The role of GATA, CArG, E-box, and a novel element in the regulation of cardiac expression of the Na+-Ca2+ exchanger gene.
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.
Cheng
G
G
Cardiology Division, Department of Medicine, and the Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina, 29425-2221, USA.
Hagen
T P
TP
Dawson
M L
ML
Barnes
K V
KV
Menick
D R
DR
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D004247:DNA; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D006321:Heart / Q000187:drug effects; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D008969:Molecular Sequence Data; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010656:Phenylephrine / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D051381:Rats; D012097:Repressor Proteins / Q000502:physiology*; D012261:Ribonucleoproteins; D019831:Sodium-Calcium Exchanger / Q000235:genetics*; D014157:Transcription Factors / Q000502:physiology*
D000316:Adrenergic alpha-Agonists; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; C121363:Hnrnpab protein, rat; D012097:Repressor Proteins; D012261:Ribonucleoproteins; D019831:Sodium-Calcium Exchanger; D014157:Transcription Factors; D010656:Phenylephrine; D004247:DNA