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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 Rap1 in reannealing of AJs and increased p120-catenin–VE-cadherin interactions during recovery after thrombin. (A) Live-cell imaging of HPAECs transfected with Rap1 siRNA or nonspecific RNA and expressing GFP-β-catenin. Snapshots depict reestablishment of β-catenin–positive AJ previously disrupted by thrombin challenge. (top panels, shown by arrow). Rap1 knockdown abolished this effect (bottom panels, shown by arrow). (B) Increased colocalization of p120-catenin and VE-cadherin in cell membrane fraction after 30 min of thrombin stimulation was abolished by Rap1 knockdown. Rap1 depletion was verified by Western blot. (C) 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 VE-cadherin 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 7: Role of Rap1 in reannealing of AJs and increased p120-catenin–VE-cadherin interactions during recovery after thrombin. (A) Live-cell imaging of HPAECs transfected with Rap1 siRNA or nonspecific RNA and expressing GFP-β-catenin. Snapshots depict reestablishment of β-catenin–positive AJ previously disrupted by thrombin challenge. (top panels, shown by arrow). Rap1 knockdown abolished this effect (bottom panels, shown by arrow). (B) Increased colocalization of p120-catenin and VE-cadherin in cell membrane fraction after 30 min of thrombin stimulation was abolished by Rap1 knockdown. Rap1 depletion was verified by Western blot. (C) 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 VE-cadherin 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: We used pulmonary ECs, transiently transfected with GFP-β-catenin, for live microscopy analysis of AJ restoration after thrombin challenge. Disappearance of GFP-β-catenin from cell–cell junction areas was observed after 5–15 min of thrombin challenge and reflects agonist-induced AJ disruption. Reappearance of a continuous GFP-β-catenin signal at the cell–cell boundary was observed after 20–30 min of thrombin challenge (Figure 7A, top panels). Rap1 depletion prevented formation of GFP-β-catenin–positive cell–cell junctions during the time of experiment (Figure 7A, bottom panels).


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 Rap1 in reannealing of AJs and increased p120-catenin–VE-cadherin interactions during recovery after thrombin. (A) Live-cell imaging of HPAECs transfected with Rap1 siRNA or nonspecific RNA and expressing GFP-β-catenin. Snapshots depict reestablishment of β-catenin–positive AJ previously disrupted by thrombin challenge. (top panels, shown by arrow). Rap1 knockdown abolished this effect (bottom panels, shown by arrow). (B) Increased colocalization of p120-catenin and VE-cadherin in cell membrane fraction after 30 min of thrombin stimulation was abolished by Rap1 knockdown. Rap1 depletion was verified by Western blot. (C) 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 VE-cadherin 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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Related In: Results  -  Collection

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Figure 7: Role of Rap1 in reannealing of AJs and increased p120-catenin–VE-cadherin interactions during recovery after thrombin. (A) Live-cell imaging of HPAECs transfected with Rap1 siRNA or nonspecific RNA and expressing GFP-β-catenin. Snapshots depict reestablishment of β-catenin–positive AJ previously disrupted by thrombin challenge. (top panels, shown by arrow). Rap1 knockdown abolished this effect (bottom panels, shown by arrow). (B) Increased colocalization of p120-catenin and VE-cadherin in cell membrane fraction after 30 min of thrombin stimulation was abolished by Rap1 knockdown. Rap1 depletion was verified by Western blot. (C) 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 VE-cadherin 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: We used pulmonary ECs, transiently transfected with GFP-β-catenin, for live microscopy analysis of AJ restoration after thrombin challenge. Disappearance of GFP-β-catenin from cell–cell junction areas was observed after 5–15 min of thrombin challenge and reflects agonist-induced AJ disruption. Reappearance of a continuous GFP-β-catenin signal at the cell–cell boundary was observed after 20–30 min of thrombin challenge (Figure 7A, top panels). Rap1 depletion prevented formation of GFP-β-catenin–positive cell–cell junctions during the time of experiment (Figure 7A, bottom panels).

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