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

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.

SUBMITTER: Punetha M

PROVIDER: S-EPMC7176634 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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

Punetha Meeti M Chouhan Vikrant S VS Sonwane Arvind A Singh Gyanendra G Bag Sadhan S Green Jonathan A JA Whitworth Kristin K Sarkar Mihir M

Scientific reports 20200422 1

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 signalin ...[more]

PMID: 32321973

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Project description:BackgroundPGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFβ1, which plays an important role during luteal regression.MethodsThe current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFβ1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3βHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFβ1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9.ResultThe present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFβ1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFβ1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFβ1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells.ConclusionThese results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFβ1 signaling for luteolysis.

Project description:In monoovulatory species like bovines, a striking diversity in regulation of the corpus luteum (CL) function exists within species during different stages of the luteal phase. The function and life span of CL appears to be regulated by the interplay of both luteotrophic and luteolytic factors, whose roles and actions are varied from one species to another. Although, studies continue to expand our current understanding of the cellular and molecular actions of prostaglandin F 2alpha (PGF2M-NM-1 - a physiological luteolysin)-induced luteolysis, knowledge of the mechanism whereby, PGF2M-NM-1 mediates its luteolytic actions remains poorly understood. The mechanism of PGF2M-NM-1-induced luteolysis is a complex process involving changes in expression of multiple genes encoding gene products that regulate steroidogenesis, stress related or apoptotic genes, factors involved in immune response and enzymes that stimulate PGF2M-NM-1 production. Thus, in the present study, efforts were made to identify the temporal changes in the global gene expression profile in the CL of buffalo cows in response to PGF2M-NM-1 treatment. The molecular signature of genome-wide transcriptional changes in the CL tissue subjected to PGF2M-NM-1-induced luteolysis will expand our knowledge on basic mechanism/s that regulates the luteolytic process. The results obtained in this study provide insight into the mechanisms underlying the PGF2M-NM-1- induced regression of CL.Keywords: CL, PGF2M-NM-1, gene expression The objective of this study was to identify the PGF2M-NM-1-induced temporal changes in transcriptome of bovine CL on day 11 of estrous cycle. In the experiments with buffalo cows, animals with history of normal estrous cyclicity were treated with Cloprostenol (an PGF2M-NM-1 analogue; 500 M-BM-5g i.m.) on day 11 of the estrous cycle to induce luteolysis and CL tissues were collected at (control; n=4 amimals/time point), 3 (n=2 animals/time point), 6 and 18 (n=3 animals/time point) h post PGF2M-NM-1 administration to examine gene expression changes associated with immediate early and delayed changes in the CL, respectively. Following retrieval of CL from the ovary, flash frozen in liquid nitrogen and stored at -70 M-BM-0C for the purpose of RNA isolation. The isolated RNA was labeled and hybridized to Affymetrix GeneChip Bovine Genome Arrays as per the manufacturerM-bM-^@M-^Ys instructions.

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

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

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