Hypoxia inducible factor-1: regulation by nitric oxide in posthypoxic microvascular endothelium.
Microvascular endothelial cells provide a critical regulatory interface between blood constituents and tissue. Hypoxia inducible factor-1 (HIF-1) is a key transcription factor required for expression of hypoxia-dependent genes. We employed a model of hypoxia and reoxygenation (H/R) using the dermal microvascular endothelial cell line HMEC-1 to examine the effects of altered oxygen concentrations on microvascular HIF-1 expression and nitric oxide (NO) formation. Hypoxia increased inducible NO synthase (iNOS) mRNA in a time-dependent manner in HMEC-1. However, endothelial NO synthase mRNA progressively declined during hypoxia. H/R promoted significant increases in cellular nitrite levels that were significantly abrogated by the specific iNOS inhibitor N6-(1-iminoethyl)-L-lysine, di hy drochloride. Exogenous NO promoted stabilization of the alpha subunit of HIF-1 and produced functional DNA binding. Exposure of HMEC-1 to H/R resulted in previously unrecognized biphasic HIF-1alpha stabilization during reoxygenation. When the iNOS gene was silenced through the use of iNOS-specific small interfering RNA, HIF-1alpha stabilization and HIF-1 activation were dramatically diminished, suggesting that inducible NOS-derived NO is a key factor sustaining HIF-1 activation during both hypoxia and reoxygenation.
Natarajan
Ramesh
R
Center for Vascular Inflammation Research, Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Box 980050, Virginia CommonwealthUniversity, Richmond, VA 23298, USA. rnataraj@hsc.vcu.edu
Jones
Drew G
DG
Fisher
Bernard J
BJ
Wallace
Timothy J
TJ
Ghosh
Shobha
S
Fowler
Alpha A
AA
D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D004730:Endothelium, Vascular / Q000201:enzymology / Q000378:metabolism / Q000503:physiopathology*; D020868:Gene Silencing; D006801:Humans; D000860:Hypoxia / Q000503:physiopathology*; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D009569:Nitric Oxide / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D051793:Hypoxia-Inducible Factor 1; D012333:RNA, Messenger; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II