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aPC/PAR1 confers endothelial anti-apoptotic activity via a discrete, β-arrestin-2-mediated SphK1-S1PR1-Akt signaling
Olivia Molinar-Inglis1, Cierra A Birch1, Dequina Nicholas2
1Department of Pharmacology, School of Medicine, University of California San Diego, La Jolla, CA 92093.
Abstract:
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.
Insights
Activated protein C (aPC) protects endothelial cells from death by activating protease-activated receptor-1 (PAR1) through a novel pathway involving β-arrestin-2, sphingosine kinase-1, S1PR1, and Akt signaling, distinct from barrier protection mechanisms.
Area of Science:
- Endothelial cell biology
- Molecular signaling pathways
- Vascular disease research
Background:
- Endothelial dysfunction contributes to vascular disease, characterized by impaired barrier function and increased apoptosis.
- Current treatments for endothelial dysfunction are limited.
- Activated protein C (aPC) shows therapeutic potential by signaling via protease-activated receptor-1 (PAR1) to stabilize endothelial barriers and prevent apoptosis.
Purpose of the Study:
- To elucidate the signaling pathways by which aPC/PAR1 mediates anti-apoptotic activities.
- To investigate the role of coreceptors in distinct aPC/PAR1 cytoprotective responses.
- To define the specific molecular axis conferring aPC/PAR1-mediated protection against endothelial cell death.
Main Methods:
- Utilized human cultured endothelial cells to study aPC/PAR1 signaling.
- Investigated protein interactions and signaling cascades using biochemical and cell biology techniques.
- Examined the role of specific proteins (β-arrestin-2, SphK1, S1PR1, Akt, Cav1, Dvl2, ERK1/2) in aPC-mediated cytoprotection.
Main Results:
- Identified a novel β-arrestin-2-mediated signaling axis: sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt, conferring aPC/PAR1-mediated protection against cell death.
- Demonstrated that aPC activates SphK1 in a β-arrestin-2-dependent manner, independent of Dvl2, leading to S1PR1-Akt transactivation.
- Showed that aPC/PAR1-induced anti-apoptotic signaling is dependent on caveolin-1 (Cav1) and distinct from the ERK1/2 pathway.
Conclusions:
- Activated protein C (aPC) utilizes discrete β-arrestin-2-driven signaling pathways within caveolae to mediate distinct cytoprotective responses, including anti-apoptosis.
- The SphK1-S1PR1-Akt axis is crucial for aPC/PAR1-mediated protection against endothelial cell death.
- These findings reveal complex signaling mechanisms underlying aPC's therapeutic potential in vascular disease.
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