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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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Peroxynitrite generation in Cav-1−/− endothelium and nitration of p190A. (a) S-nitrosylation of β- and p120-catenins, and p190A. Proteins were co-immunoprecipitated with specific Abs, and precipitates were probed with HRP-avidin; results of two independent experiments are shown; n = 4. Note that the β-catenin band is indicated by an arrow. SNO of β-catenin in VEGF-treated cells is a positive control. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) The bar plot shows nitrite accumulation in the presence of SOD and TEMPOL; mean ± SEM (error bars); *, P < 0.05 as compared with Wt control; n = 4. (c) Bar plot shows the fluorescence emission of EOH at 580 nm; mean ± SEM (error bars); *, P < 0.05 as compared with Wt control; n = 4. (d) β- and p120-catenins and (e) p190A were co-immunoprecipitated with specific Abs, and precipitates were probed for nitrotyrosine (3-N). Results of two independent experiments are shown; n = 4. (e) Cav-1 deletion induced the nitration of p190A, which was reduced by TEMPOL treatment and in cells isolated from lungs of Cav-1/eNOS double knockout mice (DKO). (e, right) Bar plot; fold increase in p190A nitration normalized to p190A loading; mean ± SEM (error bars); *, P < 0.05 as compared with Wt; n = 4.
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fig2: Peroxynitrite generation in Cav-1−/− endothelium and nitration of p190A. (a) S-nitrosylation of β- and p120-catenins, and p190A. Proteins were co-immunoprecipitated with specific Abs, and precipitates were probed with HRP-avidin; results of two independent experiments are shown; n = 4. Note that the β-catenin band is indicated by an arrow. SNO of β-catenin in VEGF-treated cells is a positive control. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) The bar plot shows nitrite accumulation in the presence of SOD and TEMPOL; mean ± SEM (error bars); *, P < 0.05 as compared with Wt control; n = 4. (c) Bar plot shows the fluorescence emission of EOH at 580 nm; mean ± SEM (error bars); *, P < 0.05 as compared with Wt control; n = 4. (d) β- and p120-catenins and (e) p190A were co-immunoprecipitated with specific Abs, and precipitates were probed for nitrotyrosine (3-N). Results of two independent experiments are shown; n = 4. (e) Cav-1 deletion induced the nitration of p190A, which was reduced by TEMPOL treatment and in cells isolated from lungs of Cav-1/eNOS double knockout mice (DKO). (e, right) Bar plot; fold increase in p190A nitration normalized to p190A loading; mean ± SEM (error bars); *, P < 0.05 as compared with Wt; n = 4.

Mentions: To address mechanisms of eNOS modulation of AJ integrity, we determined the effects of NO on modification of AJ proteins. NO can lead to S-nitrosylation of cysteine, the covalent attachment of a NO group to the thiol side chain (Lima et al., 2010), and thus influence protein function. S-nitrosylation of β-catenin was shown to facilitate destabilization of AJs in response to VEGF signaling (Thibeault et al., 2010). Therefore, we determined S-nitrosothiol (SNO) content of β- and p120-catenins and of p190RhoGAP-A (referred to as p190A), a binding partner of p120-catenin (Wildenberg et al., 2006). We reasoned that the apparent organization of actin cytoskeleton into stress fibers in Cav-1−/− MLVECs (Fig. 1 c) might result from insufficient control of RhoA activity. p190A, a GTPase-activating protein (GAP), facilitates RhoA-GTP hydrolysis (Tatsis et al., 1998) and is a major regulator of RhoA activity at the level of AJs (Mammoto et al., 2007). SNO content, determined by the biotin-switch assay (Jaffrey and Snyder, 2001), of AJ proteins was indistinguishable between Cav-1−/− and Wt endothelia, although in a control experiment we could detect SNO of β-catenin in VEGF-stimulated endothelial cells (Fig. 2 a). This finding raises the possibility that eNOS-redox signaling regulates integrity of AJs by other means.


