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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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eNOS-dependent NO-redox signaling disrupts AJ integrity. (a) Immunofluorescent staining of HPAECs for eNOS (green) and β-cat (red). DAPI (blue) is shown before and after stimulation with 50 nM α-thrombin. a1 and a2 are enlarged views of boxed regions; inter-endothelial gaps are indicated by arrowheads. Bars, 10 µm. (b) eNOS accumulation at AJs was measured as in Fig. 1 e with some modification (see Materials and methods); *, P < 0.05 as compared with Wt control; n = 6. α-Thrombin induced transient accumulation of eNOS at AJs. (c and d) α-Thrombin induced transient interaction between eNOS and p190A and nitration of p190A. eNOS and p190A were co-immunoprecipitated with specific Abs from HPAECs stimulated with 50 nM α-thrombin. Precipitates were probed for p190A, eNOS, 3-N, and p120-catenin. Molecular mass standards are indicated next to the gel blots in kilodaltons. (e) Model showing that Cav-1–mediated recruitment of eNOS into caveolar domain allows formation of stable endothelial VE-cadherin adhesions, which restricts the permeability of the paracellular route to plasma proteins. Deficiency in Cav-1 or release of eNOS from caveolae results in activation of eNOS, translocation of eNOS to AJs, and transient interaction with p190A. Peroxynitrite, a product of NO and O2•−, inhibits p190A activity by nitrating Y1105. Activation of RhoA leads to reorganization of the actin cytoskeleton, actomyosin contractility, destabilization of AJs, and increased vascular permeability via the paracellular pathway.
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fig5: eNOS-dependent NO-redox signaling disrupts AJ integrity. (a) Immunofluorescent staining of HPAECs for eNOS (green) and β-cat (red). DAPI (blue) is shown before and after stimulation with 50 nM α-thrombin. a1 and a2 are enlarged views of boxed regions; inter-endothelial gaps are indicated by arrowheads. Bars, 10 µm. (b) eNOS accumulation at AJs was measured as in Fig. 1 e with some modification (see Materials and methods); *, P < 0.05 as compared with Wt control; n = 6. α-Thrombin induced transient accumulation of eNOS at AJs. (c and d) α-Thrombin induced transient interaction between eNOS and p190A and nitration of p190A. eNOS and p190A were co-immunoprecipitated with specific Abs from HPAECs stimulated with 50 nM α-thrombin. Precipitates were probed for p190A, eNOS, 3-N, and p120-catenin. Molecular mass standards are indicated next to the gel blots in kilodaltons. (e) Model showing that Cav-1–mediated recruitment of eNOS into caveolar domain allows formation of stable endothelial VE-cadherin adhesions, which restricts the permeability of the paracellular route to plasma proteins. Deficiency in Cav-1 or release of eNOS from caveolae results in activation of eNOS, translocation of eNOS to AJs, and transient interaction with p190A. Peroxynitrite, a product of NO and O2•−, inhibits p190A activity by nitrating Y1105. Activation of RhoA leads to reorganization of the actin cytoskeleton, actomyosin contractility, destabilization of AJs, and increased vascular permeability via the paracellular pathway.

Mentions: The NO redox endothelial permeability-increasing mechanism described in this paper may contribute to deregulation of tissue fluid homeostasis during inflammation, which is known to be accompanied by loss of p190A activity and activation of RhoA (Mammoto et al., 2007). Therefore, we determined whether stimulation of endothelial cell monolayers with a pro-inflammatory mediator such as the serine protease thrombin induces p190A nitration. Thrombin proteolytically cleaves and activates the protease-activated receptor PAR-1 on the endothelial surface, leading to increased intracellular calcium concentration and activation of eNOS (Motley et al., 2007). Challenge of human pulmonary artery endothelial cells (HPAECs) with α-thrombin resulted in translocation of eNOS to AJs and a transient interaction between eNOS and p190A and nitration of p190A (Fig. 5, a–d). Nitrated p190A also remained bound to p120-catenin (Fig. 5 d). Thus, we suggest that the signaling mechanism responsible for increased vascular permeability described in this study may be important in inflammatory diseases, and hence we propose it represents a novel anti-inflammatory therapeutic target.


