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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 afadin in EC barrier restoration and reversal of Rho signaling after thrombin. (A) HPAEC monolayers were stimulated with thrombin (0.5 U/ml) for the indicated periods of time. Peripheral localization of afadin was evaluated by immunofluorescence staining with afadin antibody. (B) HPAEC monolayers transfected with control RNA or afadin-specific siRNA were stimulated with thrombin (0.5 U/ml) for the indicated periods of time. Stress fiber formation and integrity of AJs was monitored by immunofluorescence staining with Texas Red phalloidin (left) and VE-cadherin (middle) antibody, respectively. Right, merged images of F-actin and VE-cadherin staining. (C) Quantitative analysis of gap formation in control and afadin-depleted ECs at different times after thrombin treatment. Data are expressed as mean ± SD; *, p < 0.05. (D) Activation of Rho signaling was evaluated by Western blot with phospho-MYPT and diphospho-MLC antibody. Reprobing with β-actin antibody was used as the normalization control. Afadin depletion was verified by Western blot. (E) Cells were transfected with nonspecific RNA or Rap1-specific siRNA; this was followed by thrombin stimulation. Coimmunoprecipitation assays using antibody to p120-catenin were performed, and afadin and p120-catenin content in the immunoprecipitates was detected using specific antibodies. Equal protein loading was confirmed by membrane reprobing with antibodies to p120-catenin. Bottom panels depict siRNA-based depletion of endogenous Rap1. Reprobing with β-actin antibody was used as the normalization control. Bar graphs depict results of membrane densitometry analysis; data are expressed as mean ± SD; *, p < 0.05 vs. control.
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Figure 8: Role of afadin in EC barrier restoration and reversal of Rho signaling after thrombin. (A) HPAEC monolayers were stimulated with thrombin (0.5 U/ml) for the indicated periods of time. Peripheral localization of afadin was evaluated by immunofluorescence staining with afadin antibody. (B) HPAEC monolayers transfected with control RNA or afadin-specific siRNA were stimulated with thrombin (0.5 U/ml) for the indicated periods of time. Stress fiber formation and integrity of AJs was monitored by immunofluorescence staining with Texas Red phalloidin (left) and VE-cadherin (middle) antibody, respectively. Right, merged images of F-actin and VE-cadherin staining. (C) Quantitative analysis of gap formation in control and afadin-depleted ECs at different times after thrombin treatment. Data are expressed as mean ± SD; *, p < 0.05. (D) Activation of Rho signaling was evaluated by Western blot with phospho-MYPT and diphospho-MLC antibody. Reprobing with β-actin antibody was used as the normalization control. Afadin depletion was verified by Western blot. (E) Cells were transfected with nonspecific RNA or Rap1-specific siRNA; this was followed by thrombin stimulation. Coimmunoprecipitation assays using antibody to p120-catenin were performed, and afadin and p120-catenin content in the immunoprecipitates was detected using specific antibodies. Equal protein loading was confirmed by membrane reprobing with antibodies to p120-catenin. Bottom panels depict siRNA-based depletion of endogenous Rap1. Reprobing with β-actin antibody was used as the normalization control. Bar graphs depict results of membrane densitometry analysis; data are expressed as mean ± SD; *, p < 0.05 vs. control.

Mentions: A recent study shows that the AJ-associated Rap1 effector afadin promotes AJ complex assembly and mediates agonist-induced EC barrier enhancement (Birukova et al., 2012a). The potential role of afadin in EC barrier recovery after thrombin was further tested. Thrombin caused rapid disappearance of afadin from cell–cell junction areas within 5 min of stimulation, and reappearance after 30–60 min (Figure 8A). Afadin depletion prevented the reassembly of junctions and restoration of actin cytoskeletal pattern after 60 min of thrombin challenge (Figure 8B) and significantly delayed resealing of paracellular gaps in thrombin-challenged EC monolayers (Figure 8C).


