RESUMEN
Several studies have already demonstrated that micro- and milli-calpains (CAPN 1-CAPN 2), calcium-dependent intracellular cysteine-proteases are involved in many biological phenomenon including muscle growth and development. More particularly, recent studies have demonstrated that milli-calpain is implicated in myoblast fusion. Moreover, in primary muscle cells, these proteases do not appear simultaneously throughout muscle cell differentiation. Because micro- and milli-calpains do not have the same intracellular localization, it appears likely that these two calcium-dependent proteases have different biological roles during muscle cell differentiation. The goal of this study is to determine the role of micro-calpain. We therefore, have developed a muscle cell line in which micro-calpain is over-expressed, using the inducible Tet Regulated Expression System. The outcome is observed by following the behavior of different proteins, considered to be potential substrates of the protease. The present study shows important decreases in the expression level of ezrin (68%), vimentin (64%) and caveolin 3 (76%) whereas many other cytoskeletal proteins remain remarkably stable. Concerning the myogenic transcription factors, only the level of myogenin decreased (59%) after the over-expression of micro-calpain. Ultra structural studies have shown that the myofibrils formed near the cell periphery are normally oriented, lying along the longitudinal axis. This regularity is lost progressively towards the cell center where the cytoskeleton presented an increasing disorganization. All these results indicate that micro-calpain is involved in regulation pathway of myogenesis via at least its action on ezrin, vimentin, caveolin 3 and myogenin, a muscle transcription factor.
Asunto(s)
Calpaína/fisiología , Células Musculares/metabolismo , Animales , Calpaína/genética , Caveolina 3 , Caveolinas/genética , Caveolinas/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Clonación Molecular , Proteínas del Citoesqueleto , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desmina/genética , Desmina/metabolismo , Doxiciclina/farmacología , Fibronectinas/genética , Fibronectinas/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Células Musculares/fisiología , Células Musculares/ultraestructura , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteína MioD/genética , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Vimentina/genética , Vimentina/metabolismoRESUMEN
p94 belongs to the calpain family of enzymes, also called calcium-activated neutral proteases and is mainly expressed in the skeletal muscle. Mutations affecting the gene coding for p94 are responsible for a myopathy syndrome called Limb Girdle Muscular Dystrophy type 2A (LGMD2A). Although the activity of p94 seems necessary for muscle function, the biological role of the enzyme is still unknown. The goal of this study was to develop a muscle cell line in which the expression level of p94 can be regulated, by an inducible way. In this study, a biological system was developed which allowed mimicking, in vitro, of part of the events occurring in patients (i.e. a decrease of p94 activity). The first results indicate that the decrease in p94 activity results in a significant increase of myogenin level, a high specific transcription factor involved in myoblast fusion. This muscle specific inducible system is an interesting biological tool to assess specifically p94 function(s) in cultured muscle cells. According to the present results, p94 seems at least to be involved in a myogenesis regulation pathway via its action on certain proteins belonging to the myogenic regulator factor family.
Asunto(s)
Calpaína/metabolismo , Desarrollo de Músculos/fisiología , Músculo Esquelético/enzimología , Miogenina/metabolismo , Animales , Western Blotting , Calpaína/genética , Técnicas de Cultivo de Célula/métodos , Expresión Génica/fisiología , Regulación de la Expresión Génica , Isopropil Tiogalactósido/metabolismo , Ratones , Desarrollo de Músculos/genética , Músculo Esquelético/citología , Técnicas de Amplificación de Ácido Nucleico , ARN sin Sentido/genética , ARN Mensajero/genéticaRESUMEN
Many studies have demonstrated that the calcium-dependent proteolytic system (calpains and calpastatin) is involved in myoblast differentiation. It is also known that myogenic differentiation can be studied in vitro. In the present experiments, using a mouse muscle cell line (C2C12) we have analyzed both the sequences of appearance and the expression profiles of calpains 1, 2, 3 and calpastatin during the course of myoblast differentiation. Our results mainly show that the expression of ubiquitous calpains (calpain 1 and 2) and muscle-specific calpain (calpain 3) at the mRNAs level as well as at the protein level do not change significantly all along this biological process. In the same time, the specific inhibitor of ubiquitous calpains, calpastatin, presents a stable expression at mRNAs level as well as protein level, all along myoblast to myotube transition. A comparison with other myogenic cells is presented.