Basic Research in Cardiology

, Volume 106, Issue 4, pp 563–575 | Cite as

Antiproliferative effect of estrogen in vascular smooth muscle cells is mediated by Kruppel-like factor-4 and manganese superoxide dismutase

  • Derya Sivritas
  • Marc Ulrich Becher
  • Talin Ebrahimian
  • Omar Arfa
  • Stephanie Rapp
  • Annika Bohner
  • Cornelius Friedrich Mueller
  • Takashi Umemura
  • Sven Wassmann
  • Georg Nickenig
  • Kerstin Wassmann
Original Contribution


The mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) and the zinc finger transcription factor Kruppel-like factor-4 (KLF4) are involved in the regulation of redox homeostasis, apoptosis and cell proliferation. We have shown that estrogen exerts antioxidative actions via induction of MnSOD in cultured rat aortic vascular smooth muscle cells (VSMC). The purpose of the present study was to investigate whether estrogen inhibits VSMC proliferation via alteration of KLF4 and MnSOD expression. In cultured rat aortic VSMC, estrogen binding to estrogen receptor-alpha led to rapid increase in KLF4 expression and reduction of cell proliferation by 50%. Protein separation revealed that KLF4 was shifted to the nucleus when VSMC were treated with estrogen. Estrogen-mediated induction of KLF4 and the antiproliferative effect involved activation of PI-3 kinase, Akt phosphorylation and induction of NO synthase activity. Experiments in freshly isolated denuded aortic segments revealed an increase in KLF4 abundance after estrogen treatment and demonstrated that eNOS is expressed in the media at low levels. Transfection experiments showed that estrogen-induced overexpression of MnSOD required KLF4 and that both KLF4 and MnSOD were indispensable for the observed antiproliferative effect of estrogen in VSMC. To confirm these data in vivo, we investigated neointima formation after carotid artery injury in wild-type (WT) and MnSOD+/− mice. Estrogen deficiency led to enhanced neointima formation and higher numbers of Ki67-positive proliferating cells in the neointima of ovariectomized WT and MnSOD+/− mice. Moreover, MnSOD+/− mice showed more extensive neointima formation and Ki67 immunostaining. Interestingly, estrogen replacement prevented neointima formation in WT mice but failed to completely inhibit neointima formation in MnSOD+/− mice. Cultured VSMC derived from MnSOD+/− mice showed enhanced proliferation as compared to WT VSMC, and estrogen treatment failed to inhibit proliferation in MnSOD+/− VSMC. In conclusion, these data demonstrate the importance of MnSOD and KLF4 for proliferation control in VSMC. Our results provide novel insights into how proliferation of VSMC is regulated by estrogen and may help to identify novel targets for the treatment of vascular diseases such as restenosis.


Estrogen KLF4 MnSOD Proliferation Vascular smooth muscle cells 



This work was supported by the Deutsche Forschungsgemeinschaft, the BONFOR Forschungsförderung, Canada Foundation for Innovation, Canada Research Chairs and Canadian Institutes of Health Research.


