Hydrogen Peroxide Decreases Endothelial Nitric Oxide Synthase Promoter Activity through the Inhibition of Sp1 Activity

We have previously shown that endothelial nitric oxide synthase (eNOS) promoter activity is decreased in endothelial cells in response to the addition of hydrogen peroxide (H2O2), and this involves, at least in part, the inhibition of AP-1 activity. Thus, the objective of this study was to determine if other cis-element(s) and transcription factor(s) are involved in the oxidant-mediated downregulation of eNOS. Our initial experiments indicated that although H2O2 treatment increased eNOS mRNA levels in ovine pulmonary arterial endothelial cells (OPAECs), there was a significant decrease in the promoter activity of an eNOS promoter construct containing 840 bp of upstream sequence. However, a truncated promoter construct that lacked the AP-1 element (650 bp) was also inhibited by H2O2. A similar effect was observed when the 650 bp human eNOS promoter construct was transfected into human PAECs. We also found that although exposure of the cells to PEG-catalase prevented the inhibitory effect on eNOS promoter activity, the hydroxyl radical scavenger, deferoxamine myslate, did not. Nor could we identify an increase in hydroxyl radical levels in cells exposed to H2O2. Exposure of PAECs caused a significant increase in labile zinc levels in response to H2O2. As the eNOS promoter has a cis-element for Sp1 binding, we evaluated the role of Sp1 in response to H2O2. As previously reported, mutation of the Sp1 consensus lead to the complete loss of eNOS promoter activity, confirming the key role of Sp1 in regulating basal eNOS promoter activity. In addition, we found, using electrophoretic mobility and supershift assays, that H2O2 decreased Sp1 binding. Finally, using chromatin immunoprecipitation analysis, we found a significant decrease in Sp1 binding to the eNOS promoter in vivo in response to treatment with H2O2. Together, these data suggest that the inhibition of Sp1 activity, possibly through loss of zinc in the protein, plays a role in the H2O2-induced inhibition of eNOS promoter activity.