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 afadin in EC barrier restoration and reversal of Rho signaling after thrombin. (A) HPAEC monolayers were stimulated with thrombin (0.5 U/ml) for the indicated periods of time. Peripheral localization of afadin was evaluated by immunofluorescence staining with afadin antibody. (B) HPAEC monolayers transfected with control RNA or afadin-specific siRNA were stimulated with thrombin (0.5 U/ml) for the indicated periods of time. Stress fiber formation and integrity of AJs was monitored by immunofluorescence staining with Texas Red phalloidin (left) and VE-cadherin (middle) antibody, respectively. Right, merged images of F-actin and VE-cadherin staining. (C) Quantitative analysis of gap formation in control and afadin-depleted ECs at different times after thrombin treatment. Data are expressed as mean ± SD; *, p < 0.05. (D) Activation of Rho signaling was evaluated by Western blot with phospho-MYPT and diphospho-MLC antibody. Reprobing with β-actin antibody was used as the normalization control. Afadin depletion was verified by Western blot. (E) Cells were transfected with nonspecific RNA or Rap1-specific siRNA; this was followed by thrombin stimulation. Coimmunoprecipitation assays using antibody to p120-catenin were performed, and afadin and p120-catenin content in the immunoprecipitates was detected using specific antibodies. Equal protein loading was confirmed by membrane reprobing with antibodies to p120-catenin. Bottom panels depict siRNA-based depletion of endogenous Rap1. Reprobing with β-actin antibody was used as the normalization control. Bar graphs depict results of membrane densitometry analysis; data are expressed as mean ± SD; *, p < 0.05 vs. control.
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Figure 8: Role of afadin in EC barrier restoration and reversal of Rho signaling after thrombin. (A) HPAEC monolayers were stimulated with thrombin (0.5 U/ml) for the indicated periods of time. Peripheral localization of afadin was evaluated by immunofluorescence staining with afadin antibody. (B) HPAEC monolayers transfected with control RNA or afadin-specific siRNA were stimulated with thrombin (0.5 U/ml) for the indicated periods of time. Stress fiber formation and integrity of AJs was monitored by immunofluorescence staining with Texas Red phalloidin (left) and VE-cadherin (middle) antibody, respectively. Right, merged images of F-actin and VE-cadherin staining. (C) Quantitative analysis of gap formation in control and afadin-depleted ECs at different times after thrombin treatment. Data are expressed as mean ± SD; *, p < 0.05. (D) Activation of Rho signaling was evaluated by Western blot with phospho-MYPT and diphospho-MLC antibody. Reprobing with β-actin antibody was used as the normalization control. Afadin depletion was verified by Western blot. (E) Cells were transfected with nonspecific RNA or Rap1-specific siRNA; this was followed by thrombin stimulation. Coimmunoprecipitation assays using antibody to p120-catenin were performed, and afadin and p120-catenin content in the immunoprecipitates was detected using specific antibodies. Equal protein loading was confirmed by membrane reprobing with antibodies to p120-catenin. Bottom panels depict siRNA-based depletion of endogenous Rap1. Reprobing with β-actin antibody was used as the normalization control. Bar graphs depict results of membrane densitometry analysis; data are expressed as mean ± SD; *, p < 0.05 vs. control.
Mentions: A recent study shows that the AJ-associated Rap1 effector afadin promotes AJ complex assembly and mediates agonist-induced EC barrier enhancement (Birukova et al., 2012a). The potential role of afadin in EC barrier recovery after thrombin was further tested. Thrombin caused rapid disappearance of afadin from cell–cell junction areas within 5 min of stimulation, and reappearance after 30–60 min (Figure 8A). Afadin depletion prevented the reassembly of junctions and restoration of actin cytoskeletal pattern after 60 min of thrombin challenge (Figure 8B) and significantly delayed resealing of paracellular gaps in thrombin-challenged EC monolayers (Figure 8C).

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