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)

Peroxynitrite generation in Cav-1−/− endothelium and nitration of p190A. (a) S-nitrosylation of β- and p120-catenins, and p190A. Proteins were co-immunoprecipitated with specific Abs, and precipitates were probed with HRP-avidin; results of two independent experiments are shown; n = 4. Note that the β-catenin band is indicated by an arrow. SNO of β-catenin in VEGF-treated cells is a positive control. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) The bar plot shows nitrite accumulation in the presence of SOD and TEMPOL; mean ± SEM (error bars); *, P < 0.05 as compared with Wt control; n = 4. (c) Bar plot shows the fluorescence emission of EOH at 580 nm; mean ± SEM (error bars); *, P < 0.05 as compared with Wt control; n = 4. (d) β- and p120-catenins and (e) p190A were co-immunoprecipitated with specific Abs, and precipitates were probed for nitrotyrosine (3-N). Results of two independent experiments are shown; n = 4. (e) Cav-1 deletion induced the nitration of p190A, which was reduced by TEMPOL treatment and in cells isolated from lungs of Cav-1/eNOS double knockout mice (DKO). (e, right) Bar plot; fold increase in p190A nitration normalized to p190A loading; mean ± SEM (error bars); *, P < 0.05 as compared with Wt; n = 4.
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fig2: Peroxynitrite generation in Cav-1−/− endothelium and nitration of p190A. (a) S-nitrosylation of β- and p120-catenins, and p190A. Proteins were co-immunoprecipitated with specific Abs, and precipitates were probed with HRP-avidin; results of two independent experiments are shown; n = 4. Note that the β-catenin band is indicated by an arrow. SNO of β-catenin in VEGF-treated cells is a positive control. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) The bar plot shows nitrite accumulation in the presence of SOD and TEMPOL; mean ± SEM (error bars); *, P < 0.05 as compared with Wt control; n = 4. (c) Bar plot shows the fluorescence emission of EOH at 580 nm; mean ± SEM (error bars); *, P < 0.05 as compared with Wt control; n = 4. (d) β- and p120-catenins and (e) p190A were co-immunoprecipitated with specific Abs, and precipitates were probed for nitrotyrosine (3-N). Results of two independent experiments are shown; n = 4. (e) Cav-1 deletion induced the nitration of p190A, which was reduced by TEMPOL treatment and in cells isolated from lungs of Cav-1/eNOS double knockout mice (DKO). (e, right) Bar plot; fold increase in p190A nitration normalized to p190A loading; mean ± SEM (error bars); *, P < 0.05 as compared with Wt; n = 4.
Mentions: To address mechanisms of eNOS modulation of AJ integrity, we determined the effects of NO on modification of AJ proteins. NO can lead to S-nitrosylation of cysteine, the covalent attachment of a NO group to the thiol side chain (Lima et al., 2010), and thus influence protein function. S-nitrosylation of β-catenin was shown to facilitate destabilization of AJs in response to VEGF signaling (Thibeault et al., 2010). Therefore, we determined S-nitrosothiol (SNO) content of β- and p120-catenins and of p190RhoGAP-A (referred to as p190A), a binding partner of p120-catenin (Wildenberg et al., 2006). We reasoned that the apparent organization of actin cytoskeleton into stress fibers in Cav-1−/− MLVECs (Fig. 1 c) might result from insufficient control of RhoA activity. p190A, a GTPase-activating protein (GAP), facilitates RhoA-GTP hydrolysis (Tatsis et al., 1998) and is a major regulator of RhoA activity at the level of AJs (Mammoto et al., 2007). SNO content, determined by the biotin-switch assay (Jaffrey and Snyder, 2001), of AJ proteins was indistinguishable between Cav-1−/− and Wt endothelia, although in a control experiment we could detect SNO of β-catenin in VEGF-stimulated endothelial cells (Fig. 2 a). This finding raises the possibility that eNOS-redox signaling regulates integrity of AJs by other means.

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