Early growth response gene mediates in VEGF and FGF signaling as dissected by CRISPR in corpus luteum of water buffalo

Meeti Punetha,Jonathan A Green,Arvind Sonwane,Kristin Whitworth,Sadhan Bag,Mihir Sarkar,Vikrant S Chouhan,Gyanendra Singh

doi:10.1038/s41598-020-63804-z

Meeti Punetha, Jonathan A Green + Show 6 more

Open Access

https://doi.org/10.1038/s41598-020-63804-z

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Abstract

The EGR family comprises of EGR 1, EGR 2, EGR 3 and EGR 4 which are involved in the transactivation of several genes. A broad range of extracellular stimuli by growth factors is capable of activating EGR mediated transactivation of genes involved in angiogenesis and cell proliferation. However, their role in controlling VEGF A and FGF 2 signaling in the CL of water buffalo is not known. The present study was conducted to understand the role of EGR mediated regulation of VEGF A and FGF 2 signaling in buffalo luteal cells. Towards this goal, luteal cells were cultured and treated with VEGF A and FGF 2 and the mRNA expression pattern of EGR family members were documented. The EGR 1 message was found to be up-regulated in luteal cells of buffalo at 72 hours of culture. The functional validation of EGR 1 gene was accomplished by knocking out (KO) of EGR 1 in cultured luteal cells by CRISPR/Cas9 mediated gene editing technology. The EGR 1 KO cells were then cultured and stimulated with VEGF A and FGF 2. It was observed that VEGF A and FGF 2 induced angiogenesis, cell proliferation and steroidogenesis in wild type luteal cells, whereas the response of the growth factors was attenuated in the EGR 1 KO cells. Taken together our study provides evidence convincingly that both VEGF and FGF mediate their biological action through a common intermediate, EGR 1, to regulate corpus luteum function of buffalo.

Highlights

The EGR family comprises of EGR 1, EGR 2, EGR 3 and EGR 4 which are involved in the transactivation of several genes
Extensive efforts have been made to delineate the mechanisms by which vascular endothelial growth factor (VEGF) A and fibroblast growth factors (FGF) 2 induces angiogenesis, steroidogenesis and cell survivability in murine and human endothelial cells, but no information could be traced to date to understand the insight of EGR mediated regulation of VEGF and FGFs signaling in buffalo luteal cells
The effect of VEGF A and FGF 2 showed significant up regulation (p < 0.05; Fig. 3A,B) on the expression of the von Willebrand factor in treated wild type luteal cells when compared with the wild type luteal cells without treatment

Summary

Introduction

The EGR family comprises of EGR 1, EGR 2, EGR 3 and EGR 4 which are involved in the transactivation of several genes. A broad range of extracellular stimuli by growth factors is capable of activating EGR mediated transactivation of genes involved in angiogenesis and cell proliferation Their role in controlling VEGF A and FGF 2 signaling in the CL of water buffalo is not known. The transition of follicular cell to luteal cell, angiogenesis in CL and its function is controlled by FGF 2 in cattle[13] and buffalo[3] Growth factors such as VEGF and FGF family mediate their physiological role by binding with their transmembrane tyrosine kinase receptor. Extensive efforts have been made to delineate the mechanisms by which VEGF A and FGF 2 induces angiogenesis, steroidogenesis and cell survivability in murine and human endothelial cells, but no information could be traced to date to understand the insight of EGR mediated regulation of VEGF and FGFs signaling in buffalo luteal cells. The present study was aimed to explore the functional role of EGR family members on VEGF and FGF signaling in the luteal cells of water buffalo

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