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Caveolin-1-eNOS signaling promotes p190RhoGAP-A nitration and endothelial permeability.

Siddiqui MR, Komarova YA, Vogel SM, Gao X, Bonini MG, Rajasingh J, Zhao YY, Brovkovych V, Malik AB - J. Cell Biol. (2011)

Bottom Line: We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation.Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A.Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.

ABSTRACT
Endothelial barrier function is regulated by adherens junctions (AJs) and caveolae-mediated transcellular pathways. The opening of AJs that is observed in caveolin-1(-/-) (Cav-1(-/-)) endothelium suggests that Cav-1 is necessary for AJ assembly or maintenance. Here, using endothelial cells isolated from Cav-1(-/-) mice, we show that Cav-1 deficiency induced the activation of endothelial nitric oxide synthase (eNOS) and the generation of nitric oxide (NO) and peroxynitrite. We assessed S-nitrosylation and nitration of AJ-associated proteins to identify downstream NO redox signaling targets. We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation. Inhibition of eNOS or RhoA restored AJ integrity and diminished endothelial hyperpermeability in Cav-1(-/-) mice. Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A. Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.

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Cav-1 deficiency induces eNOS activation and AJ destabilization in endothelium. (a) Phosphorylation of eNOS at S1177 and Akt-1 at S473 in Wt and Cav-1−/− MLVECs; n = 3. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) Basal NO generation was measured in the absence or presence of l-NNA or NPA and 1400W in combination. Arrow, l-arginine replenishment. Scale bars are shown for 100 nM NO and 10 min. Bar plot, NO accumulation in a media over 20 min; mean ± SEM (error bars); *, P < 0.05 as compared with Wt control; n = 6. (c) Wt and Cav-1−/− endothelial monolayers stained for VE-cadherin, β-catenin (green in overlays), F-actin (red), and nuclei (blue). Inter-endothelial gaps in Cav-1−/− monolayers are indicated by arrowheads. Bar, 10 µm. (d and e) Accumulation of β-catenin at AJs was expressed as a mean pixel intensity of threshold area shown in d (threshold above intracellular background is in orange). (f) Area of inter-endothelial gaps was determined using the same set of images as in e; n = 17. Black circles and error bars in e and f indicate mean and SEM, respectively; *, P < 0.01 as compared with Wt control.
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fig1: Cav-1 deficiency induces eNOS activation and AJ destabilization in endothelium. (a) Phosphorylation of eNOS at S1177 and Akt-1 at S473 in Wt and Cav-1−/− MLVECs; n = 3. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) Basal NO generation was measured in the absence or presence of l-NNA or NPA and 1400W in combination. Arrow, l-arginine replenishment. Scale bars are shown for 100 nM NO and 10 min. Bar plot, NO accumulation in a media over 20 min; mean ± SEM (error bars); *, P < 0.05 as compared with Wt control; n = 6. (c) Wt and Cav-1−/− endothelial monolayers stained for VE-cadherin, β-catenin (green in overlays), F-actin (red), and nuclei (blue). Inter-endothelial gaps in Cav-1−/− monolayers are indicated by arrowheads. Bar, 10 µm. (d and e) Accumulation of β-catenin at AJs was expressed as a mean pixel intensity of threshold area shown in d (threshold above intracellular background is in orange). (f) Area of inter-endothelial gaps was determined using the same set of images as in e; n = 17. Black circles and error bars in e and f indicate mean and SEM, respectively; *, P < 0.01 as compared with Wt control.

Mentions: We first determined basal eNOS activity in murine lung vascular endothelial cells (MLVECs) isolated from wild-type (Wt) and Cav-1−/− mice (Fig. S1). We observed an approximately twofold increase in eNOS phosphorylation at S1177 (Fig. 1 a), the Akt-1 phosphorylation site known to regulate enzymatic activity of eNOS (Dimmeler et al., 1999). Phosphorylation of Akt-1 at S473 was also increased (Fig. 1 a), which indicates marked Akt-1 activation in Cav-1−/− endothelium. Basal nitric oxide (NO) generation determined by l-arginine replenishment was fourfold greater in Cav-1−/− MLVECs (Fig. 1 b). This finding is consistent with the reported increased plasma NO concentration in Cav-1–deficient mice (Miyawaki-Shimizu et al., 2006). Nitro-l-arginine (l-NNA) treatment at an inhibitory concentration for nNOS, and eNOS prevented NO generation, whereas treatment with 1400W and N-propyl-l-arginine (NPA) in combination (at inhibitory concentrations for iNOS and nNOS) had a marginal effect (Fig. 1 b). We concluded that Cav-1 deficiency in the endothelium resulted in Akt-1 and eNOS activation and an augmented NO production, which is in agreement with the role of Cav-1 in inhibiting eNOS activity in endothelium (García-Cardeña et al., 1997).


