aPC/PAR1 confers endothelial anti-apoptotic activity via a discrete β-arrestin-2 mediated SphK1-S1PR1-Akt signaling axis.

Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated Protein C (aPC), a promising therapeutic, signals via protease-activated receptor-1 (PAR1) and mediates several cytoprotective responses including endothelial barrier stabilization and anti-apoptotic responses. We showed that aPC-activated PAR1 signals preferentially via b-arrestin-2 (b-arr2) and dishevelled-2 (Dvl2) scaffolds rather than G proteins to promote Rac1 activation and barrier protection. However, the signaling pathways utilized by aPC/PAR1 to mediate anti-apoptotic activities are not known. APC/PAR1 cytoprotective responses also require co-receptors, however it is not clear how co-receptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. The objective of this study is to determine the function of coreceptors on aPC/PAR1-induced cytoprotective responses in endothelial cells. We hypothesize that different co-receptors distinctly regulate specific APC/PAR1 cytoprotective responses by b-arr2 recruitment and biased signaling. Here we define a novel β-arr2-mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Using human cultured endothelial cells, we found that endogenous PAR1 and S1PR1 co-exist in caveolin-1(Cav1)-rich microdomains and S1PR1 co-association with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates b-arr2-dependent SphK1 activation independent of Dvl2 and is required for transactivation of S1PR1-Akt signaling and protection against cell death. While aPC/PAR1-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation is also dependent on b-arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1-b-arr2 mediated protection against apoptosis is dependent on Cav1, the principal structural protein of endothelial caveolae. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete b-arr2-driven signaling pathways in caveolae.