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


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.


NADPH oxidase Endothelial cells Shear stress Nrf2 Oct-1 



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


Supplementary material

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


  1. 1.
    Ago T, Kitazono T, Ooboshi H, Iyama T, Han YH, Takada J, Wakisaka M, Ibayashi S, Utsumi H, Iida M (2004) Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase. Circulation 109:227–233. doi: 10.1161/01.CIR.0000105680.92873.70 PubMedCrossRefGoogle Scholar
  2. 2.
    Ambasta RK, Kumar P, Griendling KK, Schmidt HH, Busse R, Brandes RP (2004) Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J Biol Chem 279:45935–45941. doi: 10.1074/jbc.M406486200 PubMedCrossRefGoogle Scholar
  3. 3.
    Anish R, Hossain MB, Jacobson RH, Takada S (2009) Characterization of transcription from TATA-less promoters: identification of a new core promoter element XCPE2 and analysis of factor requirements. PLoS One 4:e5103. doi: 10.1371/journal.pone.0005103 PubMedCrossRefGoogle Scholar
  4. 4.
    Arlt A, Bauer I, Schafmayer C, Tepel J, Muerkoster SS, Brosch M, Roder C, Kalthoff H, Hampe J, Moyer MP, Folsch UR, Schafer H (2009) Increased proteasome subunit protein expression and proteasome activity in colon cancer relate to an enhanced activation of nuclear factor E2-related factor 2 (Nrf2). Oncogene 28:3983–3996. doi: 10.1038/onc.2009.264 PubMedCrossRefGoogle Scholar
  5. 5.
    Boon RA, Horrevoets AJ (2009) Key transcriptional regulators of the vasoprotective effects of shear stress. Hamostaseologie 29:39–40PubMedGoogle Scholar
  6. 6.
    Brandes RP, Schroder K (2008) Differential vascular functions of Nox family NADPH oxidases. Curr Opin Lipidol 19:513–518. doi: 10.1097/MOL.0b013e32830c91e3 PubMedCrossRefGoogle Scholar
  7. 7.
    Brandes RP, Weissmann N, Schroder K (2010) NADPH oxidases in cardiovascular disease. Free Radic Biol Med 49:687–706. doi: 10.1016/j.freeradbiomed.2010.04.030 PubMedCrossRefGoogle Scholar
  8. 8.
    Dai G, Vaughn S, Zhang Y, Wang ET, Garcia-Cardena G, Gimbrone MA Jr (2007) Biomechanical forces in atherosclerosis-resistant vascular regions regulate endothelial redox balance via phosphoinositol 3-kinase/Akt-dependent activation of Nrf2. Circ Res 101:723–733. doi: 10.1161/CIRCRESAHA.107.152942 PubMedCrossRefGoogle Scholar
  9. 9.
    Dhakshinamoorthy S, Jain AK, Bloom DA, Jaiswal AK (2005) Bach1 competes with Nrf2 leading to negative regulation of the antioxidant response element (ARE)-mediated NAD(P)H:quinone oxidoreductase 1 gene expression and induction in response to antioxidants. J Biol Chem 280:16891–16900. doi: 10.1161/CIRCRESAHA.107.152942 PubMedCrossRefGoogle Scholar
  10. 10.
    Duerrschmidt N, Stielow C, Muller G, Pagano PJ, Morawietz H (2006) NO-mediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells. J Physiol 576:557–567. doi: 10.1113/jphysiol.2006.111070 PubMedCrossRefGoogle Scholar
  11. 11.
    Fledderus JO, Boon RA, Volger OL, Hurttila H, Yla-Herttuala S, Pannekoek H, Levonen AL, Horrevoets AJ (2008) KLF2 primes the antioxidant transcription factor Nrf2 for activation in endothelial cells. Arterioscler Thromb Vasc Biol 28:1339–1346. doi: 10.1161/ATVBAHA.108.165811 PubMedCrossRefGoogle Scholar
  12. 12.
    Fleming I (2010) Molecular mechanisms underlying the activation of eNOS. Pflugers Arch 459:793–806. doi: 10.1007/s00424-009-0767-7 PubMedCrossRefGoogle Scholar
  13. 13.
    Frith MC, Valen E, Krogh A, Hayashizaki Y, Carninci P, Sandelin A (2008) A code for transcription initiation in mammalian genomes. Genome Res 18:1–12. doi: 10.1101/gr.6831208 PubMedCrossRefGoogle Scholar
  14. 14.
    Goettsch C, Goettsch W, Arsov A, Hofbauer LC, Bornstein SR, Morawietz H (2009) Long-term cyclic strain downregulates endothelial Nox4. Antioxid Redox Signal 11:2385–2397. doi: 10.1089/ars.2009.2561 PubMedCrossRefGoogle Scholar
  15. 15.
    Goettsch C, Goettsch W, Muller G, Seebach J, Schnittler HJ, Morawietz H (2009) Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells. Biochem Biophys Res Commun 380:355–360. doi: 10.1016/j.bbrc.2009.01.107 PubMedCrossRefGoogle Scholar