  1. 1.
    Anderson GL, Limacher M, Assaf AR, Bassford T, Beresford SA, Black H, Bonds D, Brunner R, Brzyski R, Caan B, Chlebowski R, Curb D, Gass M, Hays J, Heiss G, Hendrix S, Howard BV, Hsia J, Hubbell A, Jackson R, Johnson KC, Judd H, Kotchen JM, Kuller L, LaCroix AZ, Lane D, Langer RD, Lasser N, Lewis CE, Manson J, Margolis K, Ockene J, O’Sullivan MJ, Phillips L, Prentice RL, Ritenbaugh C, Robbins J, Rossouw JE, Sarto G, Stefanick ML, Van Horn L, Wactawski-Wende J, Wallace R, Wassertheil-Smoller S (2004) Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women’s Health Initiative randomized controlled trial. JAMA 291:1701–1712. doi:10.1001/jama.291.14.1701 PubMedCrossRefGoogle Scholar
  2. 2.
    Behrend L, Mohr A, Dick T, Zwacka RM (2005) Manganese superoxide dismutase induces p53-dependent senescence in colorectal cancer cells. Mol Cell Biol 25:7758–7769. doi:10.1128/MCB.25.17.7758-7769.2005 PubMedCrossRefGoogle Scholar
  3. 3.
    Bieker JJ (2001) Kruppel-like factors: three fingers in many pies. J Biol Chem 276:34355–34358. doi:10.1074/jbc.R100043200 PubMedCrossRefGoogle Scholar
  4. 4.
    Buchwalow IB, Podzuweit T, Bocker W, Samoilova VE, Thomas S, Wellner M, Baba HA, Robenek H, Schnekenburger J, Lerch MM (2002) Vascular smooth muscle and nitric oxide synthase. FASEB J 16:500–508. doi:10.1096/fj.01-0842com PubMedCrossRefGoogle Scholar
  5. 5.
    Cai H, Harrison DG (2000) Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 87:840–844PubMedGoogle Scholar
  6. 6.
    Chambliss KL, Shaul PW (2002) Estrogen modulation of endothelial nitric oxide synthase. Endocr Rev 23:665–686. doi:10.1210/er.2001-0045 PubMedCrossRefGoogle Scholar
  7. 7.
    Chambliss KL, Yuhanna IS, Mineo C, Liu P, German Z, Sherman TS, Mendelsohn ME, Anderson RG, Shaul PW (2000) Estrogen receptor alpha and endothelial nitric oxide synthase are organized into a functional signaling module in caveolae. Circ Res 87:E44–E52PubMedGoogle Scholar
  8. 8.
    Chen X, Whitney EM, Gao SY, Yang VW (2003) Transcriptional profiling of Kruppel-like factor 4 reveals a function in cell cycle regulation and epithelial differentiation. J Mol Biol 326:665–677. doi:10.1016/S0022-2836(02)01449-3 PubMedCrossRefGoogle Scholar
  9. 9.
    Collins P, Webb C (1999) Estrogen hits the surface. Nat Med 5:1130–1131. doi:10.1038/13453 PubMedCrossRefGoogle Scholar
  10. 10.
    Cullen JJ, Weydert C, Hinkhouse MM, Ritchie J, Domann FE, Spitz D, Oberley LW (2003) The role of manganese superoxide dismutase in the growth of pancreatic adenocarcinoma. Cancer Res 63:1297–1303PubMedGoogle Scholar
  11. 11.
    Darley-Usmar VM, McAndrew J, Patel R, Moellering D, Lincoln TM, Jo H, Cornwell T, Digerness S, White CR (1997) Nitric oxide, free radicals and cell signalling in cardiovascular disease. Biochem Soc Trans 25:925–929PubMedGoogle Scholar
  12. 12.
    Dhar SK, Xu Y, Chen Y, St Clair DK (2006) Specificity protein 1-dependent p53-mediated suppression of human manganese superoxide dismutase gene expression. J Biol Chem 281:21698–21709. doi:10.1074/jbc.M601083200 PubMedCrossRefGoogle Scholar
  13. 13.
    Feinberg MW, Cao Z, Wara AK, Lebedeva MA, Senbanerjee S, Jain MK (2005) Kruppel-like factor 4 is a mediator of proinflammatory signaling in macrophages. J Biol Chem 280:38247–38258. doi:10.1074/jbc.M509378200 PubMedCrossRefGoogle Scholar
  14. 14.
    Freudenberger T, Oppermann M, Heim HK, Mayer P, Kojda G, Schrör K, Fischer JW (2010) Proatherogenic effects of estradiol in a model of accelerated atherosclerosis in ovariectomized ApoE-deficient mice. Basic Res Cardiol 105(4):479–486. doi:10.1007/s00395-010-0091-6 PubMedCrossRefGoogle Scholar
  15. 15.
    Garg UC, Hassid A (1989) Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest 83:1774–1777. doi:10.1172/JCI114081 PubMedCrossRefGoogle Scholar
  16. 16.
    Gragasin FS, Xu Y, Arenas IA, Kainth N, Davidge ST (2003) Estrogen reduces angiotensin II-induced nitric oxide synthase and NAD(P)H oxidase expression in endothelial cells. Arterioscler Thromb Vasc Biol 23:38–44. doi:10.1161/01.ATV.0000047868.93732.B7 PubMedCrossRefGoogle Scholar
