Identification of hypoxia-response element in the human endothelial nitric-oxide synthase gene promoter.
The human endothelial nitric-oxide synthase gene (heNOS) is constitutively expressed in endothelial cells, and its expression is induced under hypoxia. The goal of this study was to search for regulatory elements of the endothelial nitric-oxide synthase (eNOS) gene responsive to hypoxia. Levels of eNOS mRNA, measured by real time reverse transcriptase-PCR analysis, were increased, and heNOS promoter activity was enhanced by hypoxia as compared with normoxia control experiments. Promoter truncation followed by footprint analysis allowed the mapping and identification of the hypoxia-responsive elements at position -5375 to -5366, closely related to hypoxia-inducible factor (HIF)-responsive element (HRE). To test whether known HIF-1 and HIF-2 are involved in hypoxia-induced heNOS promoter activation, HMEC-1 and HUVEC were transiently transfected with HIF-1alpha and HIF-1beta or HIF-2alpha and HIF-1beta expression vectors. Exogenous HIF-2 markedly increased luciferase reporter activity driven by the heNOS promoter in its native location. The induction of luciferase was conserved with the antisense construct and was increased in cotransfection experiments when this fragment was cloned 5' to the proximal 785-bp fragment of the eNOS promoter. Deletion analysis and site-directed mutagenesis demonstrated that the two contiguous HIF consensus binding sites spanning bp -5375 to -5366 relative to the transcription start site were both functional for heNOS promoter activity induction by hypoxia and by HIF-2 overexpression. In conclusion, we demonstrate that heNOS is a hypoxia-inducible gene, whose transcription is stimulated through HIF-2 interaction with two contiguous HRE sites located at -5375 to -5366 of the heNOS promoter.
Coulet
Florence
F
INSERM, Unit 525, Université Pierre et Marie Curie, Faculté de Médecine Pitié-Salpêtrière, 91 Boulevard de l'Hôpital, 75013 Paris, France.
Nadaud
Sophie
S
Agrapart
Monique
M
Soubrier
Florent
F
D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000502:physiology; D003676:Deferoxamine / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology; D004790:Enzyme Induction; D006801:Humans; D000860:Hypoxia / Q000235:genetics*; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D009687:Nuclear Proteins / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics; D020218:Response Elements; D015534:Trans-Activators / Q000378:metabolism / Q000502:physiology; D014157:Transcription Factors; D014158:Transcription, Genetic; D000099162:Endothelial PAS Domain-Containing Protein 1
D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D003676:Deferoxamine; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D009687:Nuclear Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000099162:Endothelial PAS Domain-Containing Protein 1; C497442:HIF1A protein, human; C496314:NOS3 protein, human