Caveolin-1-eNOS signaling promotes p190RhoGAP-A nitration and endothelial permeability.

Siddiqui MR, Komarova YA, Vogel SM, Gao X, Bonini MG, Rajasingh J, Zhao YY, Brovkovych V, Malik AB - J. Cell Biol. (2011)

Cav-1 deficiency induces eNOS activation and AJ destabilization in endothelium. (a) Phosphorylation of eNOS at S1177 and Akt-1 at S473 in Wt and Cav-1−/− MLVECs; n = 3. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) Basal NO generation was measured in the absence or presence of l-NNA or NPA and 1400W in combination. Arrow, l-arginine replenishment. Scale bars are shown for 100 nM NO and 10 min. Bar plot, NO accumulation in a media over 20 min; mean ± SEM (error bars); *, P < 0.05 as compared with Wt control; n = 6. (c) Wt and Cav-1−/− endothelial monolayers stained for VE-cadherin, β-catenin (green in overlays), F-actin (red), and nuclei (blue). Inter-endothelial gaps in Cav-1−/− monolayers are indicated by arrowheads. Bar, 10 µm. (d and e) Accumulation of β-catenin at AJs was expressed as a mean pixel intensity of threshold area shown in d (threshold above intracellular background is in orange). (f) Area of inter-endothelial gaps was determined using the same set of images as in e; n = 17. Black circles and error bars in e and f indicate mean and SEM, respectively; *, P < 0.01 as compared with Wt control.
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fig1: Cav-1 deficiency induces eNOS activation and AJ destabilization in endothelium. (a) Phosphorylation of eNOS at S1177 and Akt-1 at S473 in Wt and Cav-1−/− MLVECs; n = 3. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) Basal NO generation was measured in the absence or presence of l-NNA or NPA and 1400W in combination. Arrow, l-arginine replenishment. Scale bars are shown for 100 nM NO and 10 min. Bar plot, NO accumulation in a media over 20 min; mean ± SEM (error bars); *, P < 0.05 as compared with Wt control; n = 6. (c) Wt and Cav-1−/− endothelial monolayers stained for VE-cadherin, β-catenin (green in overlays), F-actin (red), and nuclei (blue). Inter-endothelial gaps in Cav-1−/− monolayers are indicated by arrowheads. Bar, 10 µm. (d and e) Accumulation of β-catenin at AJs was expressed as a mean pixel intensity of threshold area shown in d (threshold above intracellular background is in orange). (f) Area of inter-endothelial gaps was determined using the same set of images as in e; n = 17. Black circles and error bars in e and f indicate mean and SEM, respectively; *, P < 0.01 as compared with Wt control.
Mentions: We first determined basal eNOS activity in murine lung vascular endothelial cells (MLVECs) isolated from wild-type (Wt) and Cav-1−/− mice (Fig. S1). We observed an approximately twofold increase in eNOS phosphorylation at S1177 (Fig. 1 a), the Akt-1 phosphorylation site known to regulate enzymatic activity of eNOS (Dimmeler et al., 1999). Phosphorylation of Akt-1 at S473 was also increased (Fig. 1 a), which indicates marked Akt-1 activation in Cav-1−/− endothelium. Basal nitric oxide (NO) generation determined by l-arginine replenishment was fourfold greater in Cav-1−/− MLVECs (Fig. 1 b). This finding is consistent with the reported increased plasma NO concentration in Cav-1–deficient mice (Miyawaki-Shimizu et al., 2006). Nitro-l-arginine (l-NNA) treatment at an inhibitory concentration for nNOS, and eNOS prevented NO generation, whereas treatment with 1400W and N-propyl-l-arginine (NPA) in combination (at inhibitory concentrations for iNOS and nNOS) had a marginal effect (Fig. 1 b). We concluded that Cav-1 deficiency in the endothelium resulted in Akt-1 and eNOS activation and an augmented NO production, which is in agreement with the role of Cav-1 in inhibiting eNOS activity in endothelium (García-Cardeña et al., 1997).

Bottom Line: We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation.Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A.Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.

ABSTRACT
Endothelial barrier function is regulated by adherens junctions (AJs) and caveolae-mediated transcellular pathways. The opening of AJs that is observed in caveolin-1(-/-) (Cav-1(-/-)) endothelium suggests that Cav-1 is necessary for AJ assembly or maintenance. Here, using endothelial cells isolated from Cav-1(-/-) mice, we show that Cav-1 deficiency induced the activation of endothelial nitric oxide synthase (eNOS) and the generation of nitric oxide (NO) and peroxynitrite. We assessed S-nitrosylation and nitration of AJ-associated proteins to identify downstream NO redox signaling targets. We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation. Inhibition of eNOS or RhoA restored AJ integrity and diminished endothelial hyperpermeability in Cav-1(-/-) mice. Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A. Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.

Show MeSH
Related in: MedlinePlus