RESUMEN
The calcium-dependent proteolytic system is composed of cysteine proteases named calpains. They are ubiquitous or tissue-specific enzymes. The two best characterised isoforms are the ubiquitously expressed mu- and m-calpains. Besides its regulation by calcium, calpain activity is tightly controlled by calpastatin, the specific endogenous inhibitor, binding to phospholipids, autoproteolysis and phosphorylation. Calpains are responsible for limited proteolytic events. Among the multitude of substrates identified so far are cytoskeletal and membrane proteins, enzymes and transcription factors. Calpain activity is involved in a large number of physiological and pathological processes. In this review, we will particularly focus on the implication of the calcium-dependent proteolytic system in relation to muscle physiology. Because of their ability to remodel cytoskeletal anchorage complexes, calpains play a major role in the regulation of cell adhesion, migration and fusion, three key steps of myogenesis. Calcium-dependent proteolysis is also involved in the control of cell cycle. In muscle tissue, in particular, calpains intervene in the regeneration process. Another important class of calpain substrates belongs to apoptosis regulating factors. The proteases may thus play a role in muscle cell death, and as a consequence in muscle atrophy. The relationships between calcium-dependent proteolysis and muscle dysfunctions are being further developed in this review with a particular emphasis on sarcopenia.
Asunto(s)
Calcio/metabolismo , Calpaína/metabolismo , Músculo Esquelético/enzimología , Atrofia Muscular/enzimología , Animales , Calpaína/fisiología , Humanos , Músculo Esquelético/fisiología , Atrofia Muscular/etiología , Distrofias Musculares/enzimologíaRESUMEN
With the aim to identify the oncoprotein partners implicated in the c-Jun myogenic influence, we carried out stable transfection experiments of c-Jun and/or ATF2, Fra2, c-Fos overexpression in avian myoblasts. Before induction of differentiation, c-Jun repressed myoblast withdrawal from the cell cycle, as did a TPA treatment. However, after serum removal, unlike TPA, c-Jun significantly stimulated myoblast differentiation. In search for specific partners involved in this dual influence, we found that a reduction in the amounts of c-Fos and Fra2 and an increase in c-Jun proteins occurred at cell confluence, a situation likely to favor cooperation between c-Jun and ATF2 during terminal differentiation. Whereas c-Fos and Fra2 cooperated with c-Jun to abrogate myoblast withdrawal from the cell cycle and terminal differentiation, ATF2 co-expression potentiated the positive myogenic c-Jun influence. In addition, myogenin expression was a positive target of this cooperation and this regulation occurred through a stimulation of myogenin promoter activity: (1) whereas c-Fos or Fra2 co-expression abrogated c-Jun stimulatory activity on this promoter, ATF2 co-expression potentiated this influence; (2) using a dominant negative ATF2 mutant, we established that c-Jun transcriptional activity required functionality of endogenous ATF2. These data suggest that through this dual myogenic influence due to cooperations with different partners, c-Jun is involved in the control of duration of myoblast proliferation and thereafter of fusion efficiency.
Asunto(s)
Diferenciación Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Músculos/citología , Miogenina/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Codorniz , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 2 , Animales , Western Blotting , Ciclo Celular , División Celular , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Antígeno 2 Relacionado con Fos , Genes Dominantes , Microscopía Fluorescente , Músculos/metabolismo , Mutación , Miogenina/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/genética , TransfecciónRESUMEN
We have previously shown that BTG1 stimulates myoblast differentiation. In addition, this protein displays a major nuclear localization in confluent myoblasts, decreasing during the early steps of differentiation, and is essentially detected in the cytoplasm of mature myotubes. To identify the domains involved in the cellular trafficking of BTG1, we observed the localization of several BTG1 sequences fused to betaGalactosidase. The highly conserved B box among all members of the BTG family induces a significant nuclear localization of the betaGal moiety, enhanced by presence of the BTG1 carboxy-terminal sequence. In addition, a functional Nuclear Export Signal (NES) overlaps the B box. Moreover, presence of the first 43 NH(2)-terminal amino acids reduced the nuclear localization of each chimeric protein tested. Last, the BTG1 amino-terminal domain bears an LxxLL motif favouring nuclear accumulation, and another region encompassing the A box inhibiting nuclear localization. In contrast to a BTG1 mutant exclusively localized in the cytoplasm, transient expression of a mutant displaying a nuclear localization enhanced myoblasts withdrawal from the cell cycle and terminal differentiation, thus mimicking the myogenic influence of BTG1. In conclusion, several regions of BTG1 are implicated in its cellular localization, and BTG1 myogenic activity is induced at the nuclear level.
