Regulação da expressão e ativação do fator de transcrição C-JUN no miocardio de ratos submetido a sobrecarga pressora aguda

AUTOR(ES)
DATA DE PUBLICAÇÃO

2003

RESUMO

The early expression of immediate early genes is a fundamental feature of myocardial hypertrophy. The immediate early gene c-jun codifies a transcription factor that has been assigned to be an important regulator of the hypertrophic response in cardiac myocytes. In the present study we investigated whether pressure overload or phenylephrine treatment stimulated MEF2-dependent transcriptional activation of c-jun in cardiac myocytes. Westem blotting and immunohistochemistry analysis of rat myocardium demonstrated that MEF2 is high1y expressed in the rat heart and is predominantly located at nuc1ei of cardiac myocytes. Gel shift assays of myocardial nuclear extracts revealed a consistent DNA binding activation of MEF2 after 1 and 2 hours of pressure overload. We further show that pressure overload induced a progressive nuclear translocation and activation of ERK.5. Co-immunoprecipitation and in vitro kinase assays indicated that the activation of ERK.5 was paralleled by increased association ERK.5/MEF2 and by enhanced ability of ERK.5 to phosphorylate MEF2. Experiments with in vivo transfection of left ventric1e with c-jun promoter reporter gene showed that pressure overload induced a consistent increase of c-jun transcriptional activity in the rat myocardium. Rendering the MEF2 site of the c-jun plasmid inactive by mutation abolished the load-induced activation of c-jun promoter reporter gene. Mutation of MEF2 site also abolished the phenylephrine- induced c-jun promoter activation in neonatal rat ventricular myocytes (NRVM). In addition, we demonstrated that NRVM transfection with ERK.5 antisense oligodeoxynuc1eotide inhibited the phenylephrine-induced c-jun promoter activation. These findings identify MEF2 as a potential regulator of c-jun transactivation and suggest that ERK5 might be an important mediator of MEF2 and c-jun promoter activation in response to hypertrophic stimuli in cardiac myocytes. In addition to studying the regulation of c-Jun expression, we also investigated the activation of this transcription factor in the overloaded myocardium. Immunoblotting of subcellular ftactions and immunohistochemistry assays demonstrated that pressure overload induced a parallel increase in c-Jun expression and in serine-63-phosphorylation in nuc1ei cardiac myocytes nuc1ei. We also observed that load-induced c-Jun phosphorylation was concomitant to JNKl phosphorylation and translocation to cardiac myocytes nuclei. M;)reover, gel shift assays revealed that pressure overload promoted an increase in c-Jun DNA-binding activity, which correlated with the increase in c-Jun expression. These results show that c-Jun is regulated by a combination of increased expression and phosphorylation and indicate that the synergism of these effects might act as a mechanism to amplify c-Jun activity in the overloaded myocardium

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