Arterial flow reduces oxidative stress via an antioxidant response element and Oct-1 binding site within the NADPH oxidase 4 promoter in endothelial cells

Basic Res Cardiol. 2011 Jun;106(4):551-61. doi: 10.1007/s00395-011-0170-3. Epub 2011 Mar 12.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antioxidants / pharmacology*
  • Binding Sites
  • Cells, Cultured
  • Endothelial Cells / metabolism*
  • Humans
  • NADPH Oxidase 4
  • NADPH Oxidases / genetics*
  • NADPH Oxidases / physiology
  • NF-E2-Related Factor 2 / physiology
  • Octamer Transcription Factor-1 / physiology*
  • Oxidative Stress*
  • Promoter Regions, Genetic*
  • Reactive Oxygen Species / metabolism
  • Regional Blood Flow
  • Response Elements / physiology*
  • Stress, Mechanical


  • Antioxidants
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human
  • Octamer Transcription Factor-1
  • POU2F1 protein, human
  • Reactive Oxygen Species
  • NADPH Oxidase 4
  • NADPH Oxidases
  • NOX4 protein, human