cloning of the cardiac transcription factors GATA-4, -5, and -6 has greatly contributed to our understanding of how tissue-specific transcription is achieved during cardiac growth and development. Through analysis of their interacting partners, it has also become apparent that a major mechanism underlying spatial and temporal specificity within the heart as well as in the response to cardiogenic regulators is the combinatorial interaction between cardiac-restricted and inducible transcription factors. The cardiac GATA factors appear to be fundamental contributors to these regulatory networks. Two of the first targets identified for the cardiac GATA factors were the natriuretic peptide genes encoding atrial natriuretic factor (ANF) and B-type natriuretic peptide (BNP), the major heart secretory products that are also accepted clinical markers of the diseased heart. Studies using the ANF and BNP promoters as models of cardiac-specific transcription have unraveled the pivotal role that GATA proteins play in cardiac gene expression. We review the current knowledge on the modulation of the natriuretic peptide promoters by GATA factors, including examples of combinatorial interactions between GATA proteins and diverse transcription factors.
Asunto(s)
Factor Natriurético Atrial/genética , Corazón/fisiología , Péptido Natriurético Encefálico/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Animales , Factor Natriurético Atrial/biosíntesis , Proteínas de Unión al ADN/genética , Factor de Transcripción GATA4 , Factor de Transcripción GATA5 , Factor de Transcripción GATA6 , Corazón/crecimiento & desarrollo , Humanos , Miocardio/metabolismo , Péptido Natriurético Encefálico/biosíntesis , Especificidad de Órganos , Factores de Transcripción/biosíntesis , Transcripción GenéticaRESUMEN
Heterozygous Tbx5(del/+) mice were generated to study the mechanisms by which TBX5 haploinsufficiency causes cardiac and forelimb abnormalities seen in Holt-Oram syndrome. Tbx5 deficiency in homozygous mice (Tbx5(del/del)) decreased expression of multiple genes and caused severe hypoplasia of posterior domains in the developing heart. Surprisingly, Tbx5 haploinsufficiency also markedly decreased atrial natriuretic factor (ANF) and connexin 40 (cx40) transcription, implicating these as Tbx5 target genes and providing a mechanism by which 50% reduction of T-box transcription factors cause disease. Direct and cooperative transactivation of the ANF and cx40 promoters by Tbx5 and the homeodomain transcription factor Nkx2-5 was also demonstrated. These studies provide one potential explanation for Holt-Oram syndrome conduction system defects, suggest mechanisms for intrafamilial phenotypic variability, and account for related cardiac malformations caused by other transcription factor mutations.
Asunto(s)
Anomalías Múltiples/genética , Factor Natriurético Atrial/genética , Desarrollo Óseo/fisiología , Cardiopatías Congénitas/genética , Proteínas de Dominio T Box/genética , Envejecimiento , Animales , Secuencia de Bases , Sitios de Unión , Desarrollo Óseo/genética , Diferenciación Celular , Conexinas/genética , Modelos Animales de Enfermedad , Electrocardiografía , Desarrollo Embrionario y Fetal , Miembro Anterior/anomalías , Corazón/embriología , Cardiopatías Congénitas/fisiopatología , Heterocigoto , Homocigoto , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Miocardio/citología , Regiones Promotoras Genéticas , Ratas , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Ovinos , Síndrome , Proteínas de Dominio T Box/deficiencia , Proteína alfa-5 de Unión ComunicanteRESUMEN
YY1, a multifunctional protein essential for embryonic development, is a known repressor or activator of transcription. In cardiac and skeletal myocytes, YY1 has been described essentially as a negative regulator of muscle-specific genes. In this study, we report that YY1 is a transcriptional activator of the B-type natriuretic peptide (BNP) gene, which encodes one of the heart major secretory products. YY1 binds an element within the proximal cardiac BNP promoter, in close proximity to the high affinity binding sites for the zinc finger GATA proteins. We show that YY1 cooperates with GATA-4 to synergistically activate BNP transcription. Structure-function analysis revealed that the DNA binding domain of YY1 is sufficient for cooperative interaction with GATA-4, likely through corecruitment of the CREB-binding protein coactivator. The results suggest that YY1 and GATA factors are components of transcriptionally active complexes present in cardiac and other GATA-containing cells.
