Hydrogen peroxide decreases endothelial nitric oxide synthase promoter activity through the inhibition of AP-1 activity

Sanjiv Kumar, Xutong Sun, Stephen Wedgwood, Stephen M. Black

Research output: Contribution to journalArticle

34 Scopus citations

Abstract

Previously, we have reported that endothelial nitric oxide synthase (eNOS) promoter activity is decreased in pulmonary arterial endothelial cells (PAECs) in response to hydrogen peroxide (H2O2). Thus the objective of this study was to identify the cis-element(s) and transcription factor(s) responsible for oxidant-mediated downregulation of the eNOS gene. Initial promoter experiments in PAECs treated with H2O2 revealed a significant decrease in activity of a promoter fragment containing 840 bp of upstream sequence of the human eNOS gene fused to a luciferase reporter. However, a promoter construct containing only 640 bp of upstream sequence had a significantly attenuated response to H2O2 challenge. As the 840-bp promoter construct had a putative binding site for the transcription factor activator protein-1 (AP-1) that was lacking in the 640-bp construct, we evaluated the effect of H2O2 on promoter activity after mutation of the AP-1 binding sequence (TGAGTCA at -661 to TGAGTtg in the 840-bp construct). Similar to the results seen with the 640 bp, the AP-1 mutant promoter had a significantly attenuated response to H2O 2. EMSA revealed decreased binding of AP-1 during H2O 2 treatment. Supershift analysis indicated that the AP-1 complex consisted of a c-Jun and FosB heterodimer. Furthermore, in vitro EMSA analysis indicated the c-Jun binding was significantly decreased after H 2O2 exposure. Using chromatin immunoprecipitation analysis, we demonstrated decreased binding of AP-1 to the eNOS promoter in vivo in response to H2O2. These data suggest a role of decreased AP-1 binding likely through c-Jun in the H2O 2-mediated decrease in eNOS promoter activity.

Original languageEnglish (US)
Pages (from-to)L370-L377
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume295
Issue number2
DOIs
StatePublished - Aug 1 2008

Keywords

  • Gene regulation
  • Oxidative stress
  • Pulmonary hypertension

ASJC Scopus subject areas

  • Physiology
  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology

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