Regulatory elements in nepriiysin promoter regions

To further characterize as-acting regulatory elements localized in the three promoter regions of the neprilysin gene (NEP, E.G. 3.4.24.11), cell lines were analyzed by RNase protection assays to demonstrate differential expression of NEP mRNAs. While high level expression of the type 1 and type 2 mRNAs was observed in the human spinal cord HSC-2 cell line, there was essentially no NEP mRNA expressed in the human breast adenocarcinoma MCF-7 cell line. This indicates the presence of transcriptional factors in these cells which regulate the activity of NEP promoters. Rat genomic fragments upstream of the three noncoding exons were subcloned in the promoterless vector pXpl fused to a downstream luciferase gene. All fragments showed promoter activity in HSC-2 cells, but not in MCF-7 cells. Deletions from the 5' end of a L9 kb fragment containing the first promoter region were prepared. Analysis of these constructs showed that deletion of 22 bp between 137 and 115 bp upstream to the start site of the first noncoding exon eliminated >90% of the reporter gene activity. These results suggest that expression of the type 1 neprilysin mRNA is transcriptionally regulated through this 22 bp fragment.