Asunto(s)
Factor Natriurético Atrial/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Factor Natriurético Atrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA4 , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Péptido Natriurético Encefálico , Oligonucleótidos/farmacología , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Factor de Transcripción YY1RESUMEN
Endothelins are a family of biologically active peptides that are critical for development and function of neural crest-derived and cardiovascular cells. These effects are mediated by two G-protein-coupled receptors and involve transcriptional regulation of growth-responsive and/or tissue-specific genes. We have used the cardiac ANF promoter, which represents the best-studied tissue-specific endothelin target, to elucidate the nuclear pathways responsible for the transcriptional effects of endothelins. We found that cardiac-specific response to endothelin 1 (ET-1) requires the combined action of the serum response factor (SRF) and the tissue-restricted GATA proteins which bind over their adjacent sites, within a 30-bp ET-1 response element. We show that SRF and GATA proteins form a novel ternary complex reminiscent of the well-characterized SRF-ternary complex factor interaction required for transcriptional induction of c-fos in response to growth factors. In transient cotransfections, GATA factors and SRF synergistically activate atrial natriuretic factor and other ET-1-inducible promoters that contain both GATA and SRF binding sites. Thus, GATA factors may represent a new class of tissue-specific SRF accessory factors that account for muscle- and other cell-specific SRF actions.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Receptores de Superficie Celular/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Animales , Factor Natriurético Atrial/genética , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , Células Cultivadas , Cartilla de ADN/genética , Endotelina-1/farmacología , Factor de Transcripción GATA4 , Células HeLa , Humanos , Sustancias Macromoleculares , Miocardio/metabolismo , Regiones Promotoras Genéticas , Ratas , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/metabolismo , Factor de Respuesta Sérica , Transducción de SeñalRESUMEN
Understanding the molecular mechanisms controlling cardiac-specific gene transcription requires the dissection of the cis-elements that govern the complex spatio-temporal expression of these genes. The four-chambered vertebrate heart is formed during the late phases of fetal development following a series of complex morphogenetic events that require the functional presence of different proteins. The gradient-like expression of some genes, as well as the chamber-specific expression of others, is tightly regulated by combinatorial interactions of several transcription factors and their cofactors. Chamber- and stage-specific cardiac myocyte cultures have been invaluable for identifying transcription factor binding sites involved in basal, chamber-specific, and inducible expression of many cardiac promoters; these studies, which were largely confirmed in vivo in transgenic mouse models, led to the isolation of key regulators of heart development. In addition, the use of pluripotent embryonic stem cells helped elucidate the early molecular events controlling cardiomyocyte differentiation. Together, these studies point to a major role for GATA transcription factors and their interacting partners in transcriptional control of heart development. In addition, members of the T-box family of transcription factors and homeodomain containing proteins, together with chamber-restricted transcriptional repressors and co-repressors play critical roles in heart septation and chamber specification. These fine-tuned cooperative interactions between different classes of proteins are at the basis of normal cardiac function, and alteration in their expression level or function leads to cardiac pathologies.
