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Genes up-regulated by VE-cadherin expression and clustering at junctions

ABSTRACT: In order to identify genes regulated by VE-cadherin expression, we compared a mouse VE-cadherin null cell line (VEC null) with the same line reconstituted with VE-cadherin wild type cDNA (VEC positive). The morphological and functional properties of these cell lines were described previously [Lampugnani,M.G. et al. Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, beta-catenin, and the phosphatase DEP-1/CD148. J. Cell Biol. 161, 793-804 (2003)]. By Affymetrix gene expression analysis we found several genes up-regulated by VE-cadherin, among which claudin-5 reached remarkably high levels. The up-regulation of these genes required not only VE-cadherin expression but also cell confluence suggesting that VE-cadherin clustering at junctions was needed.

ORGANISM(S): Mus musculus

PROVIDER: GSE11674 | GEO | 2008/07/06

SECONDARY ACCESSION(S): PRJNA106073

REPOSITORIES: GEO

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Project description:In order to identify genes regulated by VE-cadherin expression, we compared a mouse VE-cadherin null cell line (VEC null) with the same line reconstituted with VE-cadherin wild type cDNA (VEC positive). The morphological and functional properties of these cell lines were described previously [Lampugnani,M.G. et al. Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, beta-catenin, and the phosphatase DEP-1/CD148. J. Cell Biol. 161, 793-804 (2003)]. By Affymetrix gene expression analysis we found several genes up-regulated by VE-cadherin, among which claudin-5 reached remarkably high levels. The up-regulation of these genes required not only VE-cadherin expression but also cell confluence suggesting that VE-cadherin clustering at junctions was needed. Experiment Overall Design: VEC null or positive cells in the sparse and confluent conditions have been starved in MCDB 131 medium 1% BSA for 36 h. Next, RNA has been extracted with standard guanidinium isothiocyanate lysis buffer and cesium chloride ultracentrifugation. Synthesis of biotinylated cRNA targets, array hybridization (GeneChips MG_U74Av2 and MG_U74Bv2), staining and scanning were performed according to the Affymetrix standard protocols, starting from 15 Î¼g of total RNA. Two copies of the GeneChips were hybridized with each cRNA sample. The MAS5 algorithm was used to determine the expression levels of mRNAs; the absolute analysis was performed using default parameters and scaling factor 500. Report files were extracted for each chip, and performance of labeled target was evaluated on the basis of several values (scaling factor, background and noise values, % present calls, average signal value, etc). Gene expression levels were normalized on the median over all samples. Annotation of Probe Sets is according to Affymetrix Annotation Tables (release May 31, 2007) [Vecchi,M. et al. Gene expression analysis of early and advanced gastric cancers. Oncogene 26, 4284-4294 (2007)]. We selected the genes up-regulated by VE-cadherin expression and clustering at junctions in confluent VEC positive cells.

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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Genes up-regulated by VE-cadherin expression and clustering at junctions

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