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 β-arrestin-2 (β-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 coreceptors; however, it is not clear how coreceptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. Here, we define a 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 coexist in caveolin-1 (Cav1)–rich microdomains and that S1PR1 coassociation with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates β-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 β-arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1-β-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, β-arr2–driven signaling pathways in caveolae.

Endothelial dysfunction, a hallmark of inflammation, is associated with the pathogenesis of vascular diseases and results in endothelial barrier disruption and increased sensitivity to apoptosis (1, 2). There are limited treatment options for improving endothelial dysfunction, which is prevalent in diseases such as sepsis, a condition with high morbidity and mortality (3, 4). Activated protein C (aPC) is a promising therapeutic that exhibits multiple pharmacological benefits in preclinical studies, including sepsis (5–7). In endothelial cells, protease-activated receptor-1 (PAR1), a G protein–coupled receptor (GPCR), is the central mediator of aPC cytoprotective responses, including endothelial barrier stabilization, anti-inflammatory, and anti-apoptotic activities (6). The signaling pathways by which aPC/PAR1 elicits different cytoprotective responses are poorly defined.aPC-dependent endothelial cytoprotection requires compartmentalization of PAR1 and the aPC coreceptor, endothelial protein C receptor (EPCR), in caveolin-1 (Cav1)–rich microdomains (8, 9). aPC activates PAR1 through the proteolytic cleavage of the receptor’s N-terminal arginine (R)-46 residue, which is distinct from the thrombin canonical cleavage site at (R)-41 (10). Several studies indicate that aPC/PAR1 requires β-arrestin-2 (β-arr2) to promote cytoprotection (11–13). We showed that aPC-activated PAR1 signals via β-arr2 and dishevelled-2 (Dvl2) scaffolds, and not heterotrimeric G proteins, to induce Rac1 activation and endothelial barrier protection (11). β-arr2 and Dvl2 are also required for aPC-mediated inhibition of cytokine-induced immune cell recruitment, an anti-inflammatory response (12). In addition, aPC/PAR1 stimulates Akt signaling and protects against endothelial cell death induced by tumor necrosis factor-alpha (TNF-α) and staurosporine (14, 15). However, the role of β-arr2 and Dvl2 scaffolds in mediating aPC/PAR1 anti-apoptotic responses is not known.The interaction of GPCRs with coreceptors can alter the active conformation of receptors, β-arrestin recruitment, and biased signaling (16) and is relevant to aPC/PAR1-driven endothelial cytoprotective signaling. aPC-activated PAR1 cooperates with PAR3 and sphingosine-1-phosphate receptor-1 (S1PR1) to promote cytoprotection (17–19). aPC cleaves PAR3 at a noncanonical N-terminal (R)-41 site to promote endothelial barrier protection in vitro and in vivo (19). In contrast to PAR3, aPC signals indirectly to S1PR1 to enhance basal endothelial barrier stabilization and to protect against barrier disruption (17, 18). However, the mechanism by which aPC/PAR1 transactivates S1PR1 and the role of S1PR1 in other aPC-mediated cytoprotective responses, such as cell survival, is not known.In this study, we assessed whether S1PR1 and the β-arr2 and Dvl2 scaffolds function as universal mediators of aPC/PAR1 cytoprotection by examining their function in anti-apoptotic responses. Using a combined pharmacological inhibitor and small interfering (si)RNA knockdown approach in human cultured endothelial cells, we define a β-arr2-sphingosine kinase-1 (SphK1)-S1PR1-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Our studies further demonstrate that aPC-stimulated activation of SphK1 is dependent on β-arr2 and not Dvl2, whereas neither SphK1 nor S1PR1 are required for aPC-β-arr2–induced, extracellular signal–regulated kinase 1/2 (ERK1/2) signaling. This study reveals that different aPC/PAR1 cytoprotective responses are mediated by discrete β-arr2–driven signaling pathways modulated by coreceptors localized in caveolae.