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

Human ECE-1 is expressed in four isoforms with different tissue distributio n and its mRNA and protein levels are altered under certain pathophysiologi cal conditions. To investigate the transcriptional regulation of ECE-1, we studied the regulatory region of ECE-1c, the major ECE-1 isoform, A genomic clone comprising the complete human ECE-1 gene including the putative ECE- 1c-specific promoter was obtained, Up to 968 bp upstream of the putative c- specific translation initiation start codon and several serial deletion mut ants were subcloned into a reporter vector and transfected into endothelial (BAEC, EA.hy926, ECV304) and epithelial (MDA MB435S, MCF7) cells, showing very strong promoter activity in comparison to the SV40 promoter and to the previously described ECE-1a and 1b promoters. Transfection of serial delet ion mutants indicated two positive regulatory regions within the promoter ( -142/-240 and -240/490) likely involved in binding GATA and ETS transcripti on factors, RNase protection assay (RPA) and 5'-RACE revealed multiple tran scriptional start sites located at about -110, -140 and -350 bp, Site-direc ted mutagenesis; demonstrated a crucial role for the E2F cis-element for ba sal ECE-1c promoter activity, Additionally, we found a correlation between isoform-specific ECE-1 mRNA levels and corresponding ECE-1a, 1b, 1c promote r activities. (C) 2000 Federation of European Biochemical Societies.