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Leukocyte extravasation and vascular permeability are each controlled in vivo by different tyrosine residues of VE-cadherin

Abstract

Tyrosine phosphorylation of the adhesion molecule VE-cadherin is assumed to affect endothelial junction integrity. However, it remains unclear whether tyrosine residues of VE-cadherin are required for the induction of vascular permeability and the regulation of leukocyte extravasation in vivo. We found here that knock-in mice expressing a Y685F mutant of VE-cadherin had impaired induction of vascular permeability, but those expressing a Y731F mutant did not. In contrast, mice expressing the Y731F VE-cadherin mutant showed decreased neutrophil-extravasation in cremaster tissue, but those expressing the Y685F mutant did not. Whereas inflammatory mediators induced the phosphorylation of Tyr685 in vivo, Tyr731 showed high baseline phosphorylation. Leukocytes triggered dephosphorylation of Tyr731 via the tyrosine phosphatase SHP-2, which allowed the adaptin AP-2 to bind and initiate endocytosis of VE-cadherin. Thus, Tyr685 and Tyr731 of VE-cadherin distinctly and selectively regulate the induction of vascular permeability or leukocyte extravasation.

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Figure 1: Characterization of knock-in mice expressing VEC-Y685F and VEC-Y731F.
Figure 2: Specificity of mAbs for phosphorylated Tyr685 and Tyr731 of VE-cadherin and substantial baseline phosphorylation of Tyr731 but not of Tyr685 in endothelial cells in vitro and in vivo.
Figure 3: Phosphorylation of VE-cadherin Tyr685, not of VE-cadherin Tyr731, is induced by VEGF and histamine, and only Tyr685 contributes to the proper induction of vascular permeability in vivo.
Figure 4: Selective induction of Tyr685 phosphorylation in venules but not arterioles.
Figure 5: VEGF or histamine stimulates phosphorylation of Tyr685 of VE-cadherin at sites of vascular-leak formation.
Figure 6: T cells stimulate phosphorylation of Tyr685 but substantially diminish phosphorylation of Tyr731.
Figure 7: VE-cadherin Tyr731 contributes to leukocyte extravasation in vivo, but Tyr685 does not.
Figure 8: Leukocytes induce SHP-2-mediated dephosphorylation of Tyr731 of VE-cadherin, which leads to AP-2 association and endocytosis of VE-cadherin.

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Acknowledgements

We thank B. Kempe and B. Waschk for the transfection and cultivation of embryonic stem cells; D. Brandhorst for help with intravital microscopy; and F. Kiefer for supervising work with embryonic stem cells and for critically reading the manuscript. Supported by the Max Planck Society (D.V.) and the Deutsche Forschungsgemeinschaft SFB 629 to (D.V.).

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Authors and Affiliations

Authors

Contributions

F.W. designed and did most of the experiments; M.H., M.F., R.R.-G., M.V., R.L. and U.I. designed and did experiments; A.S. and A.Z. did intravital microscopy experiments; M.W. and A.F.N. designed, supervised and did experiments; and D.V. provided overall supervision, helped design all of the experiments and prepared the manuscript.

Corresponding author

Correspondence to Dietmar Vestweber.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Specificity of newly generated mAbs for VE-cadherin phosphorylated at Tyr685 or Tyr731 in comparison with commercially available antibodies to these phosphorylated tyrosines.

(a+b) Immunoblots of VE-cadherin immunoprecipitated from lung lysates of VEC-WT and VEC-Y685F mice (a) and VEC-Y731F mice (b) that had been anesthetized and intravenously injected with PBS containing (+) or not containing (-) peroxyvanadate (PV). The same membranes were sequentially immunoblotted with the following antibodies: mp685 (a) or mp731 (b); commercial pY685 (a) or commercial pY731 (b); general anti pY antibody 4G10 (a+b) and VE-cadherin antibodies (a+b). Transfer of the complete gel with start of the separating-gel and electrophoresis front are displayed. Data are representative for 2 independent experiments.

Supplementary Figure 2 Homing of naive lymphocytes to lymph nodes is not affected in VEC-Y731F mice.

Radiolabelled naive lymphocytes from C57Bl6 mice were injected intravenously into WT and Y731F KI mice and the percentage of the total injected radioactivity in lymph nodes was determined 3 h later. Four independent experiments with at least four mice per group and experiment are shown (n=14).

