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

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 (H(2)O(2)). 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 H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) 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 H(2)O(2). EMSA revealed decreased binding of AP-1 during H(2)O(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(2)O(2) exposure. Using chromatin immunoprecipitation analysis, we demonstrated decreased binding of AP-1 to the eNOS promoter in vivo in response to H(2)O(2). These data suggest a role of decreased AP-1 binding likely through c-Jun in the H(2)O(2)-mediated decrease in eNOS promoter activity.

Brief Hyperoxia Increases Mitochondrial Oxidation and Increases Phosphodiesterase 5 Activity in Fetal Pulmonary Artery Smooth Muscle Cells

“…Thus, our data are consistent with previous studies showing that exogenous H 2 O 2 is capable of activating PDE5 in FPASMC (6). Regardless of the source, H 2 O 2 can inhibit eNOS (15), down-regulate sGC (38), and stimulate PDE5 (6) in the pulmonary vasculature.…”

Glycated human serum albumin induces NF-κB activation and endothelial nitric oxide synthase uncoupling in human umbilical vein endothelial cells

“…et al 1993). AP-1 up-regulates eNOS transcription by cyclosporine A, hypoxia, shear stress and nordihydroguaiaretic acid, whereas H 2 O 2 decreases binding of AP-1 to the eNOS promoter, leading to reduced eNOS expression (Kumar, S. et al 2008). These data suggest an…”

Cyclic stretch stimulates mitochondrial reactive oxygen species and Nox4 signaling in pulmonary artery smooth muscle cells

“…We previously reported finding an increase in H 2 O 2 signaling in the PA of PPHN lambs (48,51), which may induce abnormal vascular tone through a variety of mechanisms (40,55), thereby contributing importantly to the pathogenesis of PPHN (50). H 2 O 2 attenuates NO-mediated vasorelaxation in PPHN via inhibition of endothelial NOS (26) and extracellular superoxide dismutase (50), downregulation of soluble guanylate cyclase (51), and stimulation of cGMP-specific phosphodiesterase (12). Catalase treatment normalized vasodilator responses to NO in isolated fetal PPHN PAs (51), while intratracheal catalase improved oxygenation and vasorelaxation in ventilated PPHN lambs (50).…”