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

¹Vascular Biology Center, Medical College of Georgia, Augusta, Georgia; and ²The Department of Pediatrics, Northwestern University, Chicago, Illinois

Submitted 22 February 2008 ; accepted in final form 23 May 2008

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 H₂O₂. EMSA revealed decreased binding of AP-1 during H₂O₂ 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 H₂O₂ 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 H₂O₂-mediated decrease in eNOS promoter activity.

oxidative stress; gene regulation; pulmonary hypertension

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