ETS1, ELK1, and ETV4 Transcription Factors Regulate Angiopoietin-1 Signaling and the Angiogenic Response in Endothelial Cells.

Sharon Harel,Sabah N A Hussain,Dominique Mayaki,Javier E Sanchez-Galan,Alaa Moamer,Mathieu Blanchette,Mohammad N Abid Hussein,Veronica Sanchez

doi:10.3389/fphys.2021.683651

Abstract

BackgroundAngiopoietin-1 (Ang-1) is the main ligand of Tie-2 receptors. It promotes endothelial cell (EC) survival, migration, and differentiation. Little is known about the transcription factors (TFs) in ECs that are downstream from Tie-2 receptors.ObjectiveThe main objective of this study is to identify the roles of the ETS family of TFs in Ang-1 signaling and the angiogenic response.MethodsIn silico enrichment analyses that were designed to predict TF binding sites of the promotors of eighty-six Ang-1-upregulated genes showed significant enrichment of ETS1, ELK1, and ETV4 binding sites in ECs. Human umbilical vein endothelial cells (HUVECs) were exposed for different time periods to recombinant Ang-1 protein and mRNA levels of ETS1, ELK1, and ETV4 were measured with qPCR and intracellular localization of these transcription factors was assessed with immunofluorescence. Electrophoretic mobility shift assays and reporter assays were used to assess activation of ETS1, ELK1, and ETV4 in response to Ang-1 exposure. The functional roles of these TFs in Ang-1-induced endothelial cell survival, migration, differentiation, and gene regulation were evaluated by using a loss-of-function approach (transfection with siRNA oligos).ResultsAng-1 exposure increased ETS1 mRNA levels but had no effect on ELK1 or ETV4 levels. Immunostaining revealed that in control ECs, ETS1 has nuclear localization whereas ELK1 and ETV4 are localized to the nucleus and the cytosol. Ang-1 exposure increased nuclear intensity of ETS1 protein and enhanced nuclear mobilization of ELK1 and ETV4. Selective siRNA knockdown of ETS1, ELK1, and ETV4 showed that these TFs are required for Ang-1-induced EC survival and differentiation of cells, while ETS1 and ETV4 are required for Ang-1-induced EC migration. Moreover, ETS1, ELK1, and ETV4 knockdown inhibited Ang-1-induced upregulation of thirteen, eight, and nine pro-angiogenesis genes, respectively.ConclusionWe conclude that ETS1, ELK1, and ETV4 transcription factors play significant angiogenic roles in Ang-1 signaling in ECs.

Highlights

The TEK receptor tyrosine kinase (Tie-2) and its ligand angiopoietin-1 (ANPGT1, Ang-1) have emerged as important regulators of angiogenesis, both in adults and in embryos
Selective siRNA knockdown of E26 transformation-specific sequence-1 (ETS1), ETS like-1 protein (ELK1), and ETV4 showed that these transcription factors (TFs) are required for Ang-1-induced endothelial cell (EC) survival and differentiation of cells, while ETS1 and ETV4 are required for Ang-1-induced EC migration
We conclude that ETS1, ELK1, and ETV4 transcription factors play significant angiogenic roles in Ang-1 signaling in ECs

Summary

Introduction

The TEK receptor tyrosine kinase (Tie-2) and its ligand angiopoietin-1 (ANPGT1, Ang-1) have emerged as important regulators of angiogenesis, both in adults and in embryos. The Ang-1/Tie-2 receptor pathway stimulates vascular remodeling, vascular enlargement, wound healing, and lymphangiogenesis (Brindle et al, 2006). It inhibits endothelial cell (EC) apoptosis and stimulates proliferation, migration, and differentiation of these cells (Kolizek et al, 1998; Witzenbichler et al, 1998; Kim et al, 2000; Brindle et al, 2006). Despite the importance of the Ang-1/Tie-2 receptor pathway to vascular homeostasis and angiogenesis, relatively little progress has been made toward the identification of transcription factors (TFs) that mediate its angiogenic responses. Little is known about the transcription factors (TFs) in ECs that are downstream from Tie-2 receptors

Methods

Results

Discussion

Conclusion