title: Canonical Wnt signaling induces vascular endothelial dysfunction via p66Shc-regulated reactive oxygen species.
abstract: OBJECTIVE
  Reactive oxygen species regulate canonical Wnt signaling. However, the role of the redox regulatory protein p66(Shc) in the canonical Wnt pathway is not known. We investigated whether p66(Shc) is essential for canonical Wnt signaling in the endothelium and determined whether the canonical Wnt pathway induces vascular endothelial dysfunction via p66(Shc)-mediated oxidative stress.
  
  
  RESULTS
  The canonical Wnt ligand Wnt3a induced phosphorylation (activation) of p66(Shc) in endothelial cells. Wnt3a-stimulated dephosphorylation of β-catenin, and β-catenin-dependent transcription, was inhibited by knockdown of p66(Shc). Exogenous H2O2-induced β-catenin dephosphorylation was also mediated by p66(Shc). Moreover, p66(Shc) overexpression dephosphorylated β-catenin and increased β-catenin-dependent transcription, independent of Wnt3a ligand. P66(Shc)-induced β-catenin dephosphorylation was inhibited by antioxidants N-acetyl cysteine and catalase. Wnt3a upregulated endothelial NADPH oxidase-4, and β-catenin dephosphorylation was suppressed by knocking down NADPH oxidase-4 and by antioxidants. Wnt3a increased H2O2 levels in endothelial cells and impaired endothelium-dependent vasorelaxation in mouse aortas, both of which were rescued by p66(Shc) knockdown. P66(Shc) knockdown also inhibited adhesion of monocytes to Wnt3a-stimulated endothelial cells. Furthermore, constitutively active β-catenin expression in the endothelium increased vascular reactive oxygen species and impaired endothelium-dependent vasorelaxation. In vivo, high-fat diet feeding-induced endothelial dysfunction in mice was associated with increased endothelial Wnt3a, dephosphorylated β-catenin, and phosphorylated p66(Shc). High-fat diet-induced dephosphorylation of endothelial β-catenin was diminished in mice in which p66(Shc) was knocked down.
  
  
  CONCLUSIONS
  p66(Shc) plays a vital part in canonical Wnt signaling in the endothelium and mediates Wnt3a-stimulated endothelial oxidative stress and dysfunction.
authors.affiliation: From the Cardiovascular Division, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City (A.V., Y.-R.K., S.K., F.J.M., K.I.); Cardiovascular Institute, University of Pittsburgh, PA (A.N., A.K.); Department of Biochemistry and Molecular Biology, University of Louisville, KY (T.A.H.); and Department of Physiology, Chungnam National University, Daejeon, Korea (C.-S.K.). ajit-vikram@uiowa.edu kaikobad-irani@uiowa.edu.
authors.forename: Ajit
authors.initials: A
authors.lastname: Vikram
authors.affiliation: From the Cardiovascular Division, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City (A.V., Y.-R.K., S.K., F.J.M., K.I.); Cardiovascular Institute, University of Pittsburgh, PA (A.N., A.K.); Department of Biochemistry and Molecular Biology, University of Louisville, KY (T.A.H.); and Department of Physiology, Chungnam National University, Daejeon, Korea (C.-S.K.).
authors.forename: Young-Rae
authors.initials: YR
authors.lastname: Kim
authors.affiliation: From the Cardiovascular Division, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City (A.V., Y.-R.K., S.K., F.J.M., K.I.); Cardiovascular Institute, University of Pittsburgh, PA (A.N., A.K.); Department of Biochemistry and Molecular Biology, University of Louisville, KY (T.A.H.); and Department of Physiology, Chungnam National University, Daejeon, Korea (C.-S.K.).
authors.forename: Santosh
authors.initials: S
authors.lastname: Kumar
authors.affiliation: From the Cardiovascular Division, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City (A.V., Y.-R.K., S.K., F.J.M., K.I.); Cardiovascular Institute, University of Pittsburgh, PA (A.N., A.K.); Department of Biochemistry and Molecular Biology, University of Louisville, KY (T.A.H.); and Department of Physiology, Chungnam National University, Daejeon, Korea (C.-S.K.).
authors.forename: Asma
authors.initials: A
authors.lastname: Naqvi
authors.affiliation: From the Cardiovascular Division, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City (A.V., Y.-R.K., S.K., F.J.M., K.I.); Cardiovascular Institute, University of Pittsburgh, PA (A.N., A.K.); Department of Biochemistry and Molecular Biology, University of Louisville, KY (T.A.H.); and Department of Physiology, Chungnam National University, Daejeon, Korea (C.-S.K.).
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Hoffman
authors.affiliation: From the Cardiovascular Division, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City (A.V., Y.-R.K., S.K., F.J.M., K.I.); Cardiovascular Institute, University of Pittsburgh, PA (A.N., A.K.); Department of Biochemistry and Molecular Biology, University of Louisville, KY (T.A.H.); and Department of Physiology, Chungnam National University, Daejeon, Korea (C.-S.K.).
authors.forename: Ajay
authors.initials: A
authors.lastname: Kumar
authors.affiliation: From the Cardiovascular Division, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City (A.V., Y.-R.K., S.K., F.J.M., K.I.); Cardiovascular Institute, University of Pittsburgh, PA (A.N., A.K.); Department of Biochemistry and Molecular Biology, University of Louisville, KY (T.A.H.); and Department of Physiology, Chungnam National University, Daejeon, Korea (C.-S.K.).
authors.forename: Francis J
authors.initials: FJ
authors.lastname: Miller
authors.affiliation: From the Cardiovascular Division, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City (A.V., Y.-R.K., S.K., F.J.M., K.I.); Cardiovascular Institute, University of Pittsburgh, PA (A.N., A.K.); Department of Biochemistry and Molecular Biology, University of Louisville, KY (T.A.H.); and Department of Physiology, Chungnam National University, Daejeon, Korea (C.-S.K.).
authors.forename: Cuk-Seong
authors.initials: CS
authors.lastname: Kim
authors.affiliation: From the Cardiovascular Division, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City (A.V., Y.-R.K., S.K., F.J.M., K.I.); Cardiovascular Institute, University of Pittsburgh, PA (A.N., A.K.); Department of Biochemistry and Molecular Biology, University of Louisville, KY (T.A.H.); and Department of Physiology, Chungnam National University, Daejeon, Korea (C.-S.K.). ajit-vikram@uiowa.edu kaikobad-irani@uiowa.edu.
authors.forename: Kaikobad
authors.initials: K
authors.lastname: Irani
mesh_terms: D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000201:enzymology; D002417:Cattle; D018920:Coculture Techniques; D059305:Diet, High-Fat; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000201:enzymology; D006801:Humans; D006949:Hyperlipidemias / Q000201:enzymology / Q000503:physiopathology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress; D010766:Phosphorylation; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins / Q000235:genetics / Q000378:metabolism*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014162:Transfection; D020298:U937 Cells; D014664:Vasodilation; D014665:Vasodilator Agents / Q000494:pharmacology; D060449:Wnt Signaling Pathway; D060509:Wnt3A Protein / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism
chemical_list: C495270:CTNNB1 protein, human; C495267:CTNNB1 protein, mouse; D017382:Reactive Oxygen Species; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014665:Vasodilator Agents; C555510:WNT3A protein, human; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; D051176:beta Catenin; D019255:NADPH Oxidases
keywords: Wnt signaling pathway; endothelial cells; oxidation–reduction; p66Shc protein; reactive oxygen species