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Rap-afadin axis in control of Rho signaling and endothelial barrier recovery.

Birukova AA, Tian X, Tian Y, Higginbotham K, Birukov KG - Mol. Biol. Cell (2013)

Bottom Line: Knockdown experiments showed that Rap1 activation was essential for down-regulation of Rho signaling and actin stress fiber dissolution.Rap1 activation also enhanced interaction between adherens junction (AJ) proteins VE-cadherin and p120-catenin and stimulated AJ reannealing mediated by the Rap1 effector afadin.This mechanism also included Rap1-dependent membrane translocation of the Rac1-specific GEF Tiam1 and activation of Rac1-dependent peripheral cytoskeletal dynamics, leading to resealing of intercellular gaps.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Pulmonary and Critical Medicine, Lung Injury Center, University of Chicago, Chicago, IL 60637, USA.

ABSTRACT
Activation of the Rho GTPase pathway determines endothelial cell (EC) hyperpermeability after injurious stimuli. To date, feedback mechanisms of Rho down-regulation critical for barrier restoration remain poorly understood. We tested a hypothesis that Rho down-regulation and barrier recovery of agonist-stimulated ECs is mediated by the Ras family GTPase Rap1. Thrombin-induced EC permeability driven by rapid activation of the Rho GTPase pathway was followed by Src kinase-dependent phosphorylation of the Rap1-specific guanine nucleotide exchange factor (GEF) C3G, activation of Rap1, and initiation of EC barrier recovery. Knockdown experiments showed that Rap1 activation was essential for down-regulation of Rho signaling and actin stress fiber dissolution. Rap1 activation also enhanced interaction between adherens junction (AJ) proteins VE-cadherin and p120-catenin and stimulated AJ reannealing mediated by the Rap1 effector afadin. This mechanism also included Rap1-dependent membrane translocation of the Rac1-specific GEF Tiam1 and activation of Rac1-dependent peripheral cytoskeletal dynamics, leading to resealing of intercellular gaps. These data demonstrate that activation of the Rap1-afadin axis is a physiological mechanism driving restoration of barrier integrity in agonist-stimulated EC monolayers via negative-feedback regulation of Rho signaling, stimulation of actin peripheral dynamics, and reestablishment of cell-cell adhesive complexes.

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Role of Src and C3G phosphorylation in Rap1 activation and EC barrier restoration after thrombin. (A) Time-dependent Src activation was monitored by immunoblotting with p-Y416–specific antibody reflecting the Src-activated state. (B) ECs were stimulated with thrombin (0.5 U/ml, 5 min); this was followed by addition of vehicle or the Src kinase inhibitor PP2 (5 μM). C3G tyrosine phosphorylation was detected by Western blot with phosphospecific antibody. Reprobing with β-actin antibody was used as normalization control. (C) ECs were stimulated with thrombin (0.5 U/ml, 5 min); this was followed by addition of vehicle or the Src kinase inhibitor PP2 (5 μM). Rap1 activation was evaluated using Rap1-GTP pull-down assay and normalized to the total Rap1 content in cell lysates. (D) HPAECs plated on microelectrodes were treated with thrombin (5 min); this was followed by addition of PP2 (5 μM). Measurements of TER were performed over 3 h. Arrows indicate times of thrombin and PP2 addition.
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Figure 2: Role of Src and C3G phosphorylation in Rap1 activation and EC barrier restoration after thrombin. (A) Time-dependent Src activation was monitored by immunoblotting with p-Y416–specific antibody reflecting the Src-activated state. (B) ECs were stimulated with thrombin (0.5 U/ml, 5 min); this was followed by addition of vehicle or the Src kinase inhibitor PP2 (5 μM). C3G tyrosine phosphorylation was detected by Western blot with phosphospecific antibody. Reprobing with β-actin antibody was used as normalization control. (C) ECs were stimulated with thrombin (0.5 U/ml, 5 min); this was followed by addition of vehicle or the Src kinase inhibitor PP2 (5 μM). Rap1 activation was evaluated using Rap1-GTP pull-down assay and normalized to the total Rap1 content in cell lysates. (D) HPAECs plated on microelectrodes were treated with thrombin (5 min); this was followed by addition of PP2 (5 μM). Measurements of TER were performed over 3 h. Arrows indicate times of thrombin and PP2 addition.

Mentions: Thrombin-induced activation of the tyrosine kinase Src is also involved in control of EC permeability (Tiruppathi et al., 2001; Vouret-Craviari et al., 2002; Liu et al., 2010). Because activity of the Rap1-specific GEF C3G, is controlled by tyrosine phosphorylation (Fukuyama et al., 2005), we followed Src activation and C3G phosphorylation patterns in thrombin-stimulated pulmonary ECs. Thrombin-induced Src phosphorylation at Tyr-416, the site reflecting Src activation, was detected 5 min after stimulation and reached peak levels by 10–20 min (Figure 2A). The role of Src in the EC barrier recovery was tested in experiments with administration of the Src inhibitor PP2 5 min after thrombin addition. At this point, ECs develop maximal Rho activation and permeability response. Inhibition of Src by PP2 posttreatment after thrombin challenge abolished C3G phosphorylation (Figure 2B), which led to inhibition of Rap1 activation (Figure 2C). Inhibition of Src, C3G phosphorylation, and Rap1 activation by PP2 posttreatment after thrombin dramatically attenuated EC barrier recovery as evaluated by measurements of transendothelial electrical resistance (Figure 2D).


