RESUMEN
Gq-protein-coupled receptor (GqPCR) signalling is associated with the induction of cardiac myocyte hypertrophy, which is characterized by an increase in expression of immediate early genes via activation of pre-existing transcription factors. Here, we explore the role of MSK1 and MAPK signalling pathways in the regulation of the immediate early gene c-jun. The results provide further support for the role of MSK1 in cardiac myocyte hypertrophy and indicate that PE activates distinct signalling mechanisms which culminate with a complex activation of c-jun. ERK1/2 and JNKs are the principal kinases responsible for phosphorylation of c-Jun, whereas c-jun mRNA and protein up-regulation by PE is mediated by multiple signalling pathways that include MSK1, ERK1/2, p38-MAPK and JNKs. These signalling mechanisms seem to be critical to the phenotypic changes of cardiac myocytes in response to hypertrophic stimulation.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/enzimología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Animales , Cardiomegalia/enzimología , Aumento de la Célula , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Fenilefrina/farmacología , Fosforilación , ARN Mensajero/metabolismo , Ratas , Regulación hacia ArribaRESUMEN
H2O2, as an example of oxidative stress, induces cardiac myocyte apoptosis. Bcl-2 family proteins are key regulators of the apoptotic response while their functions can be regulated by post-translational modifications including phosphorylation, dimerization or proteolytic cleavage. In this study, we examined the role of various protein kinases in regulating total BAD protein levels in adult rat cardiac myocytes undergoing apoptosis. Stimulation with 0.1 mM H2O2, which induces apoptosis, resulted in a marked down-regulation of BAD protein, which is attributed to cleavage by caspases since it can be restored in the presence of a general caspase inhibitor. Inhibition of PKC, p38-MAPK, ERK1/2 and PI-3-K did not influence the reduced BAD protein levels observed after stimulation with H2O2. On the contrary, inhibition of PKA or specifically PKCdelta resulted in up-regulation of BAD. Decreased caspase 3 activity was observed in H2O2 treated cells after inhibition of PKA or PKCdelta whereas inhibition of PKA also resulted in improved cell survival. Furthermore, addition of okadaic acid to inhibit selected phosphatases resulted in enhanced BAD cleavage. These data suggest that, during oxidative stress-induced cardiac myocyte apoptosis, there is a caspase-dependent down-regulation of BAD protein, which seems to be regulated by coordinated action of PKA, PKCdelta and phosphatases.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Miocitos Cardíacos/enzimología , Estrés Oxidativo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteína Quinasa C-delta/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Animales , Apoptosis/efectos de los fármacos , Inhibidores de Caspasas , Supervivencia Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Peróxido de Hidrógeno/farmacología , Masculino , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Proteína Quinasa C-delta/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas WistarRESUMEN
During development of the neuromuscular junction, nerve-derived agrin and the cell substrate laminin stimulate postsynaptic nAChR clustering. This clustering is dependent on activation of the tyrosine kinase, MuSK, which signals receptor clustering via a rapsyn-dependent mechanism. Myogenin is a muscle-specific transcription factor that controls myoblast differentiation and nAChR gene expression. Here, we used RNA interference to investigate if myogenin is also necessary for nAChR clustering. We find that myogenin expression is essential for robust nAChR clustering and cannot be compensated by the muscle regulatory factors MyoD, myf5, and MRF4. In addition, we show that clustering cannot be rescued in myogenin-depleted myotubes by simply overexpressing the essential clustering molecules MuSK, rapsyn, and nAChRs. These data suggest that myogenin controls the expression of molecules crucial to nAChR clustering in addition to its role in regulating nAChR gene expression.
Asunto(s)
Fibras Musculares Esqueléticas/metabolismo , Miogenina/metabolismo , Receptores Nicotínicos/metabolismo , Agrina/metabolismo , Animales , Bungarotoxinas/metabolismo , Diferenciación Celular/fisiología , Laminina/metabolismo , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/citología , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteína MioD/genética , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico/genética , Factor 5 Regulador Miogénico/metabolismo , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Miogenina/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Receptores Nicotínicos/genética , Transducción de Señal/fisiologíaRESUMEN
During development of the neuromuscular junction (NMJ), extrajunctional expression of genes, whose products are destined for the synapse, is suppressed by muscle activity. One of the best-studied examples of activity-dependent gene regulation in muscle are those encoding nicotinic acetylcholine receptor (nAChR) subunits. We recently showed that nAChR gene expression is inhibited by calcium/calmodulin-dependent protein kinase II (CaMKII) and CaMKII inhibitors block activity-dependent suppression of these genes. Here we report results investigating the mechanism by which CaMKII suppresses nAChR gene expression. We show that the muscle helix-loop-helix transcription factor, myogenin, is necessary for activity-dependent control of nAChR gene expression in cultured rat myotubes and is a substrate for CaMKII both in vitro and in vivo. CaMKII phosphorylation of myogenin is induced by muscle activity and this phosphorylation influences DNA binding and transactivation. Thus we have identified a signaling mechanism by which muscle activity controls nAChR gene expression in developing muscle.