  17. 17.
    Harrison DG (1997) Endothelial function and oxidant stress. Clin Cardiol 20(II):11–17Google Scholar
  18. 18.
    Haynes MP, Sinha D, Russell KS, Collinge M, Fulton D, Morales-Ruiz M, Sessa WC, Bender JR (2000) Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the PI3-kinase-Akt pathway in human endothelial cells. Circ Res 87:677–682PubMedGoogle Scholar
  19. 19.
    Hu B, Wu Z, Liu T, Ullenbruch MR, Jin H, Phan SH (2007) Gut-enriched Kruppel-like factor interaction with Smad3 inhibits myofibroblast differentiation. Am J Respir Cell Mol Biol 36:78–84. doi:10.1165/rcmb.2006-0043OC PubMedCrossRefGoogle Scholar
  20. 20.
    Hulley S, Furberg C, Barrett-Connor E, Cauley J, Grady D, Haskell W, Knopp R, Lowery M, Satterfield S, Schrott H, Vittinghoff E, Hunninghake D (2002) Noncardiovascular disease outcomes during 6.8 years of hormone therapy: Heart and Estrogen/progestin Replacement Study follow-up (HERS II). JAMA 288:58–66. doi:10.1001/jama.288.1.58 PubMedCrossRefGoogle Scholar
  21. 21.
    Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E (1998) Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/progestin Replacement Study (HERS) Research Group. JAMA 280:605–613. doi:10.1001/jama.280.7.605 PubMedCrossRefGoogle Scholar
  22. 22.
    Ishida A, Sasaguri T, Kosaka C, Nojima H, Ogata J (1997) Induction of the cyclin-dependent kinase inhibitor p21(Sdi1/Cip1/Waf1) by nitric oxide-generating vasodilator in vascular smooth muscle cells. J Biol Chem 272:10050–10057. doi:10.1074/jbc.272.15.10050 PubMedCrossRefGoogle Scholar
  23. 23.
    Laufs U, Adam O, Strehlow K, Wassmann S, Konkol C, Laufs K, Schmidt W, Bohm M, Nickenig G (2003) Down-regulation of Rac-1 GTPase by estrogen. J Biol Chem 278:5956–5962. doi:10.1074/jbc.M209813200 PubMedCrossRefGoogle Scholar
  24. 24.
    Nagy N, Melchior-Becker A, Fischer JW (2010) Long-term treatment with the AT1-receptor antagonist telmisartan inhibits biglycan accumulation in murine atherosclerosis. Basic Res Cardiol 105(1):29–38. doi:10.1007/s00395-009-0051-1 PubMedCrossRefGoogle Scholar
  25. 25.
    Nickenig G, Baudler S, Muller C, Werner C, Werner N, Welzel H, Strehlow K, Bohm M (2002) Redox-sensitive vascular smooth muscle cell proliferation is mediated by GKLF and Id3 in vitro and in vivo. FASEB J 16:1077–1086. doi:10.1096/fj.01-0570com PubMedCrossRefGoogle Scholar
  26. 26.
    Reilly M, Delanty N, Lawson JA, FitzGerald GA (1996) Modulation of oxidant stress in vivo in chronic cigarette smokers. Circulation 94:19–25PubMedGoogle Scholar
  27. 27.
    Ross R (1997) Cellular and molecular studies of atherogenesis. Atherosclerosis 131(Suppl):S3–S4PubMedCrossRefGoogle Scholar
  28. 28.
    Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J (2002) Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. JAMA 288:321–333. doi:10.1001/jama.288.3.321 PubMedCrossRefGoogle Scholar
  29. 29.
    Sarsour EH, Agarwal M, Pandita TK, Oberley LW, Goswami PC (2005) Manganese superoxide dismutase protects the proliferative capacity of confluent normal human fibroblasts. J Biol Chem 280:18033–18041. doi:10.1074/jbc.M501939200 PubMedCrossRefGoogle Scholar
  30. 30.
    Strehlow K, Rotter S, Wassmann S, Adam O, Grohe C, Laufs K, Bohm M, Nickenig G (2003) Modulation of antioxidant enzyme expression and function by estrogen. Circ Res 93:170–177. doi:10.1161/01.RES.0000082334.17947.11 PubMedCrossRefGoogle Scholar
  31. 31.
    Strehlow K, Werner N, Berweiler J, Link A, Dirnagl U, Priller J, Laufs K, Ghaeni L, Milosevic M, Bohm M, Nickenig G (2003) Estrogen increases bone marrow-derived endothelial progenitor cell production and diminishes neointima formation. Circulation 107:3059–3065. doi:10.1161/01.CIR.0000077911.81151.30 PubMedCrossRefGoogle Scholar
  32. 32.
