Modulation of inducible nitric oxide synthase by hypoxia in pulmonary artery endothelial cells
Zulueta JJ, Sawhney R, Kayyali U, Fogel M, Donaldson C, Huang H, Lanzillo JJ, Hassoun PM.
Pulmonary and Critical Care Division, Department of Medicine/Tupper Research Institute, New England Medical Center, Boston, Massachusetts 02111, USA
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.
CITATION Am J Respir Cell Mol Biol. 2002 Jan;26(1):22-30