  16. 16.
    Goettsch W, Gryczka C, Korff T, Ernst E, Goettsch C, Seebach J, Schnittler HJ, Augustin HG, Morawietz H (2008) Flow-dependent regulation of angiopoietin-2. J Cell Physiol 214:491–503. doi: 10.1002/jcp.21229 PubMedCrossRefGoogle Scholar
  17. 17.
    Gorlach A, Brandes RP, Nguyen K, Amidi M, Dehghani F, Busse R (2000) A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall. Circ Res 87:26–32PubMedGoogle Scholar
  18. 18.
    Groeger G, Mackey AM, Pettigrew CA, Bhatt L, Cotter TG (2009) Stress-induced activation of Nox contributes to cell survival signalling via production of hydrogen peroxide. J Neurochem 109:1544–1554. doi: 10.1111/j.1471-4159.2009.06081.x PubMedCrossRefGoogle Scholar
  19. 19.
    Heumuller S, Wind S, Barbosa-Sicard E, Schmidt HH, Busse R, Schroder K, Brandes RP (2008) Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant. Hypertension 51:211–217. doi: 10.1161/HYPERTENSIONAHA.107.100214 PubMedCrossRefGoogle Scholar
  20. 20.
    Hosoya T, Maruyama A, Kang MI, Kawatani Y, Shibata T, Uchida K, Warabi E, Noguchi N, Itoh K, Yamamoto M (2005) Differential responses of the Nrf2-Keap1 system to laminar and oscillatory shear stresses in endothelial cells. J Biol Chem 280:27244–27250. doi: 10.1074/jbc.M502551200 PubMedCrossRefGoogle Scholar
  21. 21.
    Houston P, White BP, Campbell CJ, Braddock M (1999) Delivery and expression of fluid shear stress-inducible promoters to the vessel wall: applications for cardiovascular gene therapy. Hum Gene Ther 10:3031–3044. doi: 10.1089/10430349950016429 PubMedCrossRefGoogle Scholar
  22. 22.
    Hsieh CY, Hsiao HY, Wu WY, Liu CA, Tsai YC, Chao YJ, Wang DL, Hsieh HJ (2009) Regulation of shear-induced nuclear translocation of the Nrf2 transcription factor in endothelial cells. J Biomed Sci 16:12. doi: 10.1186/1423-0127-16-12 PubMedCrossRefGoogle Scholar
  23. 23.
    Hwang J, Ing MH, Salazar A, Lassegue B, Griendling K, Navab M, Sevanian A, Hsiai TK (2003) Pulsatile versus oscillatory shear stress regulates NADPH oxidase subunit expression: implication for native LDL oxidation. Circ Res 93:1225–1232. doi: 10.1161/01.RES.0000104087.29395.66 PubMedCrossRefGoogle Scholar
  24. 24.
    Ikeda Y, Sugawara A, Taniyama Y, Uruno A, Igarashi K, Arima S, Ito S, Takeuchi K (2000) Suppression of rat thromboxane synthase gene transcription by peroxisome proliferator-activated receptor gamma in macrophages via an interaction with NRF2. J Biol Chem 275:33142–33150. doi: 10.1074/jbc.M002319200 PubMedCrossRefGoogle Scholar
  25. 25.
    Jones CI 3rd, Zhu H, Martin SF, Han Z, Li Y, Alevriadou BR (2007) Regulation of antioxidants and phase 2 enzymes by shear-induced reactive oxygen species in endothelial cells. Ann Biomed Eng 35:683–693. doi: 10.1007/s10439-007-9279-9 PubMedCrossRefGoogle Scholar
  26. 26.
    Kronstein R (2006) Ein neuer Caveolin-1 abhängiger Mechanismus zur Regulation der paraendothelialen Barrierrefunktion. Thesis, Medical Faculty Carl Gustav Carus, Technical University of DresdenGoogle Scholar
  27. 27.
    Lauer T, Heiss C, Balzer J, Kehmeier E, Mangold S, Leyendecker T, Rottler J, Meyer C, Merx MW, Kelm M, Rassaf T (2008) Age-dependent endothelial dysfunction is associated with failure to increase plasma nitrite in response to exercise. Basic Res Cardiol 103:291–297. doi: 10.1007/s00395-008-0714-3 PubMedCrossRefGoogle Scholar
  28. 28.
    Lee BS, Kim YM, Kang HS, Kim HM, Pyun KH, Choi I (2001) Octamer binding protein-1 is involved in inhibition of inducible nitric oxide synthase expression by exogenous nitric oxide in murine liver cells. J Biochem 129:77–86PubMedGoogle Scholar
  29. 29.
    Mann GE, Rowlands DJ, Li FY, de Winter P, Siow RC (2007) Activation of endothelial nitric oxide synthase by dietary isoflavones: role of NO in Nrf2-mediated antioxidant gene expression. Cardiovasc Res 75:261–274. doi: 10.1016/j.cardiores.2007.04.004 PubMedCrossRefGoogle Scholar
  30. 30.
    McNally JS, Davis ME, Giddens DP, Saha A, Hwang J, Dikalov S, Jo H, Harrison DG (2003) Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress. Am J Physiol Heart Circ Physiol 285:H2290–H2297. doi: 10.1152/ajpheart.00515.2003 PubMedGoogle Scholar
