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

Abstract

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.

Characterization of Polymorphic Structure of Cathepsin G Gene

“…Such differences in promoter function have recently been reported for the endothelin-converting enzyme-1 gene, in which the same promoter-reporter construct displayed different activities depending on the cell type studied. 21 Second, it is obvious that in our experimental context, the basal promoter activity was relatively low so that allelic differences might be more difficult to assess. Third, it is also possible that the functionality of the allelic promoter constructs would have been more specifically tested under different in vitro conditions because it might more appropriately correspond to the physiological or pathophysiological conditions in vivo.…”

Characterization of the c‐specific promoter of the gene encoding human endothelin‐converting enzyme‐1 (ECE‐1)

“…But little is known regarding isoform‐specific ECE‐1 gene regulation in vivo and in vitro: Shao et al [16] showed different regulation of ECE‐1a, also termed ECE‐1β [11,17], and ECE‐1b (=1α) in a rat liver injury model and Corder et al studied the expression of ECE‐1a, 1b and 1c in stimulated bovine aortic endothelial cells [18]. In addition, we have recently demonstrated that ECE‐1a and ECE‐1b are regulated by alternative promoters and not by differential splicing and that members of the ETS family of transcription factors are involved in transcriptional regulation of ECE‐1a expression ([5,19,20]; submitted data). To gain further insight into isoform‐specific ECE‐1 gene expression we have cloned and analyzed the promoter of the major isoform, ECE‐1c.…”

ECV304 (endothelial) is really T24 (bladder carcinoma): Cell line cross-contamination at source

“…Ten micrograms of the respective genomic DNAs were subjected to complete HinfI digestion. The literature documents further that significant functional differences were reported between HUVECs and ECV304 with regard to various aspects, e.g., expression of endothelial cell-specific enzyme mRNA, expression of endothelial cell-specific/-associated surface antigens such as CD31, CD62E and yon Wiilebrand factor, and effects of exposure to plasma albumin (Hughes, 1996;Fuentes et al, 1997;Funke-Kaiser et al, 1998). A detailed description of the methods used will be given elsewhere (Dirks et al, in preparation).…”