Limits...
The G protein betagamma subunit mediates reannealing of adherens junctions to reverse endothelial permeability increase by thrombin.

Knezevic N, Tauseef M, Thennes T, Mehta D - J. Exp. Med. (2009)

Bottom Line: We now show that impairment of Gbetagamma function does not affect the permeability increase induced by PAR1, but prevents reannealing of adherens junctions (AJ), thereby persistently elevating endothelial permeability.Activated FAK associated with AJ and stimulated AJ reassembly in a Fyn-dependent manner.Our results demonstrate that Gbeta1-mediated Fyn activation integrates FAK with AJ, preventing persistent endothelial barrier leakiness.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Lung and Vascular Biology, Department of Pharmacology, University of Illinois, Chicago, IL 60612, USA.

ABSTRACT
The inflammatory mediator thrombin proteolytically activates protease-activated receptor (PAR1) eliciting a transient, but reversible increase in vascular permeability. PAR1-induced dissociation of Galpha subunit from heterotrimeric Gq and G12/G13 proteins is known to signal the increase in endothelial permeability. However, the role of released Gbetagamma is unknown. We now show that impairment of Gbetagamma function does not affect the permeability increase induced by PAR1, but prevents reannealing of adherens junctions (AJ), thereby persistently elevating endothelial permeability. We observed that in the naive endothelium Gbeta1, the predominant Gbeta isoform is sequestered by receptor for activated C kinase 1 (RACK1). Thrombin induced dissociation of Gbeta1 from RACK1, resulting in Gbeta1 interaction with Fyn and focal adhesion kinase (FAK) required for FAK activation. RACK1 depletion triggered Gbeta1 activation of FAK and endothelial barrier recovery, whereas Fyn knockdown interrupted with Gbeta1-induced barrier recovery indicating RACK1 negatively regulates Gbeta1-Fyn signaling. Activated FAK associated with AJ and stimulated AJ reassembly in a Fyn-dependent manner. Fyn deletion prevented FAK activation and augmented lung vascular permeability increase induced by PAR1 agonist. Rescuing FAK activation in fyn(-/-) mice attenuated the rise in lung vascular permeability. Our results demonstrate that Gbeta1-mediated Fyn activation integrates FAK with AJ, preventing persistent endothelial barrier leakiness.

Show MeSH

Related in: MedlinePlus

Fyn is required for limiting the increase in lung microvascular permeability. (A and B) Fyn deletion prevents PAR1 activation of FAK phosphorylation. Lungs from C57BL/6 (WT) and fyn −/− mice receiving control or PAR1 peptide (1 mg/kg) for indicated times were homogenized, and proteins immunoblotted with anti–phospho-Y397-FAK, anti–phospho-Y576-FAK, and anti-FAK Abs (A) or and anti–phospho-Y419Src and anti-cSrc Ab (B) to assess FAK (A) and Src (B) phosphorylation. Immunoblot with anti-Fyn and anti-cSrc show that deletion of Fyn (A) did not alter the expression of cSrc (B). Data representative of three independent experiments. (C) Changes in lung microvascular permeability in fyn−/− mice. In isogravimetric lungs intravascular pressure was raised by 10 cm H2O for 20 min and microvessel filtration coefficient (Kf) was calculated from the slope of the weight gain function normalized by lung dry weight, as described in Materials and methods. Plot gives mean ± SD of four experiments. In each experiment fyn −/− lung was paired with WT lung. * indicates significance (P < 0.05). (D–E) PAR1 agonist peptide augments the increase in lung microvascular permeability in fyn−/− mice. PAR-1 peptide (1 mg/kg) or control peptide was injected together with Evans blue albumin retroorbitally into mice (WT, grey column; fyn−/−, black bar). After 30 min, Evans blue albumin extravasation (EBAE) from lungs or plasma (D) and lung wet/dry weight ratio (E) were determined as described in Materials and methods to quantify lung microvascular protein permeability and lung edema formation, respectively. Data represent mean ± SD of four individual experiments where fyn −/− lungs were paired with WT lungs. * indicates significance from appropriate control peptide group (P < 0.05); # indicates significance from WT mice after PAR1 challenge (P < 0.05).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2806626&req=5

