title: Cyclophilin A is required for angiotensin II-induced p47phox translocation to caveolae in vascular smooth muscle cells.
abstract: OBJECTIVE
  Angiotensin II (AngII) signal transduction in vascular smooth muscle cells (VSMC) is mediated by reactive oxygen species (ROS). Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses peptidyl-prolyl cis-trans isomerase activity, scaffold function, and significantly enhances AngII-induced ROS production in VSMC. We hypothesized that CyPA regulates AngII-induced ROS generation by promoting translocation of NADPH oxidase cytosolic subunit p47phox to caveolae of the plasma membrane.
  
  
  RESULTS
  Overexpression of CyPA in CyPA-deficient VSMC (CyPA(-/-)VSMC) significantly increased AngII-stimulated ROS production. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors (VAS2870 or diphenylene iodonium) significantly attenuated AngII-induced ROS production in CyPA and p47phox-overexpressing CyPA(-/-)VSMC. Cell fractionation and sucrose gradient analyses showed that AngII-induced p47phox plasma membrane translocation, specifically to the caveolae, was reduced in CyPA(-/-)VSMC compared with wild-type-VSMC. Immunofluorescence studies demonstrated that AngII increased p47phox and CyPA colocalization and translocation to the plasma membrane. In addition, immunoprecipitation of CyPA followed by immunoblotting of p47phox and actin showed that AngII increased CyPA and p47phox interaction. AngII-induced p47phox and actin cell cytoskeleton association was attenuated in CyPA(-/-)VSMC. Mechanistically, inhibition of p47phox phosphorylation and phox homology domain deletion attenuated CyPA and p47phox interaction. Finally, cyclosporine A and CyPA-peptidyl-prolyl cis-trans isomerase mutant, R55A, inhibited AngII-stimulated CyPA and p47phox association in VSMC, suggesting that peptidyl-prolyl cis-trans isomerase activity was required for their interaction.
  
  
  CONCLUSIONS
  These findings provide the mechanism by which CyPA is an important regulator for AngII-induced ROS generation in VSMC through interaction with p47phox and cell cytoskeleton, which enhances the translocation of p47phox to caveolae.
authors.affiliation: Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.
authors.forename: Nwe Nwe
authors.initials: NN
authors.lastname: Soe
authors.forename: Mark
authors.initials: M
authors.lastname: Sowden
authors.forename: Padmamalini
authors.initials: P
authors.lastname: Baskaran
authors.forename: Elaine M
authors.initials: EM
authors.lastname: Smolock
authors.forename: Yeonghwan
authors.initials: Y
authors.lastname: Kim
authors.forename: Patrizia
authors.initials: P
authors.lastname: Nigro
authors.forename: Bradford C
authors.initials: BC
authors.lastname: Berk
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000201:enzymology; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D021941:Caveolae / Q000187:drug effects* / Q000201:enzymology; D021984:Cyclophilin A / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000201:enzymology; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D021381:Protein Transport; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D044140:Receptor, Angiotensin, Type 1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D009842:Oligopeptides; D010455:Peptides; D017382:Reactive Oxygen Species; D044140:Receptor, Angiotensin, Type 1; D011993:Recombinant Fusion Proteins; D000804:Angiotensin II; C085706:FLAG peptide; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D021984:Cyclophilin A
keywords: angiotensin II; cyclophilin A; cytoskeleton; p47phox protein, mouse; reactive oxygen species