Hypoxia induces type II NOS gene expression in pulmonary artery endothelial cells via HIF-1.
Type II nitric oxide synthase (NOS) is upregulated in the pulmonary vasculature in a chronic hypoxia model of pulmonary hypertension. In situ hybridization analysis demonstrates that type II NOS RNA is increased in the endothelium as well as in the vascular smooth muscle in the lung. The current studies examine the role of hypoxia-inducible factor (HIF)-1 in regulating type II NOS gene expression in response to hypoxia in pulmonary artery endothelial cells. Northern blot analyses demonstrate a two fold increase in HIF-1 alpha but not in HIF-1 beta RNA with hypoxia in vivo and in vitro. Electrophoretic mobility shift assays show the induction of specific DNA binding activity when endothelial cells were subjected to hypoxia. This DNA binding complex was identified as HIF-1 using antibodies directed against HIF-1 alpha and HIF-1 beta. Transient transfection of endothelial cells resulted in a 2.7-fold increase in type II NOS promoter activity in response to hypoxia compared with nonhypoxic controls. Mutation or deletion of the HIF-1 site eliminated the response to hypoxia. These results demonstrate that HIF-1 is essential for the hypoxic regulation of type II NOS gene transcription in pulmonary endothelium.
Palmer
L A
LA
Department of Anesthesiology, University of Virginia Health Sciences Center, Charlottesville 22906-0010, USA.
Semenza
G L
GL
Stoler
M H
MH
Johns
R A
RA
D000818:Animals; D002417:Cattle; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000201:enzymology*; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D018257:Helix-Loop-Helix Motifs; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D009687:Nuclear Proteins / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011651:Pulmonary Artery; D012333:RNA, Messenger / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D010100:Oxygen