fig7: Fyn is required for limiting the increase in lung microvascular permeability. (A and B) Fyn deletion prevents PAR1 activation of FAK phosphorylation. Lungs from C57BL/6 (WT) and fyn −/− mice receiving control or PAR1 peptide (1 mg/kg) for indicated times were homogenized, and proteins immunoblotted with anti–phospho-Y397-FAK, anti–phospho-Y576-FAK, and anti-FAK Abs (A) or and anti–phospho-Y419Src and anti-cSrc Ab (B) to assess FAK (A) and Src (B) phosphorylation. Immunoblot with anti-Fyn and anti-cSrc show that deletion of Fyn (A) did not alter the expression of cSrc (B). Data representative of three independent experiments. (C) Changes in lung microvascular permeability in fyn−/− mice. In isogravimetric lungs intravascular pressure was raised by 10 cm H2O for 20 min and microvessel filtration coefficient (Kf) was calculated from the slope of the weight gain function normalized by lung dry weight, as described in Materials and methods. Plot gives mean ± SD of four experiments. In each experiment fyn −/− lung was paired with WT lung. * indicates significance (P < 0.05). (D–E) PAR1 agonist peptide augments the increase in lung microvascular permeability in fyn−/− mice. PAR-1 peptide (1 mg/kg) or control peptide was injected together with Evans blue albumin retroorbitally into mice (WT, grey column; fyn−/−, black bar). After 30 min, Evans blue albumin extravasation (EBAE) from lungs or plasma (D) and lung wet/dry weight ratio (E) were determined as described in Materials and methods to quantify lung microvascular protein permeability and lung edema formation, respectively. Data represent mean ± SD of four individual experiments where fyn −/− lungs were paired with WT lungs. * indicates significance from appropriate control peptide group (P < 0.05); # indicates significance from WT mice after PAR1 challenge (P < 0.05).

Mentions: In another series of experiments, we investigated the role of Fyn in regulating endothelial barrier integrity of pulmonary microvessels using Fyn-deficient mice. First we determined whether activation of PAR1 by the specific activating peptide TFLLRN alters FAK activity in lungs. WT or fyn−/− mice received i.v. injection of either control peptide or PAR1-activating peptide (1 mg/kg). Lungs were homogenized to assess FAK phosphorylation. We observed that PAR1 agonist peptide increased FAK phosphorylation at tyrosine 397 and 576 residues in WT lungs (Fig. 7 A). However, in lungs of fyn−/− mice, FAK phosphorylation at tyrosine 397 and 576 was barely detectable under basal conditions and did not increase after PAR1 activation (Fig. 7 A), which is consistent with our findings in Fyn-depleted endothelial cells. Deletion of Fyn had no effect on either cSrc expression or cSrc phosphorylation induced by PAR1 (Fig. 7 B), implicating Fyn as the key enzyme regulating FAK activation.


The G protein betagamma subunit mediates reannealing of adherens junctions to reverse endothelial permeability increase by thrombin.

Knezevic N, Tauseef M, Thennes T, Mehta D - J. Exp. Med. (2009)

Fyn is required for limiting the increase in lung microvascular permeability. (A and B) Fyn deletion prevents PAR1 activation of FAK phosphorylation. Lungs from C57BL/6 (WT) and fyn −/− mice receiving control or PAR1 peptide (1 mg/kg) for indicated times were homogenized, and proteins immunoblotted with anti–phospho-Y397-FAK, anti–phospho-Y576-FAK, and anti-FAK Abs (A) or and anti–phospho-Y419Src and anti-cSrc Ab (B) to assess FAK (A) and Src (B) phosphorylation. Immunoblot with anti-Fyn and anti-cSrc show that deletion of Fyn (A) did not alter the expression of cSrc (B). Data representative of three independent experiments. (C) Changes in lung microvascular permeability in fyn−/− mice. In isogravimetric lungs intravascular pressure was raised by 10 cm H2O for 20 min and microvessel filtration coefficient (Kf) was calculated from the slope of the weight gain function normalized by lung dry weight, as described in Materials and methods. Plot gives mean ± SD of four experiments. In each experiment fyn −/− lung was paired with WT lung. * indicates significance (P < 0.05). (D–E) PAR1 agonist peptide augments the increase in lung microvascular permeability in fyn−/− mice. PAR-1 peptide (1 mg/kg) or control peptide was injected together with Evans blue albumin retroorbitally into mice (WT, grey column; fyn−/−, black bar). After 30 min, Evans blue albumin extravasation (EBAE) from lungs or plasma (D) and lung wet/dry weight ratio (E) were determined as described in Materials and methods to quantify lung microvascular protein permeability and lung edema formation, respectively. Data represent mean ± SD of four individual experiments where fyn −/− lungs were paired with WT lungs. * indicates significance from appropriate control peptide group (P < 0.05); # indicates significance from WT mice after PAR1 challenge (P < 0.05).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806626&req=5

