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. 2009 Sep 25;105(7):676-85, 15 p following 685.
doi: 10.1161/CIRCRESAHA.109.201673. Epub 2009 Aug 27.

Phosphorylation of caveolin-1 regulates oxidant-induced pulmonary vascular permeability via paracellular and transcellular pathways

Yu Sun  1 , Guochang Hu, Xiumei Zhang, Richard D Minshall
Affiliations

Phosphorylation of caveolin-1 regulates oxidant-induced pulmonary vascular permeability via paracellular and transcellular pathways

Yu Sun et al. Circ Res. .

Abstract

Rationale: Oxidants are important signaling molecules known to increase endothelial permeability, although the mechanisms underlying permeability regulation are not clear.

Objective: To define the role of caveolin-1 in the mechanism of oxidant-induced pulmonary vascular hyperpermeability and edema formation.

Methods and results: Using genetic approaches, we show that phosphorylation of caveolin-1 Tyr14 is required for increased pulmonary microvessel permeability induced by hydrogen peroxide (H(2)O(2)). Caveolin-1-deficient mice (cav-1(-/-)) were resistant to H(2)O(2)-induced pulmonary vascular albumin hyperpermeability and edema formation. Furthermore, the vascular hyperpermeability response to H(2)O(2) was completely rescued by expression of caveolin-1 in cav-1(-/-) mouse lung microvessels but was not restored by the phosphorylation-defective caveolin-1 mutant. The increase in caveolin-1 phosphorylation induced by H(2)O(2) was dose-dependently coupled to both increased (125)I-albumin transcytosis and decreased transendothelial electric resistance in pulmonary endothelial cells. Phosphorylation of caveolin-1 following H(2)O(2) exposure resulted in the dissociation of vascular endothelial cadherin/beta-catenin complexes and resultant endothelial barrier disruption.

Conclusions: Caveolin-1 phosphorylation-dependent signaling plays a crucial role in oxidative stress-induced pulmonary vascular hyperpermeability via transcellular and paracellular pathways. Thus, caveolin-1 phosphorylation may be an important therapeutic target for limiting oxidant-mediated vascular hyperpermeability, protein-rich edema formation, and acute lung injury.

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Conflict of interest statement

Disclosures: None.