    Takeda-Matsubara Y, Nakagami H, Iwai M, Cui TX, Shiuchi T, Akishita M, Nahmias C, Ito M, Horiuchi M (2002) Estrogen activates phosphatases and antagonizes growth-promoting effect of angiotensin II. Hypertension 39:41–45. doi:10.1161/hy1201.097197 PubMedCrossRefGoogle Scholar
  33. 33.
    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
  34. 34.
    Venkataraman S, Jiang X, Weydert C, Zhang Y, Zhang HJ, Goswami PC, Ritchie JM, Oberley LW, Buettner GR (2005) Manganese superoxide dismutase overexpression inhibits the growth of androgen-independent prostate cancer cells. Oncogene 24:77–89. doi:10.1038/sj.onc.1208145 PubMedCrossRefGoogle Scholar
  35. 35.
    Wagner AH, Schroeter MR, Hecker M (2001) 17Beta-estradiol inhibition of NADPH oxidase expression in human endothelial cells. FASEB J 15:2121–2130. doi:10.1096/fj.01-0123com PubMedCrossRefGoogle Scholar
  36. 36.
    Wassmann S, Wassmann K, Jung A, Velten M, Knuefermann P, Petoumenos V, Becher U, Werner C, Mueller C, Nickenig G (2007) Induction of p53 by GKLF is essential for inhibition of proliferation of vascular smooth muscle cells. J Mol Cell Cardiol 43:301–307. doi:10.1016/j.yjmcc.2007.06.001 PubMedCrossRefGoogle Scholar
  37. 37.
    Watanabe T, Akishita M, Nakaoka T, Kozaki K, Miyahara Y, He H, Ohike Y, Ogita T, Inoue S, Muramatsu M, Yamashita N, Ouchi Y (2003) Estrogen receptor beta mediates the inhibitory effect of estradiol on vascular smooth muscle cell proliferation. Cardiovasc Res 59:734–744. doi:10.1016/S0008-6363(03)00496-6 PubMedCrossRefGoogle Scholar
  38. 38.
    Wei D, Gong W, Kanai M, Schlunk C, Wang L, Yao JC, Wu TT, Huang S, Xie K (2005) Drastic down-regulation of Kruppel-like factor 4 expression is critical in human gastric cancer development and progression. Cancer Res 65:2746–2754. doi:10.1158/0008-5472.CAN-04-3619 PubMedCrossRefGoogle Scholar
  39. 39.
    White CR, Shelton J, Chen SJ, Darley-Usmar V, Allen L, Nabors C, Sanders PW, Chen YF, Oparil S (1997) Estrogen restores endothelial cell function in an experimental model of vascular injury. Circulation 96:1624–1630PubMedGoogle Scholar
  40. 40.
    Willert M, Augstein A, Poitz DM, Schmeisser A, Strasser RH, Braun-Dullaeus RC (2010) Transcriptional regulation of Pim-1 kinase in vascular smooth muscle cells and its role for proliferation. Basic Res Cardiol 105:267–277. doi:10.1007/s00395-009-0055-x PubMedCrossRefGoogle Scholar
  41. 41.
    Wu J, Lingrel JB (2004) KLF2 inhibits Jurkat T leukemia cell growth via upregulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1. Oncogene 23:8088–8096. doi:10.1038/sj.onc.1207996 PubMedCrossRefGoogle Scholar
  42. 42.
    Yang S, Bae L, Zhang L (2000) Estrogen increases eNOS and NOx release in human coronary artery endothelium. J Cardiovasc Pharmacol 36:242–247PubMedCrossRefGoogle Scholar
  43. 43.
    Yang Y, Goldstein BG, Chao HH, Katz JP (2005) KLF4 and KLF5 regulate proliferation, apoptosis and invasion in esophageal cancer cells. Cancer Biol Ther 4:1216–1221PubMedCrossRefGoogle Scholar
  44. 44.
    Yoon HS, Chen X, Yang VW (2003) Kruppel-like factor 4 mediates p53-dependent G1/S cell cycle arrest in response to DNA damage. J Biol Chem 278:2101–2105. doi:10.1074/jbc.M211027200 PubMedCrossRefGoogle Scholar
  45. 45.
    Yuan Y, Liao L, Tulis DA, Xu J (2002) Steroid receptor coactivator-3 is required for inhibition of neointima formation by estrogen. Circulation 105:2653–2659. doi:10.1161/01.CIR.0000018947.95555.65 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Derya Sivritas
    • 1
  • Marc Ulrich Becher
    • 1
  • Talin Ebrahimian
    • 2
  • Omar Arfa
    • 2
  • Stephanie Rapp
    • 1
  • Annika Bohner
    • 1
  • Cornelius Friedrich Mueller
    • 1
  • Takashi Umemura
    • 1
  • Sven Wassmann
    • 1
    • 2
  • Georg Nickenig
    • 1
  • Kerstin Wassmann
    • 1
  1. 1.Medizinische Klinik und Poliklinik IIUniversitätsklinikum BonnBonnGermany
  2. 2.Lady Davis Institute for Medical Research, Jewish General HospitalMcGill UniversityMontréalCanada

Personalised recommendations