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VE-cadherin interaction proteomics identifies ARVCF as a key stabilizer of endothelial adherens junctions

ABSTRACT: The endothelial cells lining the vascular wall maintain a selective barrier between blood and tissue. Structural connections between these endothelial cells are formed through vascular endothelial (VE)-cadherin-based adherens junctions. The extracellular domain of VE-cadherin forms homotypic bonds between neighboring cells, while its intracellular domain connects to the actin cytoskeleton via a conserved protein complex including α-, ß- and p120-catenins. Whether additional proteins bind to VE-cadherin and contribute to endothelial junction integrity remains unclear. By using mass spectrometry after VE-cadherin immunoprecipitations from human endothelial cells, we have determined the molecular interactions with VE-cadherin. The proteomics identified a core VE-cadherin interactome, consisting of nine proteins, which bind to VE-cadherin even in the absence of tyrosine phosphorylation of its intracellular domain. The core VE-cadherin interactome includes the known catenin proteins as well as four new interactors: ARVCF, ARHGAP23, KEAP1 and NGLY1. Co-immunoprecipitation and co-localization experiments verified that the VE-cadherin-binding protein ARVCF is an important component of endothelial adherens junctions. ARVCF binds to a selective pool of VE-cadherin proteins during junction maturation that is unbound from p120-catenin, through a mechanism involving the C-terminal intrinsically disordered regions of ARVCF. Depletion of ARVCF results in loss of endothelial barrier function and impairs collective cell migration. Accordingly, ARVCF is needed for VE-cadherin-based junction stabilization. Together, our results demonstrate that ARVCF is a key regulator of VE-cadherin to safeguard junctional stability and endothelial integrity.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Endothelial Cell Of Umbilical Vein, Umbilical Vein Endothelial Cell Line

SUBMITTER: Arie Hoogendijk

LAB HEAD: Dr Maartje van den Biggelaar

PROVIDER: PXD068012 | Pride | 2026-03-18

REPOSITORIES: Pride

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			Action	DRS
	GFPControl_GFPbeads_1.raw	Raw
	GFPControl_GFPbeads_2.raw	Raw
	GFPControl_GFPbeads_3.raw	Raw
	VECadherin1454mut_Blockedbeads_1.raw	Raw
	VECadherin1454mut_Blockedbeads_2.raw	Raw

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Project description:Vascular endothelial protein tyrosine phosphatase (VE-PTP, PTPRB) is a receptor type phosphatase that is crucial for the regulation of endothelial junctions and blood vessel development. VE-PTP regulates vascular integrity by dephosphorylating substrates which are key players in endothelial junction stability, such as the angiopoietin receptor TIE2, the endothelial adherens junction protein VE-cadherin and the vascular endothelial growth factor receptor VEGFR2. Here, we have systematically searched for novel substrates of VE-PTP in endothelial cells by utilizing two approaches. First, we studied changes in the endothelial phosphoproteome upon exposing cells to a highly VE-PTP-specific phosphatase inhibitor followed by affinity isolation and mass-spectrometric analysis of phosphorylated proteins by phosphotyrosine-specific antibodies. Second, we used a substrate trapping mutant of VE-PTP to pull down phosphorylated substrates in combination with SILAC-based quantitative mass spectrometry measurements. We identified a set of substrate candidates of VE-PTP, of which a remarkably large fraction is related to cell junctions (48/165; 29.1%). Several of those were found in both screens and displayed very high connectivity in predicted functional interaction networks. The receptor protein tyrosine kinase EPHB4 was the most prominently phosphorylated protein upon VE-PTP inhibition among those VE-PTP targets that were identified by both proteomic approaches. Further analysis revealed that EPHB4 forms a ternary complex with VE-PTP and TIE2 in endothelial cells. VE-PTP controls the phosphorylation of each of these two tyrosine kinase receptors. Despite of their simultaneous presence in a ternary complex, stimulating each of the receptors with their own specific ligand did not cross-activate the respective partner receptor. Our systematic approach has led to the identification of novel substrates of VE-PTP, of which many are relevant for the control of cellular junctions further promoting the importance of VE-PTP as a key player of junctional signalling.

Project description:Rationale: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumours, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular inter-endothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, vascular endothelial (VE)-cadherin (VEC), upregulate genes with key roles in endothelial differentiation and stability. Objective: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. Methods and Results: We compared a VEC-null cell line with the same line reconstituted with VEC wild type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, Vascular Endothelial-Protein Tyrosine Phosphatase (VE-PTP) and von Willebrand factor (vWf). Mechanistically VEC exerts this effect by inhibiting Polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 and β-catenin, which contribute to Polycomb repressive complex-2 (PRC2) binding to promoter regions of claudin-5, VE-PTP and vWf. VE-cadherin/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VE-cadherin association increases Ezh2 recruitment to claudin-5, VE-PTP and vWf promoters, causing gene downregulation. RNAseq comparison of VEC-null and VEC-positive cells suggested a more general role of VE-cadherin in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of Claudin-5 and VE-PTP. Conclusions: These data extend the knowledge of Polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the Polycomb-mediated repression system. Keywords: Polycomb, endothelial cells, VE-cadherin, vessel maturation, vascular biology, vascular permeability, cell signalling, epigenetics, gene regulation. Downloaded from http://circres.ahajour Conclusions: These data extend the knowledge of Polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the Polycomb-mediated repression system

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VE-cadherin interaction proteomics identifies ARVCF as a key stabilizer of endothelial adherens junctions

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