Abstract 700: AP-1-Mediated Downregulation of Nox4 Promoter Activity by Laminar Shear Stress in Human Endothelial Cells
Background: In endothelial cells, an NAD(P)H oxidase complex containing Nox4 has been identified as a major source of superoxide anion formation. However, the impact of long-term laminar shear stress on superoxide anion formation, Nox4 expression and the human Nox4 promoter has not been studied in detail so far.
Methods and Results: Primary cultures of human umbilical vein endothelial cells were exposed to arterial levels of laminar shear stress (30 dyne/cm2) in a cone-and-plate viscometer for up to 24 hours. Long-term arterial laminar shear stress downregulated superoxide anion formation to 56 ± 12%. Furthermore, laminar shear stress (30 dyne/cm2, 24 h) caused downregulation of Nox4 mRNA expression to 58 ± 5% of control. In order to analyze regulation of Nox4 in more detail, we cloned a 1200 bp fragment upstream of the transcription start site of the Nox4 gene. In human endothelial cells, this construct and terminal deletions of 1071 bp and 809 bp showed a 7-fold increased luciferase activity compared to pGL3-basic (dual-luciferase assay). Nox4 promoter deletions of 724 bp and 428 bp showed reduced basal luciferase activity (3.6- and 2.5-fold). After application of laminar shear stress (30 dyne/cm2), only the full-length construct of 1200 bp showed a reduced luciferase activity compared to static control. In the Nox4 promoter region between −1200 and −1071 mediating the shear stress-depended downregulation an activator protein-1 (AP-1) binding site was found. After mutation of this AP-1 binding site at position -1137, shear stress-dependent downregulation was abolished. In line with these findings, we observed a downregulation of DNA binding activity of a c-jun-containing AP-1 transcription factor in response to long-term laminar shear stress (30 dyne/cm2, 24 h: 70 ± 17% of control, EMSA).
Conclusion: This is the first report describing a functional promoter analysis of major human endothelial NAD(P)H oxidase subunit Nox4. An AP-1 binding site was found to be essential for downregulation of Nox4 and might be involved in vasoprotective downregulation of endothelial superoxide anion formation by long-term laminar shear stress. Y:Eigene Dateien AHA Nox4 promoter abstract AHA 2006.doc