  31. 31.
    Muller G, Morawietz H (2009) Nitric oxide, NAD(P)H oxidase, and atherosclerosis. Antioxid Redox Signal 11:1711–1731. doi: 10.1089/ars.2008.2403 PubMedCrossRefGoogle Scholar
  32. 32.
    Poss J, Werner C, Lorenz D, Gensch C, Bohm M, Laufs U (2010) The renin inhibitor aliskiren upregulates pro-angiogenic cells and reduces atherogenesis in mice. Basic Res Cardiol 105:725–735. doi: 10.1007/s00395-010-0120-5 PubMedCrossRefGoogle Scholar
  33. 33.
    Schluter T, Zimmermann U, Protzel C, Miehe B, Klebingat KJ, Rettig R, Grisk O (2010) Intrarenal artery superoxide is mainly NADPH oxidase-derived and modulates endothelium-dependent dilation in elderly patients. Cardiovasc Res 85:814–824. doi: 10.1093/cvr/cvp346 PubMedCrossRefGoogle Scholar
  34. 34.
    Schwachtgen JL, Remacle JE, Janel N, Brys R, Huylebroeck D, Meyer D, Kerbiriou-Nabias D (1998) Oct-1 is involved in the transcriptional repression of the von willebrand factor gene promoter. Blood 92:1247–1258PubMedGoogle Scholar
  35. 35.
    Seeger FH, Sedding D, Langheinrich AC, Haendeler J, Zeiher AM, Dimmeler S (2010) Inhibition of the p38 MAP kinase in vivo improves number and functional activity of vasculogenic cells and reduces atherosclerotic disease progression. Basic Res Cardiol 105:389–397. doi: 10.1007/s00395-009-0072-9 PubMedCrossRefGoogle Scholar
  36. 36.
    Sorescu GP, Song H, Tressel SL, Hwang J, Dikalov S, Smith DA, Boyd NL, Platt MO, Lassegue B, Griendling KK, Jo H (2004) Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress induces monocyte adhesion by stimulating reactive oxygen species production from a nox1-based NADPH oxidase. Circ Res 95:773–779. doi: 10.1161/01.RES.0000145728.22878.45 PubMedCrossRefGoogle Scholar
  37. 37.
    Tanaka Y, Aleksunes LM, Yeager RL, Gyamfi MA, Esterly N, Guo GL, Klaassen CD (2008) NF-E2-related factor 2 inhibits lipid accumulation and oxidative stress in mice fed a high-fat diet. J Pharmacol Exp Ther 325:655–664. doi: 10.1124/jpet.107.135822 PubMedCrossRefGoogle Scholar
  38. 38.
    Thum T, Borlak J (2008) LOX-1 receptor blockade abrogates oxLDL-induced oxidative DNA damage and prevents activation of the transcriptional repressor Oct-1 in human coronary arterial endothelium. J Biol Chem 283:19456–19464. doi: 10.1074/jbc.M708309200 PubMedCrossRefGoogle Scholar
  39. 39.
    Tiyerili V, Zimmer S, Jung S, Wassmann K, Naehle CP, Lutjohann D, Zimmer A, Nickenig G, Wassmann S (2010) CB1 receptor inhibition leads to decreased vascular AT1 receptor expression, inhibition of oxidative stress and improved endothelial function. Basic Res Cardiol 105:465–477. doi: 10.1007/s00395-010-0090-7 PubMedCrossRefGoogle Scholar
  40. 40.
    Wagner AH, Krzesz R, Gao D, Schroeder C, Cattaruzza M, Hecker M (2000) Decoy oligodeoxynucleotide characterization of transcription factors controlling endothelin-B receptor expression in vascular smooth muscle cells. Mol Pharmacol 58:1333–1340PubMedGoogle Scholar
  41. 41.
    Warabi E, Takabe W, Minami T, Inoue K, Itoh K, Yamamoto M, Ishii T, Kodama T, Noguchi N (2007) Shear stress stabilizes NF-E2-related factor 2 and induces antioxidant genes in endothelial cells: role of reactive oxygen/nitrogen species. Free Radic Biol Med 42:260–269. doi: 10.1016/j.freeradbiomed.2006.10.043 PubMedCrossRefGoogle Scholar
  42. 42.
    Westermann D, Riad A, Richter U, Jager S, Savvatis K, Schuchardt M, Bergmann N, Tolle M, Nagorsen D, Gotthardt M, Schultheiss HP, Tschope C (2009) Enhancement of the endothelial NO synthase attenuates experimental diastolic heart failure. Basic Res Cardiol 104:499–509. doi: 10.1007/s00395-009-0014-6 PubMedCrossRefGoogle Scholar
  43. 43.
    Zhang L, Sheppard OR, Shah AM, Brewer AC (2008) Positive regulation of the NADPH oxidase NOX4 promoter in vascular smooth muscle cells by E2F. Free Radic Biol Med 45:679–685. doi: 10.1016/j.freeradbiomed.2008.05.019 PubMedCrossRefGoogle Scholar

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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