title: Cyclophilin A is secreted by a vesicular pathway in vascular smooth muscle cells.
abstract: Reactive oxygen species (ROS) contribute to the pathogenesis of atherosclerosis in part by promoting vascular smooth muscle cell (VSMC) growth. Previously we demonstrated that cyclophilin A (CyPA) is a secreted oxidative stress-induced factor (SOXF) that promotes inflammation, VSMC growth, and endothelial cell apoptosis. However, the mechanisms that regulate CyPA secretion are unknown. In this study, we hypothesized that ROS-induced CyPA secretion from VSMC requires a highly regulated process of vesicle transport, docking, and fusion at the plasma membrane. Conditioned medium and plasma membrane sheets were prepared by exposing VSMC to 1 micromol/L LY83583, which generates intracellular superoxide. A vesicular transport mechanism was confirmed by colocalization at the plasma membrane with vesicle-associated membrane protein (VAMP). CyPA transport to the plasma membrane and secretion were significantly increased by LY83583. Reduction of VAMP-2 expression by small interfering RNA inhibited LY83583-induced CyPA secretion. Pretreatment with 3 micromol/L cytochalasin D, an actin depolymerizing agent, abrogated CyPA secretion. Infection with dominant-negative RhoA and Cdc42 adenovirus inhibited CyPA secretion by 72% and 63%, respectively, whereas dominant-negative Rac1 had a small effect (11%). Pretreatment with the Rho kinase inhibitor Y27632 (3 to 30 micromol/L) and myosin II inhibitor blebbistatin (1 to 10 micromol/L) inhibited CyPA secretion in a dose-dependent manner. Simvastatin (3 to 30 micromol/L) also dose-dependently inhibited LY83583-induced CyPA secretion likely via decreased isoprenylation of small GTPases. Our findings define a novel VSMC vesicular secretory pathway for CyPA that involves actin remodeling and myosin II activation via RhoA-, Cdc42-, and Rho kinase-dependent signaling events.
authors.affiliation: Cardiovascular Research Institute, Department of Medicine, University of Rochester, NY 14642, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Suzuki
authors.forename: Zheng-Gen
authors.initials: ZG
authors.lastname: Jin
authors.forename: David F
authors.initials: DF
authors.lastname: Meoli
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Matoba
authors.forename: Bradford C
authors.initials: BC
authors.lastname: Berk
mesh_terms: D000199:Actins / Q000378:metabolism; D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D021984:Cyclophilin A / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D024462:Myosin Type II / Q000502:physiology; D018384:Oxidative Stress; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000633:toxicity; D019821:Simvastatin / Q000494:pharmacology; D050988:Vesicle-Associated Membrane Protein 2 / Q000502:physiology; D020741:rho GTP-Binding Proteins / Q000502:physiology; D054460:rho-Associated Kinases
chemical_list: D000199:Actins; D000634:Aminoquinolines; D047908:Intracellular Signaling Peptides and Proteins; D017382:Reactive Oxygen Species; D050988:Vesicle-Associated Membrane Protein 2; C041715:6-anilino-5,8-quinolinedione; D019821:Simvastatin; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D024462:Myosin Type II; D020741:rho GTP-Binding Proteins; D021984:Cyclophilin A