Asunto(s)
Proteínas de Neoplasias/fisiología , Transporte Activo de Núcleo Celular/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Núcleo Celular/metabolismo , Secuencia Conservada , Humanos , Datos de Secuencia Molecular , Músculos/citología , Músculos/metabolismo , Músculos/fisiología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estructura Terciaria de Proteína , Codorniz , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Thyroid hormone stimulates myoblast differentiation, through an inhibition of AP-1 activity occurring at the onset of differentiation. In this study we found that the T3 nuclear receptor c-ErbAalpha1 (T3Ralpha1) is involved in a mechanism preserving the duration of myoblast proliferation. Independently of the hormone presence, T3Ralpha1 represses avian MyoD transcriptional activity. Using several mutants of T3Ralpha1, we found that the hinge region plays a crucial role in the inhibition of MyoD activity. In particular, mutations of two small basic sequences included in alpha helices abrogate the T3Ralpha1/MyoD functional interaction. Similarly, the T3 receptor also represses myogenin transcriptional activity. Therefore, despite stimulating avian myoblast differentiation by a T3-dependent pathway not involving myogenic factors, T3Ralpha1 contributes to maintain an optimal myoblast proliferation period by inhibiting MyoD and myogenin activity.
Asunto(s)
Proteína MioD/antagonistas & inhibidores , Proteína MioD/metabolismo , Codorniz , Receptores de Hormona Tiroidea/metabolismo , Transcripción Genética , Triyodotironina/metabolismo , Animales , División Celular , Línea Celular , Regulación de la Expresión Génica , Ratones , Músculos/citología , Músculos/metabolismo , Mutación , Miogenina/genética , Regiones Promotoras Genéticas/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Receptores de Hormona Tiroidea/química , Receptores de Hormona Tiroidea/genética , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Triyodotironina/farmacologíaRESUMEN
Fenofibrate and fasting are known to regulate several genes involved in lipid metabolism in a similar way. In this study measuring several mitochondrial enzyme activities, we demonstrate that, in contrast to citrate synthase and complex II, cytochrome c oxidase (COX) is a specific target of these two treatments. In mouse liver organelles, Western blot experiments indicated that mitochondrial levels of p43, a mitochondrial T3 receptor, and mitochondrial peroxisome proliferator activated receptor (mt-PPAR), previously described as a dimeric partner of p43 in the organelle, are increased by both fenofibrate and fasting. In addition, in PPAR alpha-deficient mice, this influence was abolished for mt-PPAR but not for p43, whereas the increase in COX activity was not altered. These data indicate that: (1) PPAR alpha is involved in specific regulation of mt-PPAR expression by both treatments; (2) fenofibrate and fasting regulate the mitochondrial levels of p43 and thus affect the efficiency of the direct T3 mitochondrial pathway.
