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 va sculature in a chronic hypoxia model of pulmonary hypertension. In sit u hybridization analysis demonstrates that type II NOS RNA is increase d in the endothelium as well as in the vascular smooth muscle in the l ung. The current studies examine the role of hypoxia-inducible factor (HIF)-1 in regulating type II NOS gene expression in response to hypox ia in pulmonary artery endothelial cells. Northern blot analyses demon strate a twofold increase in HIF-1 alpha but not in HIF-1 beta RNA wit h hypoxia in vivo and in vitro. Electrophoretic mobility shift assays show the induction of specific DNA binding activity when endothelial c ells were subjected to hypoxia. This DNA binding complex was identifie d as HIF-1 using antibodies directed against HIF-1 alpha and HIF-1 bet a. Transient transfection of endothelial cells resulted in a 2.7-fold increase in type II NOS promoter activity in response to hypoxia compa red with nonhypoxic controls. Mutation or deletion of the HIF-1 site e liminated the response to hypoxia. These results demonstrate that HIF- 1 is essential for the hypoxic regulation of type II NOS gene transcri ption in pulmonary endothelium.