Efficient Fine-Tuning of Endothelial Gene Expression by a Single Tyrosine (Y685) to Phenylalanine Point Mutation in the VE-Cadherin Gene.

Abstract

Background: VE (vascular endothelial)-cadherin is an endothelial cell-surface receptor that lacks intrinsic tyrosine kinase activity but can be tyrosine phosphorylated in cancer and inflammation. Previous studies have uncovered the molecular underpinnings of phosphorylation events; however, there is a need for a comprehensive analysis of the transcriptome of endothelial cells.

Methods: Using a tyrosine-to-phenylalanine (Y->F) transgenic mouse (KI), we provide the first experimental evidence that tyrosine-to phenylalanine at the site 685 (Y⁶⁸⁵F)-VE-cadherin induces a specific transcriptional program in vivo in lung tissue.

Results: RNA-sequencing analysis revealed a total of 884 differentially expressed genes (766 downregulated and 118 upregulated in endothelial cells from KI) involved in cell-cell adhesion, vascular development, and angiogenesis. The heatmap of the top 30 differentially expressed genes clearly shows 22 downregulated genes (including cell signaling enzymes, anion transport, and lipid metabolism) and 8 upregulated genes that confer significantly reduced migration, proliferation, and outgrowth capabilities to endothelial cells from KI. A central pathway in signal transduction revealed a notable increase in phosphorylation of site tyrosine 731 (Y⁷³¹) in Y⁶⁸⁵F-VE-cadherin in KI (P=0.041). This further compromised the binding of β-catenin, which was preferentially located in the nuclear fraction in KI (P=0.034) with increased transcriptional activity. One of the genes of particular interest was s1pr1 (sphingosine-1-phosphate receptor 1), which had the highest mean expression level in KI. We identified the lung endothelial-specific transcription factor FOXF1 (forkhead box protein F1) that binds to the s1pr1 promoter with a significantly higher intensity in KI (7-fold; P=0.023), as shown by chromatin immunoprecipitation assay. Consequently, increased s1pr1 expression was confirmed by reverse transcription polymerase chain reaction and Western blotting. Importantly, quantitative analysis of the pulmonary vasculature of KI revealed a significant decrease in the number of capillaries (P=0.036), with less fibrosis and no edema.

Conclusions: Overall, our results indicate a novel regulatory mechanism for transcriptional signatures in lung tissue, involving the critical site Y⁶⁸⁵ of VE-cadherin. This finding offers future insights for precision medicine applications.