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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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Role of Asef activity in HGF-induced actin remodeling. HA-tagged wild-type Asef, constitutively active (CA-Asef) or dominant-negative (DN-Asef) Asef mutants were expressed in HPAECs. After stimulation with vehicle or HGF (50 ng/ml, 10 min), cells were fixed in 4.7% formaldehyde, and F-actin was visualized by Texas red–phalloidin staining. Cells expressing recombinant Asef were visualized by immunofluorescence staining for HA tag. (A) Cells transfected with wild-type (WT) Asef were treated with vehicle or HGF. (B) Expression of constitutively active Asef mutant (ΔAPC-Asef). (C) Expression of dominant-negative Asef mutant (ΔDH-Asef). Inset, higher-magnification image of cell edge, depicting inhibition of HGF-induced formation of lamellipodia-like structures by expression of dominant-negative Asef mutant. Arrows show cells with activated peripheral actin cytoskeletal remodeling. Bar, 5 μm. (D) EC monolayers were transiently transfected with wild-type Asef or dominant-negative Asef mutant, and HGF-induced Rac1 activation was determined by Rac-GTP pull-down assay. The content of activated Rac1 was normalized to the total Rac1 content in EC lysates; *p < 0.01 vs. nonstimulated controls transfected with Asef-WT; **p < 0.05 vs. Asef-WT.
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Figure 5: Role of Asef activity in HGF-induced actin remodeling. HA-tagged wild-type Asef, constitutively active (CA-Asef) or dominant-negative (DN-Asef) Asef mutants were expressed in HPAECs. After stimulation with vehicle or HGF (50 ng/ml, 10 min), cells were fixed in 4.7% formaldehyde, and F-actin was visualized by Texas red–phalloidin staining. Cells expressing recombinant Asef were visualized by immunofluorescence staining for HA tag. (A) Cells transfected with wild-type (WT) Asef were treated with vehicle or HGF. (B) Expression of constitutively active Asef mutant (ΔAPC-Asef). (C) Expression of dominant-negative Asef mutant (ΔDH-Asef). Inset, higher-magnification image of cell edge, depicting inhibition of HGF-induced formation of lamellipodia-like structures by expression of dominant-negative Asef mutant. Arrows show cells with activated peripheral actin cytoskeletal remodeling. Bar, 5 μm. (D) EC monolayers were transiently transfected with wild-type Asef or dominant-negative Asef mutant, and HGF-induced Rac1 activation was determined by Rac-GTP pull-down assay. The content of activated Rac1 was normalized to the total Rac1 content in EC lysates; *p < 0.01 vs. nonstimulated controls transfected with Asef-WT; **p < 0.05 vs. Asef-WT.

Mentions: As a complementary approach to experiments with Asef knockdown, we performed ectopic expression of HA-tagged wild-type Asef or its constitutively activated (CA-Asef) or dominant-negative (DN-Asef) mutant (Kawasaki et al., 2003). Overexpression of wild-type Asef further enhanced EC peripheral actin remodeling in response to HGF in comparison to HGF-stimulated nontransfected cells (Figure 5A). In turn, expression of activated Asef mutant in nonstimulated cells reproduced peripheral actin cytoskeletal rearrangement observed in nontransfected ECs treated with HGF (Figure 5B). Similar to Asef knockdown experiments, the expression of dominant-negative Asef abrogated HGF-induced formation of lamellipodia-like structures and peripheral actin rim (Figure 5C). Expression of dominant-negative Asef mutant suppressed the HGF-induced activation of Rac1 GTPase in pulmonary ECs (Figure 5D).


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)

Role of Asef activity in HGF-induced actin remodeling. HA-tagged wild-type Asef, constitutively active (CA-Asef) or dominant-negative (DN-Asef) Asef mutants were expressed in HPAECs. After stimulation with vehicle or HGF (50 ng/ml, 10 min), cells were fixed in 4.7% formaldehyde, and F-actin was visualized by Texas red–phalloidin staining. Cells expressing recombinant Asef were visualized by immunofluorescence staining for HA tag. (A) Cells transfected with wild-type (WT) Asef were treated with vehicle or HGF. (B) Expression of constitutively active Asef mutant (ΔAPC-Asef). (C) Expression of dominant-negative Asef mutant (ΔDH-Asef). Inset, higher-magnification image of cell edge, depicting inhibition of HGF-induced formation of lamellipodia-like structures by expression of dominant-negative Asef mutant. Arrows show cells with activated peripheral actin cytoskeletal remodeling. Bar, 5 μm. (D) EC monolayers were transiently transfected with wild-type Asef or dominant-negative Asef mutant, and HGF-induced Rac1 activation was determined by Rac-GTP pull-down assay. The content of activated Rac1 was normalized to the total Rac1 content in EC lysates; *p < 0.01 vs. nonstimulated controls transfected with Asef-WT; **p < 0.05 vs. Asef-WT.
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Figure 5: Role of Asef activity in HGF-induced actin remodeling. HA-tagged wild-type Asef, constitutively active (CA-Asef) or dominant-negative (DN-Asef) Asef mutants were expressed in HPAECs. After stimulation with vehicle or HGF (50 ng/ml, 10 min), cells were fixed in 4.7% formaldehyde, and F-actin was visualized by Texas red–phalloidin staining. Cells expressing recombinant Asef were visualized by immunofluorescence staining for HA tag. (A) Cells transfected with wild-type (WT) Asef were treated with vehicle or HGF. (B) Expression of constitutively active Asef mutant (ΔAPC-Asef). (C) Expression of dominant-negative Asef mutant (ΔDH-Asef). Inset, higher-magnification image of cell edge, depicting inhibition of HGF-induced formation of lamellipodia-like structures by expression of dominant-negative Asef mutant. Arrows show cells with activated peripheral actin cytoskeletal remodeling. Bar, 5 μm. (D) EC monolayers were transiently transfected with wild-type Asef or dominant-negative Asef mutant, and HGF-induced Rac1 activation was determined by Rac-GTP pull-down assay. The content of activated Rac1 was normalized to the total Rac1 content in EC lysates; *p < 0.01 vs. nonstimulated controls transfected with Asef-WT; **p < 0.05 vs. Asef-WT.
Mentions: As a complementary approach to experiments with Asef knockdown, we performed ectopic expression of HA-tagged wild-type Asef or its constitutively activated (CA-Asef) or dominant-negative (DN-Asef) mutant (Kawasaki et al., 2003). Overexpression of wild-type Asef further enhanced EC peripheral actin remodeling in response to HGF in comparison to HGF-stimulated nontransfected cells (Figure 5A). In turn, expression of activated Asef mutant in nonstimulated cells reproduced peripheral actin cytoskeletal rearrangement observed in nontransfected ECs treated with HGF (Figure 5B). Similar to Asef knockdown experiments, the expression of dominant-negative Asef abrogated HGF-induced formation of lamellipodia-like structures and peripheral actin rim (Figure 5C). Expression of dominant-negative Asef mutant suppressed the HGF-induced activation of Rac1 GTPase in pulmonary ECs (Figure 5D).

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