Asunto(s)
Regulación de la Expresión Génica , Corazón/embriología , Factores de Transcripción/fisiología , Proteínas de Xenopus , Animales , Factor Natriurético Atrial/fisiología , Proteínas de Unión al ADN/fisiología , Factor de Transcripción GATA4 , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/fisiología , Humanos , Factores de Transcripción MEF2 , Factores Reguladores Miogénicos , Fosforilación , Regiones Promotoras Genéticas , Proteínas de Dominio T Box/fisiologíaRESUMEN
Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal alpha-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Miocardio/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Actinas/genética , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Factor de Transcripción GATA4 , Factor de Transcripción GATA5 , Factor de Transcripción GATA6 , Regulación de la Expresión Génica , Genes Reporteros , Haplorrinos , Músculo Esquelético/metabolismo , Mutación , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión , Elementos de Respuesta , Factor de Respuesta Sérica , Transactivadores/genética , Factores de Transcripción/genética , Transfección , Dedos de ZincRESUMEN
We examined if elevation in lysophosphatidylethanolamine acyltransferase activity was associated with elevation in phosphatidylethanolamine content during differentiation of P19 teratocarcinoma cells into cardiac myocytes. P19 cells were induced to undergo differentiation into cardiac myocytes by the addition of 1% dimethylsulfoxide to the medium. Immunofluorescence microscopy revealed the presence of striated myosin at 8 days post-dimethylsulfoxide addition confirming differentiation into cardiac cells. The content of phosphatidylethanolamine was increased 2.1-fold (P<0.05) in differentiated cells compared to undifferentiated cells, whereas the content of phosphatidylcholine was reduced 29% (P<0.05). There were no alterations in the pool sizes of other phospholipids, including cardiolipin. The relative abundance of fatty acids in phospholipids of P19 cells was 18:1 > 18:0 > 16:1 = 18:2 > 16:0 = 14:0 > 20:4 and differentiation did not affect the relative amounts of these fatty acids within individual phospholipids. When cells were incubated with [1,3-(3)H]glycerol, radioactivity incorporated into phosphatidylethanolamine was elevated 5.8-fold, whereas radioactivity incorporated into phosphatidylcholine was unaltered. Ethanolaminephosphotransferase, cholinephosphotransferase and membrane CTP:phosphocholine cytidylyltransferase activities were elevated in differentiated cells compared to undifferentiated cells, whereas membrane and cytosolic phospholipase A2 activities were unaltered. Lysophosphatidylethanolamine acyltransferase activities were elevated 2.4-fold (P<0.05). Lysophosphatidylcholine acyltransferase, monolysocardiolipin acyltransferase, acyl-Coenzyme A synthetase and acyl-Coenzyme A hydrolase activities were unaltered in differentiated cells compared to undifferentiated cells. We postulate that during cardiac cell differentiation, the observed elevation in lysophosphatidylethanolamine acyltransferase activity accompanies the elevation in phosphatidylethanolamine mass, possibly to maintain the fatty acyl composition of this phospholipid within the membrane.
Asunto(s)
Aciltransferasas/metabolismo , Miocardio/enzimología , Fosfolípidos/metabolismo , Animales , Radioisótopos de Carbono , Diferenciación Celular , Membrana Celular/enzimología , Dimetilsulfóxido , Ácidos Grasos/análisis , Glicerol/metabolismo , Miocardio/citología , Miosinas/análisis , Ácido Oléico/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolípidos/química , Tritio , Células Tumorales CultivadasRESUMEN
The myocyte enhancer factor-2 (MEF2) proteins are MADS-box transcription factors that are essential for differentiation of all muscle lineages but their mechanisms of action remain largely undefined. In mammals, the earliest site of MEF2 expression is the heart where the MEF2C isoform is detectable as early as embryonic day 7.5. Inactivation of the MEF2C gene causes cardiac developmental arrest and severe downregulation of a number of cardiac markers including atrial natriuretic factor (ANF). However, most of these promoters contain no or low affinity MEF2 binding sites and they are not significantly activated by any MEF2 proteins in heterologous cells suggesting a dependence on a cardiac-enriched cofactor for MEF2 action. We provide evidence that MEF2 proteins are recruited to target promoters by the cell-specific GATA transcription factors, and