Evidence of alternative promoters directing isoform-specific expression of human endothelin-converting enzyme-1 mRNA in cultured endothelial cells.

The endothelins, a family of closely related vasoactive and mitogenic peptides, are thought to play an important role in cardiovascular pathophysiology. The conversion of the inactive precursor "big endothelin" to the biologically active peptide is catalyzed in vitro and in vivo by endothelin-converting enzymes (ECE). Recently the cDNA cloning of two homologous proteins, termed ECE-1 and ECE-2, has been reported. ECE-1 may play a key role in the activation and regulation of the cardiovascular endothelin proteolytic cascade. ECE-1 mRNA is expressed in two isoforms, termed alpha and beta, which are identical except for the 5'-terminal regions. To investigate the transcriptional regulation of isoform-specific ECE-1 mRNA expression we isolated phage clones from a human genomic library and identified the alpha- and beta-specific exons of ECE-1. The exon/intron organization of the 5'-terminal region of the human ECE-1 gene in conjunction with putative transcription initiation start sites suggests the existence of two alternative promoters, each directing the expression of either isoform. A reverse transcription/polymerase chain reaction assay indicated differential mRNA expression of ECE-1 isoforms. Using a luciferase reporter gene assay, we found that the genomic region upstream of exon 1 alpha confers strong promoter activity in the human endothelial cell line ECV 304, which was previously shown to express predominantly ECE-1 alpha mRNA. Transfection of serial deletion mutants in ECV304 cells indicated the existence of three positive and also one negative regulating element within 2 kb of the alpha-promoter region. Luciferase reporter gene studies also revealed that the genomic region upstream of exon 3, which encodes the putative ECE-1 beta specific N-terminus, was able to direct luciferase expression in primary cultured bovine aortic endothelial cells, indicating the existence of an alternative promoter. Transfection of nested deletions spanning 1.2 kb upstream of the putative translation initiation codon of ECE-1 beta suggested the existence of three positive regulating regions within the beta-specific promoter. Both ECE-1 promoters lack TATA or CAAT boxes, and the two show different patterns of consensus sequences for transcription factors, suggesting a differential transcriptional regulation of isoform-specific ECE-1 mRNA expression.