Endothelin‐converting enzyme‐1 is a downstream target of the homeobox transcription factor Nkx2‐5

ABSTRACT The homeobox transcription factor Nkx2‐5 and the zinc metalloprotease endothelin‐converting enzyme‐1 (ECE‐1) are essential for cardiac development. Here, we demonstrate for the first time a functional link between Nkx2‐5 and ECE‐1. In transiently transfected rat H9c2 cardiomyoblasts, the alternative promoters specific for ECE‐1a, ECE‐1b, and ECE‐1c are activated by Nkx2‐5 coexpression. Lack of a consensus sequence for Nkx2‐5 binding within the ECE‐1c promoter and mutational analyses of Nkx2‐5 consensus sequences identified in the ECE‐1a and ECE‐1b promoters, respectively, reveal an indirect mechanism of activation that is supported by gel shift assays. Furthermore, we have evidence of an additional direct activation mechanism of the ECE‐1b promoter by Nkx2‐5. With the use of RNase protection assay, Northern blot, and real‐time PCR, the activating effect of Nkx2‐5 on mRNA expression of ECE‐1 isoforms was confirmed in the chromatin context of H9c2 and endothelial EA.hy926 cells, respectively, by stable Nkx2‐5 overexpression. The interaction presented in this work provides a possible explanation for distinct phenotypic aspects of patients carrying mutations in the Nkx2‐5 gene and may also be of significance for the pathophysiology of heart failure.