that MEF2 potentiates the transcriptional activity of this family of tissue-restricted zinc finger proteins. Functional MEF2/GATA-4 synergy involves physical interaction between the MEF2 DNA-binding domain and the carboxy zinc finger of GATA-4 and requires the activation domains of both proteins. However, neither MEF2 binding sites nor MEF2 DNA binding capacity are required for transcriptional synergy. The results unravel a novel pathway for transcriptional regulation by MEF2 and provide a molecular paradigm for elucidating the mechanisms of action of MEF2 in muscle and non-muscle cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Factor Natriurético Atrial/genética , Sitios de Unión , Células Cultivadas , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Sinergismo Farmacológico , Factor de Transcripción GATA4 , Células HeLa , Humanos , Proteínas de Dominio MADS , Factores de Transcripción MEF2 , Miocardio/citología , Miocardio/metabolismo , Factores Reguladores Miogénicos , Especificidad de Órganos , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Elementos de Respuesta/genética , Eliminación de Secuencia/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Transfección , Dedos de ZincRESUMEN
The biosynthesis of phosphatidylethanolamine was examined during differentiation of P19 teratocarcinoma cells into cardiac myocytes. P19 cells were induced to undergo differentiation into cardiac myocytes by the addition of dimethyl sulfoxide to the medium. Immunofluorescence labeling confirmed the expression of striated myosin 10 days postinduction of differentiation. The content of phosphatidylethanolamine increased significantly within the first 2 days of differentiation. [1,3-(3)H]Glycerol incorporation into phosphatidylethanolamine was increased 7.2-fold during differentiation, indicating an elevation in de novo synthesis from 1, 2-diacyl-sn-glycerol. The mechanism for the increase in phosphatidylethanolamine levels during cardiac cell differentiation was a 2.8-fold increase in the activity of ethanolaminephosphotransferase, the 1,2-diacyl-sn-glycerol utilizing reaction of the cytidine 5'-diphosphate-ethanolamine pathway of phosphatidylethanolamine biosynthesis. Incubation of P19 cells with the phosphatidylethanolamine biosynthesis inhibitor 8-(4-chlorophenylthio)-cAMP inhibited the differentiation-induced elevation in phosphatidylethanolamine levels but did not affect the expression of striated myosin. The results suggest that elevation in phosphatidylethanolamine is an early event of P19 cell differentiation into cardiac myocytes, but is not essential for differentiation to proceed.
Asunto(s)
Diferenciación Celular/fisiología , Miocardio/citología , Fosfatidiletanolaminas/biosíntesis , Animales , Desmosomas/metabolismo , Ratones , Microscopía Electrónica , Microscopía Fluorescente , Miocardio/metabolismo , Miocardio/ultraestructura , Miosinas/metabolismo , Plasmalógenos/biosíntesisRESUMEN
Angiotensin II (AII) is a major determinant of arterial pressure and volume homeostasis, mainly because of its vascular action via the AII type 1 receptor (AT1R). AII has also been implicated in the development of cardiac hypertrophy because angiotensin I-converting enzyme inhibitors and AT1R antagonists prevent or regress ventricular hypertrophy in animal models and in human. However, because these treatments impede the action of AII at cardiac as well as vascular levels, and reduce blood pressure, it has been difficult to determine whether AII action on the heart is direct or a consequence of pressure-overload. To determine whether AII can induce cardiac hypertrophy directly via myocardial AT1R in the absence of vascular changes, transgenic mice overexpressing the human AT1R under the control of the mouse alpha-myosin heavy chain promoter were generated. Cardiomyocyte-specific overexpression of AT1R induced, in basal conditions, morphologic changes of myocytes and nonmyocytes that mimic those observed during the development of cardiac hypertrophy in human and in other mammals. These mice displayed significant cardiac hypertrophy and remodeling with increased expression of ventricular atrial natriuretic factor and interstitial collagen deposition and died prematurely of heart failure. Neither the systolic blood pressure nor the heart rate were changed. The data demonstrate a direct myocardial role for AII in the development of cardiac hypertrophy and failure and provide a useful model to elucidate the mechanisms of action of AII in the pathogenesis of cardiac diseases.
