RESUMEN
The cardiac Na+-Ca2+ exchanger (NCX1) is the principal Ca2+ efflux mechanism in cardiocytes. The exchanger is up-regulated in both cardiac hypertrophy and failure. In this report, we identify the cis-acting elements that control cardiac expression and alpha-adrenergic up-regulation of the exchanger gene. Deletion analysis revealed that a minimal cardiac promoter fragment from -184 to +172 is sufficient for cardiac expression and alpha-adrenergic stimulation. Mutational analysis revealed that both the CArG element at -80 and the GATA element at -50 were required for cardiac expression. Gel mobility shift assay supershift analysis demonstrated that the serum response factor binds to the CArG element and GATA-4 binds to the GATA element. Point mutations in the -172 E-box demonstrated that it was required for alpha-adrenergic induction. In addition, deletion analysis revealed one or more enhancer elements in the first intron (+103 to +134) that are essential for phenylephrine up-regulation but bear no homology to any known transcription element. Therefore, this work demonstrates that SRF and GATA-4 are critical for NCX1 expression in neonatal cardiomyocytes and that the -172 E-box in addition to a novel enhancer element(s) are required for phenylephrine up-regulation of NCX1 and may mediate its hypertrophic up-regulation.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Miocardio/metabolismo , Proteínas Represoras/fisiología , Intercambiador de Sodio-Calcio/genética , Factores de Transcripción/fisiología , Agonistas alfa-Adrenérgicos/farmacología , Animales , Animales Recién Nacidos , Secuencia de Bases , Proteínas de Ciclo Celular , Células Cultivadas , ADN , Corazón/efectos de los fármacos , Datos de Secuencia Molecular , Miocardio/citología , Fenilefrina/farmacología , Regiones Promotoras Genéticas , Ratas , RibonucleoproteínasRESUMEN
The Na+-Ca2+ exchanger (NCX1) plays a major role in calcium efflux and therefore in the control and regulation of intracellular calcium in the heart. The exchanger has been shown to be regulated at several levels including transcription. NCX1 mRNA levels are up-regulated in both cardiac hypertrophy and failure. In this work, the 5'-end of the ncx1 gene has been cloned to study the mechanisms that mediate hypertrophic stimulation and cardiac expression. The feline ncx1 gene has three exons that encode 5'-untranslated sequences that are under the control of three tissue-specific promoters. The cardiac promoter drives expression in cardiocytes, but not in mouse L cells. Although it contains at least one enhancer (-2000 to -1250 base pairs (bp)) and one or more negative elements (-1250 to -250 bp), a minimum promoter (-250 to +200 bp) is sufficient for cardiac expression and alpha-adrenergic stimulation.