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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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Effects of Rap1 knockdown on functional and structural barrier restoration of pulmonary EC monolayer after thrombin. (A) ECs plated on microelectrodes were transfected with Rap1-specific siRNA or nonspecific RNA 48 h prior to TER measurements. Control and Rap1-specific siRNA-treated ECs were stimulated with thrombin at the time indicated by the arrow, and TER changes were monitored over time. (B) ECs plated on glass coverslips were transfected with Rap1-specific siRNA or nonspecific RNA prior to stimulation with thrombin. Immunofluorescence staining of F-actin (left) and VE-cadherin (middle) was performed using Texas Red phalloidin and VE-cadherin specific antibody, respectively. Right, merged images of F-actin and VE-cadherin staining. Arrows indicate areas of thrombin-induced intercellular gap formation. (C) Quantitative analysis of gap formation and cell junction VE-cadherin localization in control and Rap1-depleted ECs at different times after thrombin treatment. Data are expressed as mean ± SD; *, p < 0.05.
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Figure 3: Effects of Rap1 knockdown on functional and structural barrier restoration of pulmonary EC monolayer after thrombin. (A) ECs plated on microelectrodes were transfected with Rap1-specific siRNA or nonspecific RNA 48 h prior to TER measurements. Control and Rap1-specific siRNA-treated ECs were stimulated with thrombin at the time indicated by the arrow, and TER changes were monitored over time. (B) ECs plated on glass coverslips were transfected with Rap1-specific siRNA or nonspecific RNA prior to stimulation with thrombin. Immunofluorescence staining of F-actin (left) and VE-cadherin (middle) was performed using Texas Red phalloidin and VE-cadherin specific antibody, respectively. Right, merged images of F-actin and VE-cadherin staining. Arrows indicate areas of thrombin-induced intercellular gap formation. (C) Quantitative analysis of gap formation and cell junction VE-cadherin localization in control and Rap1-depleted ECs at different times after thrombin treatment. Data are expressed as mean ± SD; *, p < 0.05.

Mentions: Small interfering RNA (siRNA)-induced Rap1 depletion in EC monolayers significantly suppressed the restoration of transendothelial electrical resistance (TER) to control levels (Figure 3A), suggesting the direct role of Rap1 in EC barrier recovery after agonist stimulation. Functional permeability effects were linked to morphological changes. Immunofluorescence analysis showed slight but statistically significant decrease in VE-cadherin accumulation at cell junctions in Rap1-depleted EC monolayers under basal conditions (Figure 3, B and C). Thrombin induced robust stress fiber formation and disruption of continuous peripheral VE-cadherin in control cells, and Rap1 depleted ECs (Figure 3B). These changes were reversible in control ECs and returned to original patterns 1 h after thrombin challenge, thus reflecting completion of EC monolayer recovery (Figure 3B, top panels). In contrast, dissolution of stress fibers and restoration of adherens junctions (AJ) in Rap1-depleted EC monolayers was impaired (Figure 3B, bottom panels). Inhibition of EC monolayer recovery by Rap1 depletion was documented by dramatic attenuation of intercellular gap resealing and reappearance of VE-cadherin at cell junction areas (Figure 3C).


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)

Effects of Rap1 knockdown on functional and structural barrier restoration of pulmonary EC monolayer after thrombin. (A) ECs plated on microelectrodes were transfected with Rap1-specific siRNA or nonspecific RNA 48 h prior to TER measurements. Control and Rap1-specific siRNA-treated ECs were stimulated with thrombin at the time indicated by the arrow, and TER changes were monitored over time. (B) ECs plated on glass coverslips were transfected with Rap1-specific siRNA or nonspecific RNA prior to stimulation with thrombin. Immunofluorescence staining of F-actin (left) and VE-cadherin (middle) was performed using Texas Red phalloidin and VE-cadherin specific antibody, respectively. Right, merged images of F-actin and VE-cadherin staining. Arrows indicate areas of thrombin-induced intercellular gap formation. (C) Quantitative analysis of gap formation and cell junction VE-cadherin localization in control and Rap1-depleted ECs at different times after thrombin treatment. Data are expressed as mean ± SD; *, p < 0.05.
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Related In: Results  -  Collection

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Figure 3: Effects of Rap1 knockdown on functional and structural barrier restoration of pulmonary EC monolayer after thrombin. (A) ECs plated on microelectrodes were transfected with Rap1-specific siRNA or nonspecific RNA 48 h prior to TER measurements. Control and Rap1-specific siRNA-treated ECs were stimulated with thrombin at the time indicated by the arrow, and TER changes were monitored over time. (B) ECs plated on glass coverslips were transfected with Rap1-specific siRNA or nonspecific RNA prior to stimulation with thrombin. Immunofluorescence staining of F-actin (left) and VE-cadherin (middle) was performed using Texas Red phalloidin and VE-cadherin specific antibody, respectively. Right, merged images of F-actin and VE-cadherin staining. Arrows indicate areas of thrombin-induced intercellular gap formation. (C) Quantitative analysis of gap formation and cell junction VE-cadherin localization in control and Rap1-depleted ECs at different times after thrombin treatment. Data are expressed as mean ± SD; *, p < 0.05.
Mentions: Small interfering RNA (siRNA)-induced Rap1 depletion in EC monolayers significantly suppressed the restoration of transendothelial electrical resistance (TER) to control levels (Figure 3A), suggesting the direct role of Rap1 in EC barrier recovery after agonist stimulation. Functional permeability effects were linked to morphological changes. Immunofluorescence analysis showed slight but statistically significant decrease in VE-cadherin accumulation at cell junctions in Rap1-depleted EC monolayers under basal conditions (Figure 3, B and C). Thrombin induced robust stress fiber formation and disruption of continuous peripheral VE-cadherin in control cells, and Rap1 depleted ECs (Figure 3B). These changes were reversible in control ECs and returned to original patterns 1 h after thrombin challenge, thus reflecting completion of EC monolayer recovery (Figure 3B, top panels). In contrast, dissolution of stress fibers and restoration of adherens junctions (AJ) in Rap1-depleted EC monolayers was impaired (Figure 3B, bottom panels). Inhibition of EC monolayer recovery by Rap1 depletion was documented by dramatic attenuation of intercellular gap resealing and reappearance of VE-cadherin at cell junction areas (Figure 3C).

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