Serine 26 in Early Growth Response‐1 Is Critical for Endothelial Proliferation, Migration, and Network Formation

Background Vascular endothelial cell proliferation, migration, and network formation are key proangiogenic processes involving the prototypic immediate early gene product, Egr‐1 (early growth response‐1). Egr‐1 undergoes phosphorylation at a conserved Ser26 but its function is completely unknown in...

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Autores principales:	Fernando S. Santiago, Yue Li, Levon M. Khachigian
Formato:	article
Lenguaje:	EN
Publicado:	Wiley 2021
Materias:	CRISPR/Cas9 early growth response‐1 endothelial cell extracellular signal‐regulated kinase‐1 Diseases of the circulatory (Cardiovascular) system RC666-701
Acceso en línea:	https://doaj.org/article/0933ff4c1f564271b8d42dcffe695493
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spelling	oai:doaj.org-article:0933ff4c1f564271b8d42dcffe6954932021-11-23T11:36:35ZSerine 26 in Early Growth Response‐1 Is Critical for Endothelial Proliferation, Migration, and Network Formation10.1161/JAHA.120.0205212047-9980https://doaj.org/article/0933ff4c1f564271b8d42dcffe6954932021-09-01T00:00:00Zhttps://www.ahajournals.org/doi/10.1161/JAHA.120.020521https://doaj.org/toc/2047-9980Background Vascular endothelial cell proliferation, migration, and network formation are key proangiogenic processes involving the prototypic immediate early gene product, Egr‐1 (early growth response‐1). Egr‐1 undergoes phosphorylation at a conserved Ser26 but its function is completely unknown in endothelial cells or any other cell type. Methods and Results A CRISPR/Cas9 strategy was used to introduce a homozygous Ser26>Ala mutation into endogenous Egr‐1 in human microvascular endothelial cells. In the course of generating mutant cells, we produced cells with homozygous deletion in Egr‐1 caused by frameshift and premature termination. We found that Ser26 mutation in Egr‐1, or Egr‐1 deletion, perturbed endothelial cell proliferation in models of cell counting or real‐time growth using the xCELLigence System. We found that Ser26 mutation or Egr‐1 deletion ameliorated endothelial cell migration toward VEGF‐A165 (vascular endothelial growth factor‐A) in a dual‐chamber model. On solubilized basement membrane preparations, Ser26 mutation or Egr‐1 deletion prevented endothelial network (or tubule) formation, an in vitro model of angiogenesis. Flow cytometry further revealed that Ser26 mutation or Egr‐1 deletion elevated early and late apoptosis. Finally, we demonstrated that Ser26 mutation or Egr‐1 deletion increased VE‐cadherin (vascular endothelial cadherin) expression, a regulator of endothelial adhesion and signaling, permeability, and angiogenesis. Conclusions These findings not only indicate that Egr‐1 is essential for endothelial cell proliferation, migration, and network formation, but also show that point mutation in Ser26 is sufficient to impair each of these processes and trigger apoptosis as effectively as the absence of Egr‐1. This highlights the importance of Ser26 in Egr‐1 for a range of proangiogenic processes.Fernando S. SantiagoYue LiLevon M. KhachigianWileyarticleCRISPR/Cas9early growth response‐1endothelial cellextracellular signal‐regulated kinase‐1Diseases of the circulatory (Cardiovascular) systemRC666-701ENJournal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, Vol 10, Iss 18 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR/Cas9 early growth response‐1 endothelial cell extracellular signal‐regulated kinase‐1 Diseases of the circulatory (Cardiovascular) system RC666-701
spellingShingle	CRISPR/Cas9 early growth response‐1 endothelial cell extracellular signal‐regulated kinase‐1 Diseases of the circulatory (Cardiovascular) system RC666-701 Fernando S. Santiago Yue Li Levon M. Khachigian Serine 26 in Early Growth Response‐1 Is Critical for Endothelial Proliferation, Migration, and Network Formation
description	Background Vascular endothelial cell proliferation, migration, and network formation are key proangiogenic processes involving the prototypic immediate early gene product, Egr‐1 (early growth response‐1). Egr‐1 undergoes phosphorylation at a conserved Ser26 but its function is completely unknown in endothelial cells or any other cell type. Methods and Results A CRISPR/Cas9 strategy was used to introduce a homozygous Ser26>Ala mutation into endogenous Egr‐1 in human microvascular endothelial cells. In the course of generating mutant cells, we produced cells with homozygous deletion in Egr‐1 caused by frameshift and premature termination. We found that Ser26 mutation in Egr‐1, or Egr‐1 deletion, perturbed endothelial cell proliferation in models of cell counting or real‐time growth using the xCELLigence System. We found that Ser26 mutation or Egr‐1 deletion ameliorated endothelial cell migration toward VEGF‐A165 (vascular endothelial growth factor‐A) in a dual‐chamber model. On solubilized basement membrane preparations, Ser26 mutation or Egr‐1 deletion prevented endothelial network (or tubule) formation, an in vitro model of angiogenesis. Flow cytometry further revealed that Ser26 mutation or Egr‐1 deletion elevated early and late apoptosis. Finally, we demonstrated that Ser26 mutation or Egr‐1 deletion increased VE‐cadherin (vascular endothelial cadherin) expression, a regulator of endothelial adhesion and signaling, permeability, and angiogenesis. Conclusions These findings not only indicate that Egr‐1 is essential for endothelial cell proliferation, migration, and network formation, but also show that point mutation in Ser26 is sufficient to impair each of these processes and trigger apoptosis as effectively as the absence of Egr‐1. This highlights the importance of Ser26 in Egr‐1 for a range of proangiogenic processes.
format	article
author	Fernando S. Santiago Yue Li Levon M. Khachigian
author_facet	Fernando S. Santiago Yue Li Levon M. Khachigian
author_sort	Fernando S. Santiago
title	Serine 26 in Early Growth Response‐1 Is Critical for Endothelial Proliferation, Migration, and Network Formation
title_short	Serine 26 in Early Growth Response‐1 Is Critical for Endothelial Proliferation, Migration, and Network Formation
title_full	Serine 26 in Early Growth Response‐1 Is Critical for Endothelial Proliferation, Migration, and Network Formation
title_fullStr	Serine 26 in Early Growth Response‐1 Is Critical for Endothelial Proliferation, Migration, and Network Formation
title_full_unstemmed	Serine 26 in Early Growth Response‐1 Is Critical for Endothelial Proliferation, Migration, and Network Formation
title_sort	serine 26 in early growth response‐1 is critical for endothelial proliferation, migration, and network formation
publisher	Wiley
publishDate	2021
url	https://doaj.org/article/0933ff4c1f564271b8d42dcffe695493
work_keys_str_mv	AT fernandossantiago serine26inearlygrowthresponse1iscriticalforendothelialproliferationmigrationandnetworkformation AT yueli serine26inearlygrowthresponse1iscriticalforendothelialproliferationmigrationandnetworkformation AT levonmkhachigian serine26inearlygrowthresponse1iscriticalforendothelialproliferationmigrationandnetworkformation
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Serine 26 in Early Growth Response‐1 Is Critical for Endothelial Proliferation, Migration, and Network Formation

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