Supplementary Figure 3 Knock down of the expression of VE-PTP or DEP-1 by siRNA does not interfere with leukocyte-induced dephosphorylation of VE-cadherin Tyr731.

Immunoblots of VE-cadherin immunoprecipitated from TNF-activated bEnd.5 cells that had been either treated with VE-PTP siRNA (a) or with DEP-1 siRNA (b) and control siRNA (as indicated above). Blots were incubated with mp731 or anti VE-cadherin antibodies (left panels). Knock-down efficiency of VE-PTP and DEP-1 was confirmed in total cell lysates by immunoblotting with anti VE-PTP or anti DEP-1 antibodies and equal loading was controlled with anti α-tubulin antibodies (right panels). Data are representative for 2 (a) and 3 (b) independent experiments.

Supplementary Figure 4 Endocytosis of VE-cadherin induced by single T cells.

(a) T cells (labeled with cell tracker orange) were added for 15 min to TNF-α-stimulated primary isolated mouse endothelial cells at a ratio of 1:5 (one T cell per five endothelial cells). Uptake of VE-cadherin was monitored with mAb BV13 as described under Material & Methods (red). After 15 min, endothelial cells were carefully washed to avoid removal of T cells. VE-cadherin at cell contacts was stained with a polyclonal antibody (green). F-actin was stained with phalloidin (magenta) and nuclei were stained with Hoechst 33342 (blue). Note that VE-cadherin-containing endocytic vesicles have accumulated close to the single associated lymphocyte. VE-cadherin junctional staining reveals a gap at the site where the lymphocyte is probing the cell contact (white arrow). (b) Serial optical sections of the same cells displaying only the staining for VE-cadherin at cell contacts, the lymphocyte and nuclei to highlight the gap (white arrow) in VE-cadherin staining at junctions. The leukocyte is polarized towards the site of junctional loss of VE-cadherin. Scale bar 20 μm. Data are representative for 2 independent experiments with more than 100 lymphocytes analyzed.

Supplementary Figure 5 Induction of endothelial permeability relies exclusively on phosphorylation of VE-cadherin Tyr685, whereas efficient extravasation of leukocytes depends exclusively on the dephosphorylation of Tyr731.

Upper panel: Under resting conditions VE-cadherin is not significantly phosphorylated at Y685, but strongly phosphorylated at Y731. Vascular permeability-inducing factors such as VEGF or histamine strongly enhance Y685 phosphorylation whereas high baseline phosphorylation of Y731 is unaltered. Only Y685, but not Y731 is required for proper induction of vascular permeability in vivo. Lower panel: In contrast to permeability-inducing stimuli, leukocytes rapidly dephosphorylate Y731. This is mediated by the phosphatase SHP2 and followed by binding to AP-2 and subsequent rapid endocytosis. Only Y731, but not Y685 supports extravasation of leukocytes in vivo. Collectively, the induction of permeability and the extravasation of leukocytes regulate the phosphorylation of different tyrosine residues on VE-cadherin in different ways resulting in different signatures of phosphorylation sites. Each of the two sites is exclusively involved in only one of the two processes.

Supplementary Figure 6 VE-PTP counteracts phosphorylation of Tyr685 but not of Tyr731.

(a) 24 h after downregulation of VE-PTP expression by siRNA in bEnd.5 cells, VE-cadherin was immunoprecipitated and precipitates were immunoblotted with the mAb mp685, mp731 or VE-cadherin antibodies (left panels). Knock-down of VE-PTP was confirmed in total cell lysates by immunoblotting with anti VE-PTP and gel loading was controlled with anti α-tubulin antibodies (right panels). (b) VE-PTP-associated VE-cadherin is phosphorylated at Y731, but not at Y685. VE-PTP was immunoprecipitated from highly confluent bEnd.5 cells. Phosphorylation of co-precipitated VE-cadherin was checked in immunoblots using the mAb mp731 (left panels) and mp685 (right panels). Co-precipitated VE-cadherin-levels and precipitated VE-PTP was analyzed in immunoblots with the respective antibodies. Data in (a) and (b) are representative for 3 independent experiments.

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Wessel, F., Winderlich, M., Holm, M. et al. Leukocyte extravasation and vascular permeability are each controlled in vivo by different tyrosine residues of VE-cadherin. Nat Immunol 15, 223–230 (2014). https://doi.org/10.1038/ni.2824

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