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Asef controls vascular endothelial permeability and barrier recovery in the lung.

Tian X, Tian Y, Gawlak G, Meng F, Kawasaki Y, Akiyama T, Birukova AA - Mol. Biol. Cell (2014)

Bottom Line: Molecular inhibition of Asef attenuated HGF-induced peripheral accumulation of cortactin, formation of lamellipodia-like structures, and enhancement of VE-cadherin adherens junctions and compromised HGF-protective effect against thrombin-induced RhoA GTPase activation, Rho-dependent cytoskeleton remodeling, and EC permeability.This effect was lost in Asef(-/-) mice.This study shows for the first time the role of Asef in HGF-mediated protection against endothelial hyperpermeability and lung injury.

View Article: PubMed Central - PubMed

Affiliation: Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, IL 60637.

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Asef knockdown attenuates HGF-induced enhancement of adherens junctions. HPAECs were transfected with Asef-specific siRNA or nonspecific RNA and stimulated with HGF (50 ng/ml). Sparse (A) or dense (B) HPAEC cultures were stimulated with HGF, followed by isolation of membrane fraction. HGF-induced VE-cadherin accumulation in the membrane fraction was detected with specific antibodies. VE-cadherin content in corresponding total cell lysates was used as a normalization control. (C) Visualization of cell–cell contacts was performed by immunofluorescence staining of formaldehyde-fixed HPAECs with VE-cadherin antibody. Bar, 10 μm. Magnified images (insets) show details of adherens junction structures. (D) HGF-treated HPAECs were used for reciprocal coimmunoprecipitation assays with VE-cadherin (top) and p120-catenin (bottom) antibodies, followed by Western blot detection of p120-catenin and VE-cadherin; *p <0.01 vs. nonstimulated cells treated with nonspecific RNA; **p <0.05 vs. nonspecific RNA. Results are representative of three independent experiments.
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Figure 6: Asef knockdown attenuates HGF-induced enhancement of adherens junctions. HPAECs were transfected with Asef-specific siRNA or nonspecific RNA and stimulated with HGF (50 ng/ml). Sparse (A) or dense (B) HPAEC cultures were stimulated with HGF, followed by isolation of membrane fraction. HGF-induced VE-cadherin accumulation in the membrane fraction was detected with specific antibodies. VE-cadherin content in corresponding total cell lysates was used as a normalization control. (C) Visualization of cell–cell contacts was performed by immunofluorescence staining of formaldehyde-fixed HPAECs with VE-cadherin antibody. Bar, 10 μm. Magnified images (insets) show details of adherens junction structures. (D) HGF-treated HPAECs were used for reciprocal coimmunoprecipitation assays with VE-cadherin (top) and p120-catenin (bottom) antibodies, followed by Western blot detection of p120-catenin and VE-cadherin; *p <0.01 vs. nonstimulated cells treated with nonspecific RNA; **p <0.05 vs. nonspecific RNA. Results are representative of three independent experiments.

Mentions: VE-cadherin accumulation at the cell membrane compartment is essential for initiation adherens junction protein complex assembly. Using subcellular fractionation assay, we examined VE-cadherin membrane translocation in confluent (Figure 6A) and sparse (Figure 6B) EC cultures after HGF stimulation. HGF induced robust VE-cadherin membrane accumulation in dense (confluent) EC monolayers but not in sparse EC cultures. Observed VE-cadherin membrane accumulation was significantly attenuated in HGF-stimulated ECs with Asef knockdown. Immunofluorescence staining of VE-cadherin shows a dramatic increase of VE-cadherin–positive immunofluorescence signal at the cell junction area of HGF-stimulated EC monolayers. This effect was abolished by Asef knockdown (Figure 6C).


Asef controls vascular endothelial permeability and barrier recovery in the lung.

Tian X, Tian Y, Gawlak G, Meng F, Kawasaki Y, Akiyama T, Birukova AA - Mol. Biol. Cell (2014)

Asef knockdown attenuates HGF-induced enhancement of adherens junctions. HPAECs were transfected with Asef-specific siRNA or nonspecific RNA and stimulated with HGF (50 ng/ml). Sparse (A) or dense (B) HPAEC cultures were stimulated with HGF, followed by isolation of membrane fraction. HGF-induced VE-cadherin accumulation in the membrane fraction was detected with specific antibodies. VE-cadherin content in corresponding total cell lysates was used as a normalization control. (C) Visualization of cell–cell contacts was performed by immunofluorescence staining of formaldehyde-fixed HPAECs with VE-cadherin antibody. Bar, 10 μm. Magnified images (insets) show details of adherens junction structures. (D) HGF-treated HPAECs were used for reciprocal coimmunoprecipitation assays with VE-cadherin (top) and p120-catenin (bottom) antibodies, followed by Western blot detection of p120-catenin and VE-cadherin; *p <0.01 vs. nonstimulated cells treated with nonspecific RNA; **p <0.05 vs. nonspecific RNA. Results are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 6: Asef knockdown attenuates HGF-induced enhancement of adherens junctions. HPAECs were transfected with Asef-specific siRNA or nonspecific RNA and stimulated with HGF (50 ng/ml). Sparse (A) or dense (B) HPAEC cultures were stimulated with HGF, followed by isolation of membrane fraction. HGF-induced VE-cadherin accumulation in the membrane fraction was detected with specific antibodies. VE-cadherin content in corresponding total cell lysates was used as a normalization control. (C) Visualization of cell–cell contacts was performed by immunofluorescence staining of formaldehyde-fixed HPAECs with VE-cadherin antibody. Bar, 10 μm. Magnified images (insets) show details of adherens junction structures. (D) HGF-treated HPAECs were used for reciprocal coimmunoprecipitation assays with VE-cadherin (top) and p120-catenin (bottom) antibodies, followed by Western blot detection of p120-catenin and VE-cadherin; *p <0.01 vs. nonstimulated cells treated with nonspecific RNA; **p <0.05 vs. nonspecific RNA. Results are representative of three independent experiments.
Mentions: VE-cadherin accumulation at the cell membrane compartment is essential for initiation adherens junction protein complex assembly. Using subcellular fractionation assay, we examined VE-cadherin membrane translocation in confluent (Figure 6A) and sparse (Figure 6B) EC cultures after HGF stimulation. HGF induced robust VE-cadherin membrane accumulation in dense (confluent) EC monolayers but not in sparse EC cultures. Observed VE-cadherin membrane accumulation was significantly attenuated in HGF-stimulated ECs with Asef knockdown. Immunofluorescence staining of VE-cadherin shows a dramatic increase of VE-cadherin–positive immunofluorescence signal at the cell junction area of HGF-stimulated EC monolayers. This effect was abolished by Asef knockdown (Figure 6C).

Bottom Line: Molecular inhibition of Asef attenuated HGF-induced peripheral accumulation of cortactin, formation of lamellipodia-like structures, and enhancement of VE-cadherin adherens junctions and compromised HGF-protective effect against thrombin-induced RhoA GTPase activation, Rho-dependent cytoskeleton remodeling, and EC permeability.This effect was lost in Asef(-/-) mice.This study shows for the first time the role of Asef in HGF-mediated protection against endothelial hyperpermeability and lung injury.

View Article: PubMed Central - PubMed

Affiliation: Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, IL 60637.

Show MeSH
Related in: MedlinePlus