Asunto(s)
Cardiomegalia/genética , Miocardio/patología , Receptores de Angiotensina/genética , Remodelación Ventricular/genética , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , Animales , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Unión Competitiva/efectos de los fármacos , Northern Blotting , Cardiomegalia/patología , Regulación de la Expresión Génica , Atrios Cardíacos/química , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Ventrículos Cardíacos/química , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Humanos , Imidazoles/farmacología , Inmunohistoquímica , Losartán/farmacología , Ratones , Ratones Transgénicos , Miocardio/citología , Miocardio/metabolismo , Fenotipo , Piridinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ensayo de Unión Radioligante , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/metabolismo , Distribución Tisular , Transgenes/genéticaRESUMEN
The GATA family of zinc finger proteins are transcriptional regulators with critical functions in lineage differentiation and embryonic development. Based on structural and expression pattern comparisons, the GATA proteins have been subdivided into two groups. The first subgroup consists of GATA-1, -2, and -3, which are all highly expressed in the hematopoietic system, whereas GATA-4, -5, and -6 are present essentially in the heart and gut. We have isolated and functionally characterized the rat GATA-5 cDNA, which encodes a 45-kDa protein with 71%, 73%, and 97% homology to its amphibian, avian, and murine homologs, respectively. Northern blot analysis showed that rat GATA-5 is expressed in a dynamic pattern during embryonic and postnatal development. In the midgestation embryo, GATA-5 transcripts are most abundant in the heart and decrease dramatically in the postnatal heart; in contrast, GATA-5 expression is upregulated in the lung and gut during postnatal development. Functional studies with recombinant GATA-4, -5, and -6 proteins show that GATA-5 has preferential affinity for a subset of GATA elements found on cardiac promoters and differentially activate cardiac gene transcription. Structure-function analysis revealed the presence of an activation domain within the carboxy terminal region of GATA-5 that is essential for transcriptional regulation of target promoters. Linkage analysis localized Gata5 to distal mouse Chromosome (Chr) 2 in a conserved linkage group with genes localized to rat Chr 3q43 and human Chr 20q13.2-q13.3. The results suggest that GATA-5 may have specific downstream targets and that GATA-4, -5, and -6 can only partially substitute for each other in cardiogenesis. Thus, Gata5 probably plays a specialized evolutionary conserved role in cardiac development.
Asunto(s)
Mapeo Cromosómico , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Diferenciación Celular/genética , Línea Celular , Cromosomas Humanos Par 20 , Clonación Molecular , Proteínas de Unión al ADN/química , Factor de Transcripción GATA5 , Regulación del Desarrollo de la Expresión Génica , Ligamiento Genético , Humanos , Ratones , Datos de Secuencia Molecular , Miocardio/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Activación Transcripcional , Dedos de Zinc/genéticaRESUMEN
Although accumulating evidence suggests that the heart develops in a segmental fashion, the molecular mechanisms that control regional specification of cardiomyocytes in the developing heart remain largely unknown. In this study, we have used the mouse cardiac-restricted ankyrin repeat protein (CARP) gene as a model system to study these mechanisms. The CARP gene encodes a nuclear co-regulator for cardiac gene expression, which lies downstream of the cardiac homeobox gene, Nkx 2.5, and is an early marker of the cardiac muscle cell lineage. We have demonstrated that the expression of the gene is developmentally down regulated and dramatically induced as part of the embryonic gene program during cardiac hypertrophy. Using a lacZ/knock-in mouse and three lines of transgenic mouse harboring various CARP promoter/lacZ reporters, we have identified distinct 5' cis regulatory elements of the gene that can direct heart segment-specific transgene expression, such as atrial versus ventricular and left versus right. Most interestingly, a 213 base pair sequence element of the gene was found to confer conotruncal segment-specific transgene expression. Using the transgene as a conotruncal segment-specific marker, we were able to document the developmental fate of a subset of cardiomyocytes in the conotruncus during cardiogenesis. In addition, we have identified an essential GATA-4 binding site in the proximal upstream regulatory region of the gene and cooperative transcriptional regulation mediated by Nkx2.5 and GATA-4. We have shown that this cooperative regulation is dependent on binding of GATA-4 to its cognate DNA sequence in the promoter, which suggests that Nkx2.5 controls CARP expression, at least in part, through GATA-4.
