Abstract
The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. 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. 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 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC 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. 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.
Highlights
We found that VEC expression and clustering at cell–cell contacts relieve the inhibitory effect of the FoxO1 (Forkhead box protein O1)/β-catenin complex on the expression of claudin-5, an endothelial-specific TJ protein,[5] acting via the removal of a transcriptional repression mechanism
VEC Clustering Triggers an Endothelial-Specific Transcription Program To investigate whether VEC was able to upregulate other endothelial-specific genes besides claudin-5,5 we performed an RNA sequencing comparative analysis of a mouse VEC-null cell line (VEC-null) and the same line reconstituted with VEC wild-type cDNA (VEC-positive)
Further validation of RNA sequencing data was performed by quantitative real-time polymerase chain reaction of vascular endothelial-protein tyrosine phosphatase (VE-PTP),[13] von Willebrand factor (vWf),[14] T-cell lymphoma invasion and metastasis-1 (Tiam1),[15] LIM domain only-2 (Lmo2),[16] signal transducer and activator of transcription-6 (Stat6)[17] and Elk[3] (ETS domain containing protein)[18,19] among VECinduced genes, hes-related family bHLH transcription factor with YRPW motif-1 (Hey1)[20] and adrenomedullin (Adm)[21] among VEC-repressed genes, and platelet/endothelial cell adhesion molecule-1 (Pecam1)[22] and SRY-box-18 (Sox18)[19] as genes not influenced by VEC expression (Online Figures IA and IIA through IIC)
Summary
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). VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/β-catenin complex sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh[2] recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP
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