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

J. Yamashita, T. Yoshimasa, H. Arai, J. Hiraoka, K. Takaya, Y. Miyamoto, Y. Ogawa, H. Itoh, K. Nakao

研究成果: Article査読

28 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)15993-15999
ページ数7
ジャーナルJournal of Biological Chemistry
273
26
DOI
出版ステータスPublished - 1998 6 26
外部発表はい

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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