Human endothelin-converting enzyme-1 beta mRNA expression is regulated by an alternative promoter.
The central step in endothelin biosynthesis is site-specific cleavage of big endothelins by endothelin-converting enzymes (ECEs). ECE-1 is a membrane-bound metalloprotease, predominantly but not exclusively expressed in endothelial cells. ECE-1 is expressed in two mRNA isoforms, termed alpha and beta, which differ only in the 5'-terminal regions but are functionally very similar when expressed in vitro. The structure of the human ECE-1 gene suggests either alternative splicing or alternative promoters as underlying mechanisms of mRNA isoform expression. We have previously shown that the alpha-upstream region exerts promoter activity in endothelial cells. To clarify whether the 5'-untranslated region upstream of exon 3, which contains the beta-specific sequence, acts as an alternative transcriptional promoter, we sequenced and cloned 1,206 bp upstream of the beta-specific translation initiation codon in a luciferase reporter vector. After transfection, we detected strong promoter activity in primary cultured endothelial cells (HU-VECs, BAECs) but only marginal activity in the endothelial cell line ECV304 and in CHO cells. Maximal promoter activity was observed with the full-length construct, 1206 (136% of the SV40 promoter activity in BAECs). Transfection of serial deletion mutants indicated at least three major regulatory regions within the promoter. Our results are consistent with cell type-restricted action of the beta-promoter and, in conjunction with the previously reported transcriptional start sites, clearly prove the existence of an alternative beta-specific promoter located in intron 2 of the human ECE-1 gene.
Funke-Kaiser
H
H
Institute of Clinical Pharmacology and Toxicology, Benjamin Franklin Hospital, Free University of Berlin, Germany.
Orzechowski
H D
HD
Richter
M
M
Paul
M
M
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D000072396:Endothelin-Converting Enzymes; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D015723:Gene Library; D006801:Humans; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis*
D012333:RNA, Messenger; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes