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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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HGF induces activation of Asef and Rac1. (A–D) HPAECs were stimulated with HGF (50 ng/ml) for the indicated periods of time. (A) Rac1 activation was determined by Rac-GTP pull-down assay. Content of activated Rac1 was normalized to the total Rac1 content in EC lysates. *p < 0.05 vs. vehicle. (B) Asef activation was determined by pull-down assay with immobilized Rac1G15A and evaluated by increased Asef association with Rac1G15A. Content of activated Asef was normalized to the total Asef content in EC lysates. *p < 0.05 vs. vehicle. (C) HPAECs were preincubated with vehicle or c-Met inhibitor (carboxamide 50 nM, 30 min), followed by stimulation with HGF (2 and 5 min). Asef activation was determined by pull-down assay with immobilized Rac1G15A and normalized to the total Asef. *p < 0.05 vs. cMet inh + HGF. (D) HPAECs were stimulated with HGF, followed by isolation of cytosolic and membrane fractions. Time-dependent, HGF-induced accumulation of Asef in membrane fraction was detected with specific antibodies. Asef content in corresponding total cell lysates was used as a normalization control. *p <0.05 vs. vehicle. (E) Endothelial cells grown on glass coverslips were stimulated with HGF (50 ng/ml, 30 min). Intracellular redistribution of endogenous Asef was examined by immunofluorescence staining with Asef antibody. Representative results of three to five independent experiments. Bar, 5 μm.
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Figure 1: HGF induces activation of Asef and Rac1. (A–D) HPAECs were stimulated with HGF (50 ng/ml) for the indicated periods of time. (A) Rac1 activation was determined by Rac-GTP pull-down assay. Content of activated Rac1 was normalized to the total Rac1 content in EC lysates. *p < 0.05 vs. vehicle. (B) Asef activation was determined by pull-down assay with immobilized Rac1G15A and evaluated by increased Asef association with Rac1G15A. Content of activated Asef was normalized to the total Asef content in EC lysates. *p < 0.05 vs. vehicle. (C) HPAECs were preincubated with vehicle or c-Met inhibitor (carboxamide 50 nM, 30 min), followed by stimulation with HGF (2 and 5 min). Asef activation was determined by pull-down assay with immobilized Rac1G15A and normalized to the total Asef. *p < 0.05 vs. cMet inh + HGF. (D) HPAECs were stimulated with HGF, followed by isolation of cytosolic and membrane fractions. Time-dependent, HGF-induced accumulation of Asef in membrane fraction was detected with specific antibodies. Asef content in corresponding total cell lysates was used as a normalization control. *p <0.05 vs. vehicle. (E) Endothelial cells grown on glass coverslips were stimulated with HGF (50 ng/ml, 30 min). Intracellular redistribution of endogenous Asef was examined by immunofluorescence staining with Asef antibody. Representative results of three to five independent experiments. Bar, 5 μm.

Mentions: The HGF-induced endothelial barrier protective response is associated with activation of Rac1 GTPase signaling, but the precise mechanisms of Rac1 activation remain elusive. We found that HGF induced activation of Rac1 (Figure 1A), which was associated with rapid stimulation of Asef nucleotide exchange activity toward Rac1 observed at 2–10 min after HGF addition (Figure 1B). Western blot analysis showed comparable levels of Asef expression in both human lung microvascular and pulmonary artery EC cultures. These data suggest the role for Asef-dependent signaling mechanisms in ECs from different vascular beds. HGF-induced activation of Asef guanine nucleotide exchange activity toward Rac1 was abrogated by cell pretreatment with the pharmacological inhibitor of HGF receptor c-Met (Figure 1C). Activation of Asef by HGF also led to Asef translocation to the cell membrane fraction (Figure 1D), with preferential accumulation at the cell periphery as visualized by immunofluorescence staining with Asef antibody (Figure 1E).


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)

HGF induces activation of Asef and Rac1. (A–D) HPAECs were stimulated with HGF (50 ng/ml) for the indicated periods of time. (A) Rac1 activation was determined by Rac-GTP pull-down assay. Content of activated Rac1 was normalized to the total Rac1 content in EC lysates. *p < 0.05 vs. vehicle. (B) Asef activation was determined by pull-down assay with immobilized Rac1G15A and evaluated by increased Asef association with Rac1G15A. Content of activated Asef was normalized to the total Asef content in EC lysates. *p < 0.05 vs. vehicle. (C) HPAECs were preincubated with vehicle or c-Met inhibitor (carboxamide 50 nM, 30 min), followed by stimulation with HGF (2 and 5 min). Asef activation was determined by pull-down assay with immobilized Rac1G15A and normalized to the total Asef. *p < 0.05 vs. cMet inh + HGF. (D) HPAECs were stimulated with HGF, followed by isolation of cytosolic and membrane fractions. Time-dependent, HGF-induced accumulation of Asef in membrane fraction was detected with specific antibodies. Asef content in corresponding total cell lysates was used as a normalization control. *p <0.05 vs. vehicle. (E) Endothelial cells grown on glass coverslips were stimulated with HGF (50 ng/ml, 30 min). Intracellular redistribution of endogenous Asef was examined by immunofluorescence staining with Asef antibody. Representative results of three to five independent experiments. Bar, 5 μm.
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Figure 1: HGF induces activation of Asef and Rac1. (A–D) HPAECs were stimulated with HGF (50 ng/ml) for the indicated periods of time. (A) Rac1 activation was determined by Rac-GTP pull-down assay. Content of activated Rac1 was normalized to the total Rac1 content in EC lysates. *p < 0.05 vs. vehicle. (B) Asef activation was determined by pull-down assay with immobilized Rac1G15A and evaluated by increased Asef association with Rac1G15A. Content of activated Asef was normalized to the total Asef content in EC lysates. *p < 0.05 vs. vehicle. (C) HPAECs were preincubated with vehicle or c-Met inhibitor (carboxamide 50 nM, 30 min), followed by stimulation with HGF (2 and 5 min). Asef activation was determined by pull-down assay with immobilized Rac1G15A and normalized to the total Asef. *p < 0.05 vs. cMet inh + HGF. (D) HPAECs were stimulated with HGF, followed by isolation of cytosolic and membrane fractions. Time-dependent, HGF-induced accumulation of Asef in membrane fraction was detected with specific antibodies. Asef content in corresponding total cell lysates was used as a normalization control. *p <0.05 vs. vehicle. (E) Endothelial cells grown on glass coverslips were stimulated with HGF (50 ng/ml, 30 min). Intracellular redistribution of endogenous Asef was examined by immunofluorescence staining with Asef antibody. Representative results of three to five independent experiments. Bar, 5 μm.
Mentions: The HGF-induced endothelial barrier protective response is associated with activation of Rac1 GTPase signaling, but the precise mechanisms of Rac1 activation remain elusive. We found that HGF induced activation of Rac1 (Figure 1A), which was associated with rapid stimulation of Asef nucleotide exchange activity toward Rac1 observed at 2–10 min after HGF addition (Figure 1B). Western blot analysis showed comparable levels of Asef expression in both human lung microvascular and pulmonary artery EC cultures. These data suggest the role for Asef-dependent signaling mechanisms in ECs from different vascular beds. HGF-induced activation of Asef guanine nucleotide exchange activity toward Rac1 was abrogated by cell pretreatment with the pharmacological inhibitor of HGF receptor c-Met (Figure 1C). Activation of Asef by HGF also led to Asef translocation to the cell membrane fraction (Figure 1D), with preferential accumulation at the cell periphery as visualized by immunofluorescence staining with Asef antibody (Figure 1E).

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