Identification of cis-elements of the human endothelin-A receptor gene and inhibition of the gene expression by the decoy strategy

Abstract

Previously, we succeeded in molecular cloning of the cDNA and the gene for human endothelin-A receptor (ET-AR). In the present study, we define cis- elements in the 5'-flanking region of the ET-AR gene. Deletion analyses were performed in A7r5 cells, rat vascular smooth muscle cell line, and Chinese hamster ovary cells using ET-AR promoter-luciferase gene constructs including 5 kilobases of the 5'-flanking region. These analyses demonstrated the existence of one negative regulatory element (-2.0 kilobases to -857 bases) and two positive regulatory elements (-137 to -53 and -53 to +251). Gel mobility shift assay revealed a nuclear protein binding to the region (-104 to -78) (R1). DNase I footprinting analysis showed a footprint spanning from -91 to -83 whose sequence is CCCCACCTT (ETA-P1). When a plasmid including R1 fragments (R1 decoy) was co-transfected into A7r5 cells with ET-AR (-137 to +251)-luciferase gene construct, it significantly reduced the luciferase activity in a dose-dependent manner. Moreover, R1 decoy down-regulated the endogenous ET-AR mRNA in A7r5 cells by a maximum of 75%. Thus, we identified cis-elements that regulate basal transcriptional activity of the ET-AR gene and proved the feasibility to suppress the expression of the ET-AR gene by the DNA decoy strategy using the positive regulatory element we identified.