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Basic Research in Cardiology

, Volume 106, Issue 4, pp 551–561 | Cite as

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

  • Claudia Goettsch
  • Winfried Goettsch
  • Melanie Brux
  • Claudia Haschke
  • Coy Brunssen
  • Gregor Muller
  • Stefan R. Bornstein
  • Nicole Duerrschmidt
  • Andreas H. Wagner
  • Henning Morawietz
Original Contribution

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.

Keywords

NADPH oxidase Endothelial cells Shear stress Nrf2 Oct-1 

Notes

Acknowledgments

This work was supported by the German Federal Ministry of Education and Research program, NBL3, of the University of Technology Dresden (PhD program Metabolism and Endothelium to C.G.; Professorship of Vascular Endothelium and Microcirculation to H.M.), the MeDDrive program of the Medical Faculty Carl Gustav Carus of the University of Technology Dresden, Germany (to C.G. and W.G.), the Doktor Robert Pfleger Foundation, Bamberg, Germany (to H.M. and W.G), and the Deutsche Forschungsgemeinschaft (SFB/TR2 to H.M. and A.H.W., GO 1801/4–1 to C.G. and MO 1695/4–1 to H.M.).

Conflict of interest

None.

Supplementary material

395_2011_170_MOESM1_ESM.doc (1.3 mb)
Supplementary material 1 (DOC 1,356 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Claudia Goettsch
    • 1
    • 2
  • Winfried Goettsch
    • 1
  • Melanie Brux
    • 1
  • Claudia Haschke
    • 1
  • Coy Brunssen
    • 1
  • Gregor Muller
    • 1
  • Stefan R. Bornstein
    • 3
  • Nicole Duerrschmidt
    • 4
  • Andreas H. Wagner
    • 5
  • Henning Morawietz
    • 1
  1. 1.Division of Vascular Endothelium and Microcirculation, Department of Medicine IIIUniversity of Technology DresdenDresdenGermany
  2. 2.Division of Endocrinology, Diabetes, and Metabolic Bone Diseases, Department of Medicine III University of Technology DresdenDresdenGermany
  3. 3.Division of Endocrinology, Diabetes and Metabolism, Department of Medicine III University of Technology DresdenDresdenGermany
  4. 4.Department of Cardiac Surgery, Heart Center LeipzigUniversity of LeipzigLeipzigGermany
  5. 5.Institute of Physiology and Pathophysiology, University of HeidelbergHeidelbergGermany

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