Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae.
The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its NH(2) terminus. PAR1 activation stimulates the RhoA/Rho kinase signaling cascade, leading to myosin light chain (MLC) phosphorylation, actin stress fiber formation, and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5 mug/ml, 5 min) to ablate lipid rafts before thrombin (100 nM) or PAR agonist stimulation. We found that diphosphorylation of MLC and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used a small interferring RNA approach to knockdown caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.
Carlile-Klusacek
Maryellen
M
Cardiovascular Research Center and Dept. of Anatomy & Cell Biology, Temple University School of Medicine, 3420 N. Broad Street, Philadelphia, PA 19140, USA.
Rizzo
Victor
V
D000818:Animals; D002417:Cattle; D021941:Caveolae / Q000378:metabolism; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure*; D042783:Endothelial Cells / Q000378:metabolism* / Q000648:ultrastructure*; D021962:Membrane Microdomains / Q000378:metabolism* / Q000648:ultrastructure*; D044463:Receptor, PAR-1 / Q000378:metabolism*
D044463:Receptor, PAR-1