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

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

doi:10.3389/fphys.2021.683651

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

Front Physiol. 2021 Jul 26:12:683651. doi: 10.3389/fphys.2021.683651. eCollection 2021.

Authors

Sharon Harel^{1

2

3}, Veronica Sanchez^{1

2

3}, Alaa Moamer^{1

2

3}, Javier E Sanchez-Galan⁴, Mohammad N Abid Hussein⁵, Dominique Mayaki^{1

2

3}, Mathieu Blanchette⁴, Sabah N A Hussain^{1

2

3}

Affiliations

¹ Translational Research in Respiratory Diseases Program, Research Institute of the McGill University Health Centre, Montreal, QC, Canada.
² Department of Critical Care, McGill University Health Centre, Montreal, QC, Canada.
³ Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, QC, Canada.
⁴ School of Computer Science, McGill Centre for Bioinformatics, McGill University, Montreal, QC, Canada.
⁵ School of Engineering and Technology (SET), Aldar University College, Dubai, United Arab Emirates.

Abstract

Background: Angiopoietin-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.

Objective: The main objective of this study is to identify the roles of the ETS family of TFs in Ang-1 signaling and the angiogenic response.

Methods: In 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).

Results: Ang-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.

Conclusion: We conclude that ETS1, ELK1, and ETV4 transcription factors play significant angiogenic roles in Ang-1 signaling in ECs.

Keywords: Tie-2 receptors; angiogenesis; angiopoietins; cell migration; cell signaling; endothelial cells; proliferation; transcription factors.