Endothelin-converting enzyme-1 (ECE-1) is a downstream target of the homeobox transcription factor Nkx2-5.
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.
Funke-Kaiser
H
H
Institute of Clinical Pharmacology and Toxicology, Benjamin Franklin Medical Center, Department of Clinical Pharmacology, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany.
Lemmer
J
J
Langsdorff
C V
CV
Thomas
A
A
Kovacevic
S D
SD
Strasdat
M
M
Behrouzi
T
T
Zollmann
F S
FS
Paul
M
M
Orzechowski
H-D
HD
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D001665:Binding Sites; D002460:Cell Line; D002843:Chromatin / Q000235:genetics; D016384:Consensus Sequence; D000072396:Endothelin-Converting Enzymes; D004790:Enzyme Induction; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000378:metabolism*; D008666:Metalloendopeptidases; D008957:Models, Genetic; D032386:Myoblasts, Cardiac / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D029867:Xenopus Proteins
D002843:Chromatin; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C095552:NKX2-5 protein, human; D020033:Protein Isoforms; D014157:Transcription Factors; D029867:Xenopus Proteins; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes