title: Antiproliferative effect of estrogen in vascular smooth muscle cells is mediated by Kruppel-like factor-4 and manganese superoxide dismutase.
abstract: 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.
authors.affiliation: Medizinische Klinik und Poliklinik II, Universitätsklinikum Bonn, Sigmund-Freud-Strasse 25, Bonn, Germany.
authors.forename: Derya
authors.initials: D
authors.lastname: Sivritas
authors.forename: Marc Ulrich
authors.initials: MU
authors.lastname: Becher
authors.forename: Talin
authors.initials: T
authors.lastname: Ebrahimian
authors.forename: Omar
authors.initials: O
authors.lastname: Arfa
authors.forename: Stephanie
authors.initials: S
authors.lastname: Rapp
authors.forename: Annika
authors.initials: A
authors.lastname: Bohner
authors.forename: Cornelius Friedrich
authors.initials: CF
authors.lastname: Mueller
authors.forename: Takashi
authors.initials: T
authors.lastname: Umemura
authors.forename: Sven
authors.initials: S
authors.lastname: Wassmann
authors.forename: Georg
authors.initials: G
authors.lastname: Nickenig
authors.forename: Kerstin
authors.initials: K
authors.lastname: Wassmann
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D004958:Estradiol / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000502:physiology; D005260:Female; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000502:physiology; D058426:Neointima; D019001:Nitric Oxide Synthase / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013482:Superoxide Dismutase / Q000502:physiology*
chemical_list: D004958:Estradiol; D047628:Estrogen Receptor alpha; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D019001:Nitric Oxide Synthase; D019869:Phosphatidylinositol 3-Kinases; D013482:Superoxide Dismutase; C000715468:Klf4 protein, mouse; C000715469:Klf4 protein, rat