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Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Proteínas Nucleares/genética , Proteínas Represoras/genética , Transcripción Genética , Proteínas de Xenopus , Factores de Edad , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cardiomegalia/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Exones , Factor de Transcripción GATA4 , Genes Reguladores/genética , Corazón/anatomía & histología , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Ratones , Ratones Transgénicos , Modelos Genéticos , Proteínas Musculares , Plásmidos/metabolismo , Ratas , Factores de Tiempo , Factores de Transcripción/genética , TransfecciónRESUMEN
Three members of the GATA family of transcription factors, GATA-4, -5, and -6, are expressed in the developing heart. One family member, GATA-5, is restricted to the endocardium while the other two, GATA-4 and -6, are present in the myocardium where they apparently fulfil distinct functions. The mechanisms underlying GATA factor specificity are not fully understood but may involve interaction with other tissue-restricted or ubiquitous co-factors. Thus, combinatorial interaction among GATA factors or between GATA factors and other co-factors may differentially control various stages of cardiogenesis.
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Proteínas de Unión al ADN/fisiología , Corazón/embriología , Factores de Transcripción/fisiología , Proteínas de Xenopus , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/fisiología , Cardiomegalia/genética , Pollos , Proteínas de Unión al ADN/genética , Factor de Transcripción GATA4 , Factor de Transcripción GATA5 , Factor de Transcripción GATA6 , Regulación del Desarrollo de la Expresión Génica , Corazón/crecimiento & desarrollo , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Humanos , Ratones , Miocardio/metabolismo , Ratas , Transducción de Señal/genética , Factores de Transcripción/genética , XenopusRESUMEN
There is little information on the molecular events that control the subcellular distribution of protein kinase C during cardiac cell differentiation. We examined protein kinase C activity and the subcellular distribution of representatives of the "classical," "novel," and "atypical" protein kinase C's in P19 murine teratoma cells induced to undergo differentiation into cardiac myocytes by the addition of dimethylsulfoxide to the medium (Grepin et al., Development 124, 2387-2395, 1997). Differentiation was assessed by the presence of striated myosin, a morphological marker for cardiac cells. Addition of dimethyl sulfoxide to the medium resulted in the appearance of striated myosin by 10 days postincubation. Immunolocalization and Western blot studies revealed that a significant proportion of protein kinase Calpha, -epsilon, and -zeta were associated with the particulate fraction in P19 cells prior to differentiation. Differentiation into cardiac cells resulted in a translocation of protein kinase C activity from the particulate fraction to cytosol and localization of most of protein kinase Calpha, -epsilon, and -zeta to the cytoplasmic compartment. The total cellular protein kinase C activity was unaltered during differentiation. The translocation of protein kinase C activity during differentiation of P19 cells into cardiac myocytes was associated with a decrease in the levels of cellular 1, 2-diacyl-sn-glycerol. The cellular levels of phosphatidylserine and phosphatidylinositol did not change during differentiation. Addition of 1,2-dioctanoyl-sn-glycerol, a cell-permeant 1, 2-diacyl-sn-glycerol analog, reversed the differentiation-induced switch in the relative distribution of protein kinase C activity and dramatically increased the association of protein kinase Calpha with the particulate fraction. Addition of 1,2-dioctanoyl-sn-glycerol did not reverse the pattern of distribution for protein kinase Cepsilon or -zeta. The results indicate that protein kinase C activity and protein kinase Calpha, -epsilon and -zeta isoforms are redistributed from the particulate to the cytosolic fraction during differentiation of P19 cells into cardiomyocytes. The mechanism for the redistribution of protein kinase Calpha may be related to the reduction in the cellular 1,2-diacyl-sn-glycerol levels that accompany differentiation.