The role of lipid rafts / caveolae in PAR‐induced signaling

The vasoactive protease thrombin, a known mediator of inflammation and endothelial barrier function, activates the protinase‐activated receptor‐1 (PAR‐1) via cleavage of its N‐terminus. PAR‐1 activation stimulates the RhoA / Rho kinase signaling cascade, leading to myosin light chain phosphorylation, actin stress fiber formation and hyperpermeability in endothelial cells. Previous studies suggest that some elements of this signaling pathway are localized to caveolin‐containing cholesterol‐rich membrane domains. Thus we hypothesize that the compartmentalization of PAR‐1 and its immediate downstream effectors into lipid rafts / caveolae serves to regulate thrombin‐induced signaling leading to changes in endothelial barrier function. Here we show that key components of the PAR‐associated signaling cascade localize to lipid rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5μg/mL, 5 min) to ablate lipid rafts prior to thrombin (100nM) or PAR agonist stimulation. We found that the myosin light chain (MLC) phosphorylation and actin stress fiber formation normally induced by PAR activation was attenuated after lipid raft disruption. To target caveolae specifically, we used an siRNA approach to knock‐down caveolin‐1 expression. Surprisingly, thrombin‐induced MLC phosphorylation was not altered in caveolin‐1 depleted cells. These findings suggest that lipid rafts, but not necessarily caveolae, modulate thrombin‐activated signaling pathways in endothelial cells. This work was supported by NIH HL66301.