Rap-afadin axis in control of Rho signaling and endothelial barrier recovery.

Birukova AA, Tian X, Tian Y, Higginbotham K, Birukov KG - Mol. Biol. Cell (2013)

Role of Src and C3G phosphorylation in Rap1 activation and EC barrier restoration after thrombin. (A) Time-dependent Src activation was monitored by immunoblotting with p-Y416–specific antibody reflecting the Src-activated state. (B) ECs were stimulated with thrombin (0.5 U/ml, 5 min); this was followed by addition of vehicle or the Src kinase inhibitor PP2 (5 μM). C3G tyrosine phosphorylation was detected by Western blot with phosphospecific antibody. Reprobing with β-actin antibody was used as normalization control. (C) ECs were stimulated with thrombin (0.5 U/ml, 5 min); this was followed by addition of vehicle or the Src kinase inhibitor PP2 (5 μM). Rap1 activation was evaluated using Rap1-GTP pull-down assay and normalized to the total Rap1 content in cell lysates. (D) HPAECs plated on microelectrodes were treated with thrombin (5 min); this was followed by addition of PP2 (5 μM). Measurements of TER were performed over 3 h. Arrows indicate times of thrombin and PP2 addition.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756920&req=5

Figure 2: Role of Src and C3G phosphorylation in Rap1 activation and EC barrier restoration after thrombin. (A) Time-dependent Src activation was monitored by immunoblotting with p-Y416–specific antibody reflecting the Src-activated state. (B) ECs were stimulated with thrombin (0.5 U/ml, 5 min); this was followed by addition of vehicle or the Src kinase inhibitor PP2 (5 μM). C3G tyrosine phosphorylation was detected by Western blot with phosphospecific antibody. Reprobing with β-actin antibody was used as normalization control. (C) ECs were stimulated with thrombin (0.5 U/ml, 5 min); this was followed by addition of vehicle or the Src kinase inhibitor PP2 (5 μM). Rap1 activation was evaluated using Rap1-GTP pull-down assay and normalized to the total Rap1 content in cell lysates. (D) HPAECs plated on microelectrodes were treated with thrombin (5 min); this was followed by addition of PP2 (5 μM). Measurements of TER were performed over 3 h. Arrows indicate times of thrombin and PP2 addition.
Mentions: Thrombin-induced activation of the tyrosine kinase Src is also involved in control of EC permeability (Tiruppathi et al., 2001; Vouret-Craviari et al., 2002; Liu et al., 2010). Because activity of the Rap1-specific GEF C3G, is controlled by tyrosine phosphorylation (Fukuyama et al., 2005), we followed Src activation and C3G phosphorylation patterns in thrombin-stimulated pulmonary ECs. Thrombin-induced Src phosphorylation at Tyr-416, the site reflecting Src activation, was detected 5 min after stimulation and reached peak levels by 10–20 min (Figure 2A). The role of Src in the EC barrier recovery was tested in experiments with administration of the Src inhibitor PP2 5 min after thrombin addition. At this point, ECs develop maximal Rho activation and permeability response. Inhibition of Src by PP2 posttreatment after thrombin challenge abolished C3G phosphorylation (Figure 2B), which led to inhibition of Rap1 activation (Figure 2C). Inhibition of Src, C3G phosphorylation, and Rap1 activation by PP2 posttreatment after thrombin dramatically attenuated EC barrier recovery as evaluated by measurements of transendothelial electrical resistance (Figure 2D).

Bottom Line: Knockdown experiments showed that Rap1 activation was essential for down-regulation of Rho signaling and actin stress fiber dissolution.Rap1 activation also enhanced interaction between adherens junction (AJ) proteins VE-cadherin and p120-catenin and stimulated AJ reannealing mediated by the Rap1 effector afadin.This mechanism also included Rap1-dependent membrane translocation of the Rac1-specific GEF Tiam1 and activation of Rac1-dependent peripheral cytoskeletal dynamics, leading to resealing of intercellular gaps.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Pulmonary and Critical Medicine, Lung Injury Center, University of Chicago, Chicago, IL 60637, USA.

ABSTRACT
Activation of the Rho GTPase pathway determines endothelial cell (EC) hyperpermeability after injurious stimuli. To date, feedback mechanisms of Rho down-regulation critical for barrier restoration remain poorly understood. We tested a hypothesis that Rho down-regulation and barrier recovery of agonist-stimulated ECs is mediated by the Ras family GTPase Rap1. Thrombin-induced EC permeability driven by rapid activation of the Rho GTPase pathway was followed by Src kinase-dependent phosphorylation of the Rap1-specific guanine nucleotide exchange factor (GEF) C3G, activation of Rap1, and initiation of EC barrier recovery. Knockdown experiments showed that Rap1 activation was essential for down-regulation of Rho signaling and actin stress fiber dissolution. Rap1 activation also enhanced interaction between adherens junction (AJ) proteins VE-cadherin and p120-catenin and stimulated AJ reannealing mediated by the Rap1 effector afadin. This mechanism also included Rap1-dependent membrane translocation of the Rac1-specific GEF Tiam1 and activation of Rac1-dependent peripheral cytoskeletal dynamics, leading to resealing of intercellular gaps. These data demonstrate that activation of the Rap1-afadin axis is a physiological mechanism driving restoration of barrier integrity in agonist-stimulated EC monolayers via negative-feedback regulation of Rho signaling, stimulation of actin peripheral dynamics, and reestablishment of cell-cell adhesive complexes.

Show MeSH
Related in: MedlinePlus