Human Endothelin-Converting Enzyme-1β mRNA Expression Is Regulated by an Alternative Promoter

Publisher Website
Google Scholar
Abstract
The central step in endothelin biosynthesis is sitespecific 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 α and β, 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 α-upstream region exerts promoter activity in endothelial cells. To clarify whether the 5′-untranslated region upstream of exon 3, which contains the β-specific sequence, acts as an alternative transcriptional promoter, we sequenced and cloned 1,206 bp upstream of the β-specific translation initiation codon in a luciferase reporter vector. After transfection, we detected strong promoter activity in primary cultured endothelial cells (HUVECs, 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 typerestricted action of the β-promoter and, in conjunction with the previously reported transcriptional start sites, clearly prove the existence of an alternative β-specific promoter located in intron 2 of the human ECE-1 gene.