Identification of the hypoxia-responsive element in the 5′ flanking region of human endothelin-1 gene

Academic Article

Abstract

  • We have previously demonstrated that exposure to hypoxia selectively increases endothelin-1 (ET-1) gene expression in rat lung, and in cultured human pulmonary microvessel endothelial cells and hepatocytes (HepSB). The current study was designed to identify the cis-regulatory element(s) of the human ET-1 gene which mediate hypoxia induced ET-1 gene expression in cultured Hep3B cells. Fragments of 5'-flanking promoter of human ET-1 gene were subcloned into a pGL vector with a luciferase (Luc) reporter gene. The constructed plasmids were transfected into HepSB cells by the lipofection method. A CMV promoter driven β-galactosidase (β-Gal) gene was cotransfected for assessment of transfection efficiency. Transfected Hep3B cells were placed in hypoxic (1%O2-5%CO2) or normoxic (21%O2-5%CO2) incubators for 24 hrs. ET-1 promoter activities were expressed as Luc/β-Gal ratios in cell extracts. Results (mean±SEM. n=6, expressed as % of -2459/+165 normoxic controls) are: -2459/+165# -1514/+165 -645/+165 -138/+165 Normoxia 100±15 1000± 53 84±6 96±11 Hypoxia 288±601t 2597+418 256±98 118±10 # Numbers (relative to transcription start site) represent the length of the ET-1 promoter fragment . p<0.05, compared to their respective normoxic groups. These data indicate that there are positive hypoxia-inducible element(s) located between -645 and the transcription start site of the 5′-flanking region of the ET-1 promoter, supporting the hypothesis that ET-1 is a hypoxia response gene.
  • Authors

    Published In

  • The FASEB Journal  Journal
  • Author List

  • Li HB; Chen YF; Oparil S
  • Volume

  • 10
  • Issue

  • 3