Figures

Figure 1
Figure 1. H2O2-induced activation of Src and c-Abl and resultant phosphorylation of Cav-1
(A) H2O2 induced the activation of Src and phosphorylation of Cav-1 in a concentration-dependent manner. RLMVECs were exposed to different concentrations of H2O2 (0.01-0.8 mmol/L) for 30 min. (B) Time course of H2O2-induced Cav-1 phosphorylation. Cells were stimulated with 0.6 mmol/L H2O2 for indicated times. (C) Effect of PP2 on Cav-1 phosphorylation induced by different concentrations of H2O2. (D) Dose-dependent c-Abl phosphorylation induced by H2O2. Cells were exposed to vehicle or H2O2 (0.1-0.6 mmol/L) for 30 min. c-Abl phosphorylation was measured by immunoprecipitation with c-Abl antibody and immunoblotting with phosphotyrosine antibody. Immunoblot of total c-Abl is shown as a loading control. (E) Effect of c-Abl siRNA on Cav-1 phosphorylation induced by the different concentrations of H2O2. (F) Effect of PP2 on H2O2-stimulated c-Abl activation. Cells were pretreated with PP2 or vehicle for 15 min and then stimulated with H2O2 (0.6 mmol/L) for 30 min. All blots are representative of 3 separate experiments.
Figure 2
Figure 2. Phosphorylation of caveolin-1 is required for H2O2-induced endocytosis and transendothelial albumin transport
(A) Confocal images showing H2O2-induced concentration-dependent increase in the uptake of Alexa 488-labeled albumin (green). The nucleus (blue) was stained with DAPI. Scale bars = 10 μm. Results are typical of 3 experiments. (B) H2O2 increased 125I-albumin endocytosis in a concentration-dependent manner. (C) H2O2 induced a concentration-dependent increase in transendothelial transport of 125I-albumin. (D,E) Effect of H2O2 on 125I-albumin endocytosis (D) and transendothelial albumin permeability (E) in cells stably expressing WT and phosphorylation-defective Y14F-Cav-1 mutant. (F,G) Effect of Src inhibition and downregulation of c-Abl kinase on H2O2-induced increase in 125I-albumin endocytosis (F) and transcytosis (G). n = 4-6 for each group. The baseline permeability values for WT and control groups were 7.8±1.2 μl·min-1·cm-2×10-2 (E) and 6.1±1.1 μl·min-1·cm-2×10-2 (G), respectively. *P <0.05 compared with control (B, C, F, and G) and WT (D, E) groups, †P <0.05 compared with respective (D,E) and control (F,G) groups.
Figure 3
Figure 3. Phosphorylation of Cav-1 is required for signaling H2O2-induced endothelial barrier disruption
RLMVECs were grown to confluence, treated with H2O2 at the concentration indicated, and TER was recorded. In the panels on the left, the original TER recordings are shown, and in the panels on the right, the mean value (±SEM) of the peak TER responses to H2O2 (relative to the starting value) is plotted. (A) Dose-response relationship of H2O2-induced decrease in TER; note there was no effect of ∼0.2 mmol/L H2O2. (B) Phospho-defective Y14F Cav-1 mutant blocks the effect of H2O2 on TER. Over-expression of WT-Cav-1 in RLMVECs reduced the threshold of H2O2 needed to decrease TER. (C) Effect of Src inhibitor PP2 and downregulation of c-Abl kinase on H2O2-induced decrease in TER. n = 4-6 for each group. *P < 0.05, compared to control (untreated) groups; †P < 0.05 compared with respective H2O2 groups.
Figure 4
Figure 4. Caveolin-1 phosphorylation mediates H2O2-induced dissociation of caveolin-1 from β-catenin
(A) Effects of H2O2 on the co-localization of caveolin-1 (green) and β-catenin (red) in naïve RLMVECs. Cells were exposed to different concentrations of H2O2 for 30 min. The nucleus (blue) was stained with DAPI. Scale bars =10 μm. (B, C) The association of endogenous caveolin-1 (B) or over-expressed Myc-tagged WT-Cav-1 or Y14F-Cav-1 mutant (C) with β-catenin in naïve endothelial cells and stable endothelial cell lines was determined by immunoprecipitation and immunoblot (IP/IB) with anti-caveolin-1, anti-Myc, or anti-β-catenin antibodies. Left, representative Western blots for β-catenin and caveolin-1 (or Myc); Right, protein quantification by densitometry. The density of proteins in each untreated control group was used as a standard (1 arbitrary unit) to compare the relative density of the other groups. *P < 0.05 compared to control (untreated) groups; † P < 0.05 compared with H2O2 (0.2 mmol/L) groups.
Figure 5
Figure 5. Caveolin-1 phosphorylation mediates H2O2-induced dissociation of VE-cadherin and β-catenin
The association of VE-cadherin with β-catenin was determined by immunoprecipitation and immunoblot analysis using anti-β-catenin or anti-VE-cadherin antibodies. Left, representative Western blots for VE-cadherin and β-catenin; Right, protein quantification by densitometry. The density of proteins in each untreated (control) group was used as a standard (1 arbitrary unit) to compare the relative density in the other groups. (A) Effect of H2O2 on the association of VE-cadherin with β-catenin in naïve endothelial cells. Note dissociation upon exposure to 0.6 mmol/L H2O2. (B) Effect of H2O2 on the association of VE-cadherin with β-catenin in cells stably expressing WT and phosphorylation-defective Y14F-Cav-1 mutant. *P < 0.05, compared to control (untreated) group (A) or WT alone group (B); n = 3/each group.
Figure 6
Figure 6. Caveolin-1 phosphorylation is required for H2O2-induced β-catenin redistribution
Cells were exposed to the indicated concentrations of H2O2 for 30 min and lysates separated into cytosolic and membrane fractions followed by Western blotting with β–catenin antibody to determine β–catenin localization. Top panel shows a representative Western blot for β-catenin and β-actin loading control; bottom panels show protein quantification by densitometry. The density of proteins in each untreated control group was used as a standard (1 arbitrary unit) to compare relative densities in the other groups. (A) H2O2 at 0.6 mmol/L concentration induced β-catenin redistribution from the membrane to cytosol in RLMVECs. (B) H2O2 (0.2 mmol/L) induced the translocation of β-catenin from cytosol to membrane in a manner dependent on Cav-1 phosphorylation in Cav-1 over-expressing cells. The redistribution of β-catenin was observed in cells stably expressing WT but not the phosphorylation-defective Y14F-Cav-1 mutant. n = 3. *P < 0.05, compared to control (untreated) group (A) or WT alone group (B).
Figure 7
Figure 7. H2O2-induced increase in lung microvessel albumin permeability and pulmonary edema formation requires caveolin-1 phosphorylation
Cav-1-/- mice were injected intravenously with liposomes containing WT- or Y14F-Cav-1 cDNA. After 48 h, lungs were isolated and perfused with H2O2 (0.5 mmol/L) for 30 min. (A) Western blots show increase in Cav-1 phosphorylation in whole lung homogenates induced by H2O2, the absence of Cav-1 in null mice, and exogenous expression of Myc-tagged WT-Cav-1 and Y14F-Cav-1 in cav-1-/- lungs. Note that H2O2 induced phosphorylation of reconstituted WT-Cav-1 in the isolated mouse lung. (B) Effect of H2O2 on pulmonary microvessel 125I-albumin permeability (PS product) in wild-type (cav-1+/+) and cav-1-/- lungs with or without rescue with WT-Cav-1 or Y14F-Cav-1 mutant cDNA. (C) H2O2 induced and increase in lung wet/dry (W/D) weight ratio in cav-1+/+ lungs but not in cav-1-/- lungs. Rescue of Cav-1 expression with WT-Cav-1 but not Y14F-Cav-1 cDNA restored the lung edema response to H2O2. n = 6/each group. * P < 0.05 compared with control groups (cav-1+/+ mouse without H2O2 treatment); †P < 0.05 compared with cav-1+/+ mouse with H2O2 treatment group.
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