title: Arterial flow reduces oxidative stress via an antioxidant response element and Oct-1 binding site within the NADPH oxidase 4 promoter in endothelial cells.
abstract: The main sources of oxidative stress in the vessel wall are nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes. The endothelium mainly expresses the Nox4-containing complex; however, the mechanism by which shear stress in endothelial cells regulates Nox4 is not well understood. This study demonstrates that long-term application of arterial laminar shear stress using a cone-and-plate viscometer reduces endothelial superoxide anion formation and Nox4 expression. In primary human endothelial cells, we identified a 47 bp 5'-untranslated region of Nox4 mRNA by 5'-rapid amplification of cDNA ends (5'-RACE) PCR. Cloning and functional analysis of human Nox4 promoter revealed a range between -1,490 and -1,310 bp responsible for flow-dependent downregulation. Mutation of an overlapping antioxidative response element (ARE)-like and Oct-1 binding site at -1,376 bp eliminated shear stress-dependent Nox4 downregulation. Consistent with these observations, electrophoretic mobility shift assays (EMSA) demonstrated an enhanced shear stress-dependent binding of Nox4 oligonucleotide containing the ARE-like/Oct-1 binding site, which could be inhibited by specific antibodies against the transcription factors nuclear factor erythroid 2-related factor 2 (Nrf2) and octamer transcription factor 1 (Oct-1). Furthermore, shear stress caused the translocation of Nrf2 and Oct-1 from the cytoplasm to the nucleus. Knockdown of Nrf2 by short hairpin RNA (shRNA) increased Nox4 expression twofold, indicating a direct cross-talk between Nrf2 and Nox4. In conclusion, an ARE-like/Oct-1 binding site was noticed to be essential for shear stress-dependent downregulation of Nox4. This novel mechanism may be involved in the flow-dependent downregulation of endothelial superoxide anion formation.
authors.affiliation: Division of Vascular Endothelium and Microcirculation, Department of Medicine III, University of Technology Dresden, Fetscherstrasse 74, Dresden, Germany. claudia.goettsch@uniklinikum-dresden.de
authors.forename: Claudia
authors.initials: C
authors.lastname: Goettsch
authors.forename: Winfried
authors.initials: W
authors.lastname: Goettsch
authors.forename: Melanie
authors.initials: M
authors.lastname: Brux
authors.forename: Claudia
authors.initials: C
authors.lastname: Haschke
authors.forename: Coy
authors.initials: C
authors.lastname: Brunssen
authors.forename: Gregor
authors.initials: G
authors.lastname: Muller
authors.forename: Stefan R
authors.initials: SR
authors.lastname: Bornstein
authors.forename: Nicole
authors.initials: N
authors.lastname: Duerrschmidt
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Wagner
authors.forename: Henning
authors.initials: H
authors.lastname: Morawietz
mesh_terms: D000975:Antioxidants / Q000494:pharmacology*; D001665:Binding Sites; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics* / Q000502:physiology; D051267:NF-E2-Related Factor 2 / Q000502:physiology; D050811:Octamer Transcription Factor-1 / Q000502:physiology*; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D017382:Reactive Oxygen Species / Q000378:metabolism; D012039:Regional Blood Flow; D020218:Response Elements / Q000502:physiology*; D013314:Stress, Mechanical
chemical_list: D000975:Antioxidants; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; D017382:Reactive Oxygen Species; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human