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)

eNOS-dependent NO-redox signaling disrupts AJ integrity. (a) Immunofluorescent staining of HPAECs for eNOS (green) and β-cat (red). DAPI (blue) is shown before and after stimulation with 50 nM α-thrombin. a1 and a2 are enlarged views of boxed regions; inter-endothelial gaps are indicated by arrowheads. Bars, 10 µm. (b) eNOS accumulation at AJs was measured as in Fig. 1 e with some modification (see Materials and methods); *, P < 0.05 as compared with Wt control; n = 6. α-Thrombin induced transient accumulation of eNOS at AJs. (c and d) α-Thrombin induced transient interaction between eNOS and p190A and nitration of p190A. eNOS and p190A were co-immunoprecipitated with specific Abs from HPAECs stimulated with 50 nM α-thrombin. Precipitates were probed for p190A, eNOS, 3-N, and p120-catenin. Molecular mass standards are indicated next to the gel blots in kilodaltons. (e) Model showing that Cav-1–mediated recruitment of eNOS into caveolar domain allows formation of stable endothelial VE-cadherin adhesions, which restricts the permeability of the paracellular route to plasma proteins. Deficiency in Cav-1 or release of eNOS from caveolae results in activation of eNOS, translocation of eNOS to AJs, and transient interaction with p190A. Peroxynitrite, a product of NO and O2•−, inhibits p190A activity by nitrating Y1105. Activation of RhoA leads to reorganization of the actin cytoskeleton, actomyosin contractility, destabilization of AJs, and increased vascular permeability via the paracellular pathway.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105546&req=5

fig5: eNOS-dependent NO-redox signaling disrupts AJ integrity. (a) Immunofluorescent staining of HPAECs for eNOS (green) and β-cat (red). DAPI (blue) is shown before and after stimulation with 50 nM α-thrombin. a1 and a2 are enlarged views of boxed regions; inter-endothelial gaps are indicated by arrowheads. Bars, 10 µm. (b) eNOS accumulation at AJs was measured as in Fig. 1 e with some modification (see Materials and methods); *, P < 0.05 as compared with Wt control; n = 6. α-Thrombin induced transient accumulation of eNOS at AJs. (c and d) α-Thrombin induced transient interaction between eNOS and p190A and nitration of p190A. eNOS and p190A were co-immunoprecipitated with specific Abs from HPAECs stimulated with 50 nM α-thrombin. Precipitates were probed for p190A, eNOS, 3-N, and p120-catenin. Molecular mass standards are indicated next to the gel blots in kilodaltons. (e) Model showing that Cav-1–mediated recruitment of eNOS into caveolar domain allows formation of stable endothelial VE-cadherin adhesions, which restricts the permeability of the paracellular route to plasma proteins. Deficiency in Cav-1 or release of eNOS from caveolae results in activation of eNOS, translocation of eNOS to AJs, and transient interaction with p190A. Peroxynitrite, a product of NO and O2•−, inhibits p190A activity by nitrating Y1105. Activation of RhoA leads to reorganization of the actin cytoskeleton, actomyosin contractility, destabilization of AJs, and increased vascular permeability via the paracellular pathway.
Mentions: The NO redox endothelial permeability-increasing mechanism described in this paper may contribute to deregulation of tissue fluid homeostasis during inflammation, which is known to be accompanied by loss of p190A activity and activation of RhoA (Mammoto et al., 2007). Therefore, we determined whether stimulation of endothelial cell monolayers with a pro-inflammatory mediator such as the serine protease thrombin induces p190A nitration. Thrombin proteolytically cleaves and activates the protease-activated receptor PAR-1 on the endothelial surface, leading to increased intracellular calcium concentration and activation of eNOS (Motley et al., 2007). Challenge of human pulmonary artery endothelial cells (HPAECs) with α-thrombin resulted in translocation of eNOS to AJs and a transient interaction between eNOS and p190A and nitration of p190A (Fig. 5, a–d). Nitrated p190A also remained bound to p120-catenin (Fig. 5 d). Thus, we suggest that the signaling mechanism responsible for increased vascular permeability described in this study may be important in inflammatory diseases, and hence we propose it represents a novel anti-inflammatory therapeutic target.

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