Activated protein C promotes protease-activated receptor-1 cytoprotective signaling through β-arrestin and dishevelled-2 scaffolds

Abstract

Protease-activated receptor-1 (PAR1) is a guanine nucleotide-binding (G) protein-coupled receptor that elicits cellular responses to coagulant and anticoagulant proteases. Activation of PAR1 by the coagulant protease thrombin results in Ras homolog gene family member A (RhoA) activation, disassembly of adherens junctions, and disruption of the endothelial barrier. In contrast, activation of PAR1 with the anticoagulant protease activated protein C (APC) results in activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and endothelial barrier protection. We previously showed that APC cytoprotective signaling requires the compartmentalization of PAR1 in caveolar microdomains. However, the mechanism by which APC-activated PAR1 promotes cytoprotective signaling in human endothelial cells remains poorly understood. Here we show that APC-activated PAR1 cytoprotective signaling is mediated by β-arrestin recruitment and activation of the dishevelled-2 (Dvl-2) scaffold and not by G protein α inhibiting activity polypeptide 2 (Gα(i)) signaling. In human endothelial cells, PAR1 and β-arrestins form a preassembled complex and cosegregate in caveolin-1-enriched fractions. Remarkably, we found that depletion of β-arrestin expression by RNA interference resulted in the loss of APC-induced Rac1 activation but not of thrombin-stimulated RhoA signaling. APC also failed to protect against thrombin-induced endothelial barrier permeability in cells deficient in β-arrestin expression. We further demonstrate that APC activation of PAR1 results in β-arrestin-dependent recruitment of Dvl-2, which is critical for Rac1 signaling and endothelial barrier protection but not for thrombin-induced RhoA signaling. Our findings identify a role for β-arrestin and Dvl-2 scaffolds in APC-activated PAR1 cytoprotective signaling in human endothelial cells.

Fig. 1.

PAR1, Gα_i, and β-arrestins cosegregate in caveolin-1–enriched fractions. Endothelial EA.hy926 cells were left untreated (Control) (A) or were treated at 37 °C with 20 nM APC for 5 min (B) or with 20 nM APC for 180 min (C). Cells were lysed, and caveolin-1–enriched fractions were isolated by detergent-free sucrose-gradient centrifugation. Aliquots representing each of the 12 fractions were immunoblotted with antibodies that recognize PAR1, β-arrestins (A1CT), Gα_i, Gα₁₂, Gα₁₃, caveolin-1 (Cav-1), and EEA-1.

Fig. 2.

APC-activated PAR1 signaling is independent of Gα_i protein. (A) Confluent endothelial cell (EC) monolayers were preincubated with or without (Ctrl) 100 ng/mL PTX for 16 h at 37 °C, and basal endothelial barrier permeability was monitored over time. The data (mean ± SEM) are representative of three independent experiments. (B) Control (Ctrl) and PTX-pretreated endothelial cells were incubated with 20 nM APC or 100 ng/mL SDF-1α for various times at 37 °C, and the activation of ERK1,2 was determined by immunoblotting (IB). Membranes were reprobed with an anti-ERK1,2 antibody to control for loading. Data shown are representative of three independent experiments. (C) (Upper) Control and PTX-pretreated endothelial cells were stimulated with or without 20 nM APC for 5 min or 180 min at 37 °C and were lysed. Equivalent amounts of cell lysates were incubated with GST-PBD, and the amount of activated Rac1 was determined by immunoblotting. Total cell lysates were immunoblotted with an anti-Rac1 antibody as a control. (Lower) Data (mean ± SEM) are expressed as the fold over untreated control (Ctrl) from three independent experiments. The differences in Rac1 activity measured in APC-stimulated cells versus untreated control was significant (*P < 0.05; **P < 0.01).

Fig. 3.