Asunto(s)
Fenofibrato/farmacología , Mitocondrias Hepáticas/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Citrato (si)-Sintasa/metabolismo , Cruzamientos Genéticos , Proteínas de Unión al ADN/metabolismo , Dimerización , Complejo II de Transporte de Electrones , Complejo IV de Transporte de Electrones/metabolismo , Ayuno , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Complejos Multienzimáticos/metabolismo , Orgánulos/efectos de los fármacos , Orgánulos/fisiología , Oxidorreductasas/metabolismo , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genética , Succinato Deshidrogenasa/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
Besides their involvement in the control of nuclear gene expression by activating several peroxisome proliferator-activated receptors (PPARs), peroxisome proliferators influence mitochondrial activity. By analogy with the previous characterization of a mitochondrial T3 receptor (p43), we searched for the presence of a peroxisome proliferator target in the organelle. Using several antisera raised against different domains of PPARs, we demonstrated by Western blotting, immunoprecipitation and electron microscopy experiments, that a 45 kDa protein related to PPARgamma2 (mt-PPAR) is located in the matrix of rat liver mitochondria. In addition, we found that the amounts of mt-PPAR are increased by clofibrate treatment. Moreover, in EMSA experiments mt-PPAR bound to a DR2 sequence located in the mitochondrial D-loop, by forming a complex with p43. Last, studies of tissue-specific expression indicated that mt-PPAR is detected in mitochondria of all tissues tested except the brain in amounts positively related to p43 abundance. Besides their involvement in the control of nuclear gene expression by activating several peroxisome proliferator-activated receptors (PPARs), peroxisome proliferators influence mitochondrial activity. By analogy with the previous characterization of a mitochondrial T3 receptor (p43), we searched for the presence of a peroxisome proliferator target in the organelle. Using several antisera raised against different domains of PPARs, we demonstrated by Western blotting, immunoprecipitation and electron microscopy experiments, that a 45 kDa protein related to PPARgamma2 (mt-PPAR) is located in the matrix of rat liver mitochondria. In addition, we found that the amounts of mt-PPAR are increased by clofibrate treatment. Moreover, in EMSA experiments mt-PPAR bound to a DR2 sequence located in the mitochondrial D-loop, by forming a complex with p43. Last, studies of tissue-specific expression indicated that mt-PPAR is detected in mitochondria of all tissues tested except the brain in amounts positively related to p43 abundance.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Mitocondrias Hepáticas/química , Mitocondrias Hepáticas/efectos de los fármacos , Proliferadores de Peroxisomas/farmacología , Receptores Citoplasmáticos y Nucleares/química , Factores de Transcripción/química , Regulación hacia Arriba/efectos de los fármacos , Animales , Clofibrato/farmacología , Secuencia de Consenso/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/química , Masculino , Microscopía Electrónica , Mitocondrias Hepáticas/genética , Mitocondrias Hepáticas/metabolismo , Peso Molecular , Especificidad de Órganos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Ratas , Ratas Wistar , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
To characterize the regulatory pathways involved in the inhibition of cell differentiation induced by the impairment of mitochondrial activity, we investigated the relationships occurring between organelle activity and myogenesis using an avian myoblast cell line (QM7). The inhibition of mitochondrial translation by chloramphenicol led to a potent block of myoblast differentiation. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone and oligomycin, which affect the organelle at different levels, exerted a similar influence. In addition, we provided evidence that this phenomenon was not the result of an alteration in cell viability. Conversely, overexpression of the mitochondrial T3 receptor (p43) stimulated organelle activity and strongly potentiated myoblast differentiation. The involvement of mitochondrial activity in an actual regulation of myogenesis is further supported by results demonstrating that the muscle regulatory gene myogenin, in contrast to CMD1 (chicken MyoD) and myf5, is a specific transcriptional target of mitochondrial activity. Whereas myogenin mRNA and protein levels were down-regulated by chloramphenicol treatment, they were up-regulated by p43 overexpression, in a positive relationship with the expression level of the transgene. We also found that myogenin or CMD1 overexpression in chloramphenicol-treated myoblasts did not restore differentiation, thus indicating that an alteration in mitochondrial activity interferes with the ability of myogenic factors to induce terminal differentiation.