RESUMEN
Autophagy allows cell survival during starvation through the bulk degradation of proteins and organelles by lysosomal enzymes. However, the mechanisms responsible for the induction and regulation of the autophagy program are poorly understood. Here we show that the FoxO3 transcription factor, which plays a critical role in muscle atrophy, is necessary and sufficient for the induction of autophagy in skeletal muscle in vivo. Akt/PKB activation blocks FoxO3 activation and autophagy, and this effect is not prevented by rapamycin. FoxO3 controls the transcription of autophagy-related genes, including LC3 and Bnip3, and Bnip3 appears to mediate the effect of FoxO3 on autophagy. This effect is not prevented by proteasome inhibitors. Thus, FoxO3 controls the two major systems of protein breakdown in skeletal muscle, the ubiquitin-proteasomal and autophagic/lysosomal pathways, independently. These findings point to FoxO3 and Bnip3 as potential therapeutic targets in muscle wasting disorders and other degenerative and neoplastic diseases in which autophagy is involved.
Asunto(s)
Autofagia/fisiología , Factores de Transcripción Forkhead/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Animales , Autofagia/genética , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Serina-Treonina Quinasas TOR , Ubiquitina/metabolismoRESUMEN
BACKGROUND: We reported previously that the cardiac troponin I (cTnI) promoter drives cardiac-specific expression of reporter genes in cardiac muscle cells and in transgenic mice, and that disruption of GATA elements inactivates the cTnI promoter in cultured cardiomyocytes. We have now examined the role of cTnI promoter GATA elements in skeletal muscle cells. RESULTS: Mutation or deletion of GATA elements induces a strong transcriptional activation of the cTnI promoter in regenerating skeletal muscle and in cultured skeletal muscle cells. Electrophoretic mobility shift assays show that proteins present in nuclear extracts of C2C12 muscle cells bind the GATA motifs present in the cTnI promoter. However, GATA protein complex formation is neither reduced nor supershifted by antibodies specific for GATA-2, -3 and -4, the only GATA transcripts present in muscle cells. CONCLUSION: These findings indicate that the cTnI gene promoter is repressed in skeletal muscle cells by GATA-like factors and open the way to further studies aimed at identifying these factors.
Asunto(s)
Factores de Transcripción GATA/metabolismo , Músculo Esquelético/metabolismo , Regiones Promotoras Genéticas , Troponina I/genética , Animales , Animales Recién Nacidos , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/análisis , Factores de Transcripción GATA/genética , Eliminación de Gen , Genes Reporteros , Masculino , Mutación , Ratas , Ratas Wistar , Transcripción Genética , Transfección , beta-Galactosidasa/análisisRESUMEN
OBJECTIVES: The aim of this study was to investigate the contribution of host-derived circulating cells to cardiac repair after tissue damage using the model of heterotopic heart transplantation between transgenic recipient rats expressing green fluorescent protein (GFP) and wild-type donors. METHODS: Unlabeled donor rat hearts, some of which underwent prolonged cold ischemia pretreatment, were transplanted into the abdominal cavity of GFP+ transgenic recipient rats and were analyzed 15 and 90 days after surgery. An additional experimental group underwent heart transplantation following administration of granulocyte-colony stimulatory factor (G-CSF) to mobilize bone marrow cells. RESULTS: Most transplants contained GFP+ mature cardiomyocytes. However, systematic counting in the transplants showed that the proportion of GFP+ cardiomyocytes was only 0.0005% to 0.008% of all cardiomyocytes. These relative proportions did not change after G-CSF treatment, despite evidence for sustained marrow cell mobilization. Confocal image analysis showed that the majority of GFP+ cardiomyocytes contained a high number of nuclei, suggesting that these cells may derive from fusion events. Very rarely, small GFP+ undifferentiated cells, expressing GATA-4, were also identified. Occasionally, GFP+ endothelial cells, but not smooth muscle cells, were detected in blood vessels of some transplants. CONCLUSIONS: Our results demonstrate that cardiomyocytes expressing a host transgenic marker are detectable in heterotopic heart transplants; however, they do not significantly contribute to repopulation of the damaged myocardium.
Asunto(s)
Trasplante de Corazón , Miocitos Cardíacos/citología , Regeneración , Animales , Animales Modificados Genéticamente , Biomarcadores/análisis , Células de la Médula Ósea/citología , Recuento de Células , Fusión Celular , ADN/análisis , Células Endoteliales/patología , Femenino , Citometría de Flujo , Factor de Transcripción GATA4/análisis , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Movilización de Célula Madre Hematopoyética , Procesamiento de Imagen Asistido por Computador , Masculino , Microscopía Confocal , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratas , Ratas Sprague-Dawley , Trasplante Heterotópico/patologíaRESUMEN
The SH3 binding glutamic acid-rich (SH3BGR) gene was cloned in an effort to identify genes located to human chromosome 21, within the congenital heart disease region, and expressed in the developing heart. After the identification of SH3BGR, two human homologous genes, SH3BGRL and SH3BGRL3, were identified and mapped to chromosome Xq13.3 and 1p34.3-35, respectively. SH3BGRL and SH3BGRL3 code for small proteins similar to the N-terminal region of the SH3BGR protein. SH3BGRL3 protein shows a significant similarity to Glutaredoxin 1 of Escherichia coli, and all the three proteins are predicted to belong to Thioredoxin-like protein Superfamily. Here we describe the identification and characterization of an additional human homologue of SH3BGR, named SH3BGRL2. The SH3BGRL2 gene maps to chromosome 6q13-15 and its messenger RNA has a large 3' untranslated region containing several AUUUA repeats. SH3BGRL2 codes for a protein of 107 amino acids, which, like SH3BGRL and SH3BGRL3 proteins, is highly homologous to the N-terminal region of the SH3BGR protein and appears to be related to Glutaredoxins and to PKC-interacting cousin of thioredoxin homology domain. We propose that the identification of SH3BGRL2 establishes a novel family of human genes, coding for highly conserved small proteins belonging to Thioredoxin-like protein Superfamily.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Musculares/genética , Tiorredoxinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Células COS , Núcleo Celular/metabolismo , Clonación Molecular , Secuencia Conservada/genética , ADN Complementario/química , ADN Complementario/genética , Femenino , Expresión Génica , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Congenital heart disease (CHD) is the most common birth defect in humans and is present in 40% of newborns affected by Down syndrome (DS). The SH3BGR gene maps to the DS-CHD region and is a potential candidate for the pathogenesis of CHD, since it is selectively expressed in cardiac and skeletal muscle. To determine whether overexpression of Sh3bgr in the murine heart may cause abnormal cardiac development, we have generated transgenic mice using a cardiac- and skeletal-muscle-specific promoter to drive the expression of a Sh3bgr transgene. We report here that heart morphogenesis is not affected by overexpression of Sh3bgr.