β-Arrestins associate with PAR1 and mediate APC-activated PAR1 signaling in human endothelial cells. (A) Endothelial cells were incubated with or without 20 nM APC or 10 nM thrombin (Th) for various times at 37 °C, lysed, and immunoprecipitated (IP) with IgG or anti-PAR1 WEDE antibody. Immunoprecipitates were examined for the presence of β-arrestins using an anti–β-arrestin A1CT antibody or for PAR1 using an anti-PAR1 polyclonal antibody. Total cell lysates were immunoblotted with a β-arrestin A1CT antibody or with anti-actin antibody as controls. (B) Endothelial cells were transfected with 100 nM nonspecific (ns), β-arrestin-1 (β-Arr1), β-arrestin-2 (β-Arr2), or both β-arrestin-1 and -2 siRNAs. Cells were lysed, and equivalent amounts of cell lysates were immunoblotted with an anti-A2CT antibody (which detects β-arrestin-2 over β-arrestin-1), anti-A1CT (which detects β-arrestin-1 over β-arrestin-2), or anti-actin antibody to control for loading. The asterisk indicates a nonspecific band. (C) Serum-deprived endothelial cells transfected with nonspecific or β-arrestin siRNAs were incubated with 10 nM thrombin (Th) or 20 nM APC for 5 min at 37 °C. Cell lysates were prepared, and ERK1,2 activity was determined by immunoblotting using anti–phospho-ERK1,2 antibody. Membranes were reprobed for total ERK1,2 to control for loading. Data (mean ± SEM) are expressed as the fold over untreated control (Ctrl) and are representative of three independent experiments. The differences in ERK1,2 activation induced by agonist compared with untreated control were significant (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 4.

β-Arrestins are critical for APC-induced Rac1 activation but not for thrombin-induced RhoA activation. (A and B) Endothelial cells transfected with 100 nM nonspecific (ns) or β-arrestin siRNAs were stimulated with 10 nM thrombin (Th) or 20 nM APC for 5 min or 180 min at 37 °C. Cells were lysed. Equivalent amounts of cell lysates were incubated with GST-PBD or GST-RBD, and the amount of activated Rac1 or RhoA was determined by immunoblotting. Total cell lysates were immunoblotted with an anti-Rac1 or anti-RhoA antibody as a control. Data (mean ± SEM) are expressed as the fold increase over untreated control (Ctrl) of three independent experiments. The differences between Rac1 or RhoA activity observed in agonist-treated cells versus untreated control cells were significant (*P < 0.05; **P < 0.01).

Fig. 5.

APC-induced endothelial barrier protection is mediated by β-arrestins. (A and B) Serum-deprived endothelial cells (EC) transfected with 100 nM nonspecific (ns) or β-arrestin siRNAs were preincubated with or without 20 nM APC for 3 h at 37 °C. Cells were washed and then were stimulated with 10 nM thrombin (Th) or 20 nM APC for 10 min at 37 °C, and endothelial barrier permeability was assessed. Data (mean ± SEM) are expressed as the fraction of response compared with thrombin-induced permeability determined at 10 min from three separate experiments. The differences in thrombin-stimulated endothelial barrier permeability observed in APC-pretreated cells versus untreated control cells were significant (**P < 0.01; ***P < 0.001).

Fig. 6.

APC induces Dvl-2 association with β-arrestins and Dvl-2 polymerization. (A) Endothelial cells transfected with FLAG-tagged β-arrestin-1 and -2 were incubated with 20 nM APC for various times at 37 °C. Cells were lysed and immunoprecipitated with anti-FLAG M2 antibody. Immunoprecipitates were examined for the presence of endogenous Dvl-2 using an anti–Dvl-2 polyclonal antibody or for FLAG–β-arrestins using an anti-FLAG antibody. Cell lysates were immunoblotted with anti–Dvl-2 and anti-actin antibodies as a control. Untransfected (UT) and FLAG–β-arrestin cell lysates were immunoblotted with anti–β-arrestin A1CT or A2CT antibodies to detect individual β-arrestin isoform expression. The asterisk indicates a nonspecific band. These data are representative of three separate experiments. (B) Endothelial cells were incubated with 20 nM APC for various times at 37 °C. Cells were lysed, and fractions representing soluble proteins or total cell lysates were prepared and immunoblotted with anti–Dvl-2 antibody. Membranes were reprobed with an anti-actin antibody to control for loading. The data (mean ± SEM) are expressed as the fraction of untreated 0-min control and are representative of four independent experiments. The differences in soluble Dvl-2 detected in APC-stimulated cells versus untreated control cells were significant (*P < 0.05; **P < 0.01). (C) Serum-starved endothelial cells were left untreated (Control) or were treated with 20 nM APC for 60 min at 37 °C. Cells were fixed, processed, and stained for filamentous actin (red) and immunostained for Dvl-2 (green) and were imaged by confocal microscopy. Images shown are representative fields of three independent experiments. Magnifications of boxed areas in merged images are shown on the right. (Scale bars, 20 μm.)

