Cyclophilin A is secreted by a vesicular pathway in vascular smooth muscle cells.
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.
Suzuki
Jun
J
Cardiovascular Research Institute, Department of Medicine, University of Rochester, NY 14642, USA.
Jin
Zheng-Gen
ZG
Meoli
David F
DF
Matoba
Tetsuya
T
Berk
Bradford C
BC
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
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