Modulation of inducible nitric oxide synthase by hypoxia in pulmonary artery endothelial cells.
The effects of hypoxia on the regulation of inducible nitric oxide synthase (NOS) 2 expression were examined in cultured rat pulmonary microvascular endothelial cells (EC). EC did not express NOS 2 mRNA or protein when exposed to normoxia or hypoxia unless they were pretreated with interleukin (IL)-1beta and/or tumor necrosis factor (TNF)-alpha for 24 h. Induction of NOS 2 by IL-1beta+TNF-alpha was significantly attenuated by concomitant exposure of EC to hypoxia or treatment of EC with antioxidants such as tiron, diphenyliodonium, and catalase, suggesting that NOS 2 expression is dependent on the production of reactive oxygen species. Degradation of IkappaB and activation of NF-kappaB, which were both induced by treatment of EC with cytokines, were not altered when the cells were exposed to hypoxia, suggesting that the modulation of NOS 2 expression by hypoxia is unrelated to NF-kappaB activation. Following stimulation with IL-1beta+TNF-alpha for 24 h, incubation of EC in normoxia resulted in a progressive decline in NOS 2 expression and a calculated half-life of approximately 6 h for NOS 2 mRNA. Hypoxia significantly prolonged the half-life of NOS 2 mRNA (17 h, P < 0.05 versus normoxic EC). The half-life of NOS 2 mRNA was also prolonged by actinomycin D treatment (19.5 and 29.5 h for normoxic and hypoxic EC, respectively), suggesting that transcription of an RNA destabilizing factor or RNAse contributes to NOS 2 mRNA degradation. In EC transiently transfected with the rat NOS 2 promoter, hypoxia and the combination of IL-1beta+TNF-alpha independently increased promoter activity 2.2- and 3-fold, respectively. As opposed to the attenuating effect that hypoxia had on IL-1beta+TNF-alpha- dependent induction of NOS 2 gene expression, the concomitant treatment with IL-1beta+TNF-alpha and hypoxia synergistically increased NOS 2 promoter activity 17.6-fold. Taken together, these results suggest that hypoxia alone does not induce NOS 2 expression in cultured pulmonary microvascular EC, but may modulate cytokine induction of this enzyme at pretranscriptional, transcriptional, and posttranscriptional levels.
Zulueta
Javier J
JJ
Pulmonary and Critical Care Division, Department of Medicine/Tupper Research Institute, New England Medical Center, Boston, Massachusetts 02111, USA.
Sawhney
Raneeta
R
Kayyali
Usamah
U
Fogel
Michael
M
Donaldson
Cameron
C
Huang
Hailu
H
Lanzillo
Joseph J
JJ
Hassoun
Paul M
PM
D000818:Animals; D015153:Blotting, Western; D002374:Catalase / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis; D003609:Dactinomycin / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology* / Q000201:enzymology*; D004789:Enzyme Activation; D000860:Hypoxia; D007375:Interleukin-1 / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010100:Oxygen / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011651:Pulmonary Artery / Q000166:cytology / Q000201:enzymology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016207:Cytokines; D007375:Interleukin-1; D016328:NF-kappa B; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin; D002374:Catalase; D008156:Luciferases; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D010100:Oxygen