Fig. 7.

β-Arrestins mediate APC-induced Dvl-2 redistribution and polymerization. (A and B) Endothelial cells transfected with 100 nM nonspecific (ns) or β-arrestin siRNAs were incubated with 20 nM APC for various times at 37 °C and lysed. Cell fractions representing soluble proteins or total cell lysates were immunoblotted with an anti–Dvl-2 antibody. Membranes were reprobed with an anti-actin antibody as a control. Data (mean ± SEM) shown are expressed as the fraction of untreated 0-min control and are representative of three independent experiments. The difference in soluble Dvl-2 detected in APC-stimulated cells transfected with nonspecific siRNA was significant (*P < 0.05). (C) Endothelial cells transfected with 100 nM nonspecific or β-arrestin siRNAs were left untreated (Control) or were treated with 20 nM APC for 60 min at 37 °C. Cells were fixed, processed, and stained for filamentous actin (red) or immunostained for Dvl-2 (green) and imaged by confocal microscopy. Images shown are representative fields from three independent experiments. Magnifications of boxed areas in the merged images are shown on the right. (Scale bars, 20 μm.)

Fig. 8.

APC-induced cytoprotective signaling is mediated by Dvl-2. (A and B) Endothelial cells transfected with 50 nM nonspecific (ns) or Dvl-2 siRNAs were incubated with 10 nM thrombin (Th) or 20 nM APC for 5 or 180 min at 37 °C. Cells were lysed and incubated with GST-PBD or GST-RBD, and the amount of activated Rac1 or RhoA was detected by immunoblotting. Total cell lysates were immunoblotted with anti-Rac1 or anti-RhoA antibody as a control. Inset shows an anti–Dvl-2 and anti-actin antibody immunoblot of total cell lysates. The data (mean ± SEM) are expressed as the fold increase over untreated control (Ctrl) from three independent experiments. The differences between Rac1 or RhoA activation observed in agonist-treated cells versus untreated control cells were significant (*P < 0.05; **P < 0.01). (C) Serum-deprived endothelial cells (EC) transfected with 50 nM nonspecific or Dvl-2–specific siRNAs were preincubated with or without 20 nM APC for 3 h at 37 °C. Inset shows a total cell lysate immunoblot using the anti–Dvl-2 and anti-actin antibodies. Cells then were stimulated with 10 nM Th or 20 nM APC for 10 min at 37 °C, and endothelial barrier permeability was assessed. Data (mean ± SEM) are expressed as the fraction of thrombin-induced endothelial barrier permeability measured at 10 min and are representative of three independent experiments. The differences in thrombin-induced endothelial barrier permeability in cells transfected with nonspecific siRNA versus cells transfected with Dvl-2 siRNA was significant (**P < 0.01).

Fig. P1.

Model of thrombin versus APC activation of PAR1 and endothelial barrier regulation. Thrombin binds to and activates PAR1, which results in preferential coupling to the heterotrimeric Gα_q and Gα_12/13 proteins, stimulation of Ca²⁺ mobilization, and PKC and RhoA activation, resulting in endothelial barrier disruption. In contrast, APC binds to its coreceptor endothelial protein C receptor (EPCR) and activates PAR1, resulting in Rac1 signaling and endothelial barrier protection. We now show that PAR1 and β-arrestins exist in a preassembled complex and cosegregate in caveolar-enriched fractions. APC stimulation results in β-arrestin–dependent recruitment and activation of the Dvl-2 scaffold, which is critical for Rac1 activation and endothelial barrier protection.