fig7: Fyn is required for limiting the increase in lung microvascular permeability. (A and B) Fyn deletion prevents PAR1 activation of FAK phosphorylation. Lungs from C57BL/6 (WT) and fyn −/− mice receiving control or PAR1 peptide (1 mg/kg) for indicated times were homogenized, and proteins immunoblotted with anti–phospho-Y397-FAK, anti–phospho-Y576-FAK, and anti-FAK Abs (A) or and anti–phospho-Y419Src and anti-cSrc Ab (B) to assess FAK (A) and Src (B) phosphorylation. Immunoblot with anti-Fyn and anti-cSrc show that deletion of Fyn (A) did not alter the expression of cSrc (B). Data representative of three independent experiments. (C) Changes in lung microvascular permeability in fyn−/− mice. In isogravimetric lungs intravascular pressure was raised by 10 cm H2O for 20 min and microvessel filtration coefficient (Kf) was calculated from the slope of the weight gain function normalized by lung dry weight, as described in Materials and methods. Plot gives mean ± SD of four experiments. In each experiment fyn −/− lung was paired with WT lung. * indicates significance (P < 0.05). (D–E) PAR1 agonist peptide augments the increase in lung microvascular permeability in fyn−/− mice. PAR-1 peptide (1 mg/kg) or control peptide was injected together with Evans blue albumin retroorbitally into mice (WT, grey column; fyn−/−, black bar). After 30 min, Evans blue albumin extravasation (EBAE) from lungs or plasma (D) and lung wet/dry weight ratio (E) were determined as described in Materials and methods to quantify lung microvascular protein permeability and lung edema formation, respectively. Data represent mean ± SD of four individual experiments where fyn −/− lungs were paired with WT lungs. * indicates significance from appropriate control peptide group (P < 0.05); # indicates significance from WT mice after PAR1 challenge (P < 0.05).
Mentions: In another series of experiments, we investigated the role of Fyn in regulating endothelial barrier integrity of pulmonary microvessels using Fyn-deficient mice. First we determined whether activation of PAR1 by the specific activating peptide TFLLRN alters FAK activity in lungs. WT or fyn−/− mice received i.v. injection of either control peptide or PAR1-activating peptide (1 mg/kg). Lungs were homogenized to assess FAK phosphorylation. We observed that PAR1 agonist peptide increased FAK phosphorylation at tyrosine 397 and 576 residues in WT lungs (Fig. 7 A). However, in lungs of fyn−/− mice, FAK phosphorylation at tyrosine 397 and 576 was barely detectable under basal conditions and did not increase after PAR1 activation (Fig. 7 A), which is consistent with our findings in Fyn-depleted endothelial cells. Deletion of Fyn had no effect on either cSrc expression or cSrc phosphorylation induced by PAR1 (Fig. 7 B), implicating Fyn as the key enzyme regulating FAK activation.

Bottom Line: We now show that impairment of Gbetagamma function does not affect the permeability increase induced by PAR1, but prevents reannealing of adherens junctions (AJ), thereby persistently elevating endothelial permeability.Activated FAK associated with AJ and stimulated AJ reassembly in a Fyn-dependent manner.Our results demonstrate that Gbeta1-mediated Fyn activation integrates FAK with AJ, preventing persistent endothelial barrier leakiness.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Lung and Vascular Biology, Department of Pharmacology, University of Illinois, Chicago, IL 60612, USA.

ABSTRACT
The inflammatory mediator thrombin proteolytically activates protease-activated receptor (PAR1) eliciting a transient, but reversible increase in vascular permeability. PAR1-induced dissociation of Galpha subunit from heterotrimeric Gq and G12/G13 proteins is known to signal the increase in endothelial permeability. However, the role of released Gbetagamma is unknown. We now show that impairment of Gbetagamma function does not affect the permeability increase induced by PAR1, but prevents reannealing of adherens junctions (AJ), thereby persistently elevating endothelial permeability. We observed that in the naive endothelium Gbeta1, the predominant Gbeta isoform is sequestered by receptor for activated C kinase 1 (RACK1). Thrombin induced dissociation of Gbeta1 from RACK1, resulting in Gbeta1 interaction with Fyn and focal adhesion kinase (FAK) required for FAK activation. RACK1 depletion triggered Gbeta1 activation of FAK and endothelial barrier recovery, whereas Fyn knockdown interrupted with Gbeta1-induced barrier recovery indicating RACK1 negatively regulates Gbeta1-Fyn signaling. Activated FAK associated with AJ and stimulated AJ reassembly in a Fyn-dependent manner. Fyn deletion prevented FAK activation and augmented lung vascular permeability increase induced by PAR1 agonist. Rescuing FAK activation in fyn(-/-) mice attenuated the rise in lung vascular permeability. Our results demonstrate that Gbeta1-mediated Fyn activation integrates FAK with AJ, preventing persistent endothelial barrier leakiness.

Show MeSH
Related in: MedlinePlus