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

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.