Transcriptional and Posttranscriptional Regulation of Endothelial Nitric Oxide Synthase Expression by Hydrogen Peroxide

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Abstract
—Diverse stimuli, including shear stress, cyclic strain, oxidized LDL, hyperglycemia, and cell growth, modulate endothelial nitric oxide synthase (eNOS) expression. Although seemingly unrelated, these may all alter cellular redox state, suggesting that reactive oxygen intermediates might modulate eNOS expression. The present study was designed to test this hypothesis. Exposure of bovine aortic endothelial cells for 24 hours to paraquat, a superoxide (O 2 −· )–generating compound, did not affect eNOS mRNA levels. However, cotreatment with paraquat and either Cu 2+ /Zn 2+ superoxide dismutase or the superoxide dismutase mimetic tetrakis(4-benzoic acid)porphyrin chloride increased eNOS mRNA by 2.3- and 2.2-fold, respectively, implicating a role for H 2 O 2 . Direct addition of 100 and 150 μmol/L H 2 O 2 caused increases in bovine aortic endothelial cell eNOS mRNA that were dependent on concentration (ie, 3.1- and 5.2-fold increases) and time, and elevated eNOS protein expression and enzyme activity, accordingly. Nuclear run-on and 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole–chase studies showed that H 2 O 2 caused a 3.0-fold increase in eNOS gene transcription and a 2.8-fold increase in eNOS mRNA half-life. Induction of eNOS by H 2 O 2 was not affected by the hydroxyl radical scavenger DMSO, mannitol, or N-tert-butyl -α-phenylnitrone, but it was inhibited by the antioxidants N -acetylcysteine, ebselen, and exogenously added catalase. Unlike H 2 O 2 , the 4.0-fold induction of eNOS by shear stress (15 dyne/cm 2 for 6 hours) was not inhibited by N -acetylcysteine or exogenous catalase. In conclusion, H 2 O 2 increases eNOS expression through transcriptional and post-transcriptional mechanisms. Although H 2 O 2 does not mediate shear-dependent eNOS regulation, it is likely to be involved in regulation of eNOS expression in response to other physiological and/or pathophysiological stimuli.

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