Regulation of the major isoform of human endothelin-converting enzyme-1 by a strong housekeeping promoter modulated by polymorphic microsatellites.
BACKGROUND
Human endothelin-converting enzyme (ECE)-1, the key enzyme in endothelin biosynthesis, shows broad cell and tissue expression within the cardiovascular system. Expression of ECE-1c, which represents the major ECE-1 isoform, is directed by an alternative promoter, but the mechanisms of ECE-1c promoter regulation are largely unknown. As ECE-1c transcription is initiated from several start sites, we hypothesized that the ECE-1c promoter functions as a housekeeping promoter.


OBJECTIVE
To investigate the putative housekeeping function of the ECE-1c promoter in vascular endothelial cells, which represent a main site of its expression.


RESULTS
Using promoter reporter assays, gel shift and supershift assays, we have demonstrated, in human endothelial EA.hy926 cells, functionality of cis-acting elements for binding of the CAAT-box binding protein NF-YB, GATA-2) E2F-2, and a GC-box binding factor, which are spatially associated with transcriptional start sites of ECE-1c. In the more upstream promoter region we have identified three highly polymorphic dinucleotide repeats, 5'-(CA)n, (CG)n and 3'-(CA)n, which strongly affected promoter function in endothelial EA.hy926 cells (2.7-fold activation comparing the most active to the least active allele) and, in a similar manner, in human neuronal KELLY cells. Finally, by in-vitro methylation, we were able to achieve strong suppression of the ECE-1c promoter activity in endothelial cells.


CONCLUSIONS
Our results provide a molecular explanation for constitutive expression of ECE-1c mRNA. Modulation by genetic and epigenetic mechanisms as revealed in our study may account for interindividual variation of the constitutive endothelin system activity in humans and thus influence individual predisposition to cardiovascular disease.
Institute of Clinical Pharmacology and Toxicology, Charité - Campus Benjamin Franklin, Berlin, Germany.
Heiko
H
Funke-Kaiser
Alexander
A
Thomas
Juliane
J
Bremer
Slobodan D
SD
Kovacevic
Kathrin
K
Scheuch
Juliane
J
Bolbrinker
Steffen
S
Theis
Julia
J
Lemmer
Andreas
A
Zimmermann
Frank S
FS
Zollmann
Stefan-Martin
SM
Herrmann
Martin
M
Paul
Hans-Dieter
HD
Orzechowski
D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D023081:CCAAT-Binding Factor / Q000502:physiology; D002460:Cell Line; D016384:Consensus Sequence / Q000502:physiology; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000502:physiology; D018900:Dinucleotide Repeats; D050703:E2F2 Transcription Factor; D000072396:Endothelin-Converting Enzymes; D050989:GATA2 Transcription Factor; D005786:Gene Expression Regulation / Q000502:physiology; D016678:Genome; D006801:Humans; D008666:Metalloendopeptidases; D018895:Microsatellite Repeats / Q000502:physiology*; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000235:genetics; D014157:Transcription Factors / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology
D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins; D050703:E2F2 Transcription Factor; D050989:GATA2 Transcription Factor; C494711:GATA2 protein, human; C492063:NFYB protein, human; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes