GRP78 regulates milk biosynthesis and the proliferation of bovinemammaryepithelial cells through the mTOR signaling pathway

Abstract Background Glucose-regulated protein 78 (GRP78) is a member of the HSP70 protein family and a key endoplasmic reticulum chaperone. It has been revealed to play important roles both in the maturation, folding and transport of proteins and in cellproliferation. However, its involvement in mil...

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Main Authors: Ying Liu, Xuemei Wang, Zhen Zhen, Yanbo Yu, Youwen Qiu, Wensheng Xiang
Format: Article
Language:English
Published: BMC 2019-10-01
Series:Cellular & Molecular Biology Letters
Subjects:
Online Access:http://link.springer.com/article/10.1186/s11658-019-0181-x
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author Ying Liu
Xuemei Wang
Zhen Zhen
Yanbo Yu
Youwen Qiu
Wensheng Xiang
author_facet Ying Liu
Xuemei Wang
Zhen Zhen
Yanbo Yu
Youwen Qiu
Wensheng Xiang
author_sort Ying Liu
collection DOAJ
description Abstract Background Glucose-regulated protein 78 (GRP78) is a member of the HSP70 protein family and a key endoplasmic reticulum chaperone. It has been revealed to play important roles both in the maturation, folding and transport of proteins and in cellproliferation. However, its involvement in milk biosynthesis or the proliferation of bovine primary mammary epithelial cells (BMECs) has yet to be established. Methods The expressions of GRP78 in BMECs stimulated with methionine, leucine, estrogen and prolactin were determined using western blotting and immunofluorescence assays. To explore the function of GRP78 in BMECs, the protein was overexpressed or knocked down, respectively using an overexpression vector or an siRNA mixture transfected into cells cultured in vitro. Flow cytometry was used to analyze cell proliferation and cell activity. The contents of lactose and triglyceride (TG) secreted from the treated BMECs were measured using lactose and TG assay kits, respectively. Western blotting analysis was used to measure the β-casein content and the protein levels of the signaling molecules known to be involved in milk biosynthesis and cell proliferation. Results GRP78overexpression significantly stimulated milk protein and milk fat synthesis, enhanced cell proliferation, positively regulated the phosphorylation of mammalian target of rapamycin (mTOR), and increased the amount of protein of cyclinD1andsterol regulatory element-binding protein 1c (SREBP-1c). GRP78 knockdown after siRNA transfection had the opposite effects. We further found that GRP78 was located in the cytoplasm of BMECs, and that stimulating methionine, leucine, estrogen and prolactin expression led to a significant increase in the protein expression of GRP78 in BMECs. Conclusions These data reveal that GRP78 is an important regulator of milk biosynthesis and the proliferation of BMECs through the mTOR signaling pathway.
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spelling doaj.art-78174062cf1e4b00b5895b836950f5b02022-12-21T22:30:32ZengBMCCellular & Molecular Biology Letters1425-81531689-13922019-10-0124111210.1186/s11658-019-0181-xGRP78 regulates milk biosynthesis and the proliferation of bovinemammaryepithelial cells through the mTOR signaling pathwayYing Liu0Xuemei Wang1Zhen Zhen2Yanbo Yu3Youwen Qiu4Wensheng Xiang5The Key Laboratory of Dairy Science of Education MinistryHaiNanUniversityThe Key Laboratory of Dairy Science of Education MinistryThe Key Laboratory of Dairy Science of Education MinistryThe Key Laboratory of Dairy Science of Education MinistryThe Key Laboratory of Dairy Science of Education MinistryAbstract Background Glucose-regulated protein 78 (GRP78) is a member of the HSP70 protein family and a key endoplasmic reticulum chaperone. It has been revealed to play important roles both in the maturation, folding and transport of proteins and in cellproliferation. However, its involvement in milk biosynthesis or the proliferation of bovine primary mammary epithelial cells (BMECs) has yet to be established. Methods The expressions of GRP78 in BMECs stimulated with methionine, leucine, estrogen and prolactin were determined using western blotting and immunofluorescence assays. To explore the function of GRP78 in BMECs, the protein was overexpressed or knocked down, respectively using an overexpression vector or an siRNA mixture transfected into cells cultured in vitro. Flow cytometry was used to analyze cell proliferation and cell activity. The contents of lactose and triglyceride (TG) secreted from the treated BMECs were measured using lactose and TG assay kits, respectively. Western blotting analysis was used to measure the β-casein content and the protein levels of the signaling molecules known to be involved in milk biosynthesis and cell proliferation. Results GRP78overexpression significantly stimulated milk protein and milk fat synthesis, enhanced cell proliferation, positively regulated the phosphorylation of mammalian target of rapamycin (mTOR), and increased the amount of protein of cyclinD1andsterol regulatory element-binding protein 1c (SREBP-1c). GRP78 knockdown after siRNA transfection had the opposite effects. We further found that GRP78 was located in the cytoplasm of BMECs, and that stimulating methionine, leucine, estrogen and prolactin expression led to a significant increase in the protein expression of GRP78 in BMECs. Conclusions These data reveal that GRP78 is an important regulator of milk biosynthesis and the proliferation of BMECs through the mTOR signaling pathway.http://link.springer.com/article/10.1186/s11658-019-0181-xCyclin D1GRP78Bovine mammary epithelial cellsmTORSREBP-1c
spellingShingle Ying Liu
Xuemei Wang
Zhen Zhen
Yanbo Yu
Youwen Qiu
Wensheng Xiang
GRP78 regulates milk biosynthesis and the proliferation of bovinemammaryepithelial cells through the mTOR signaling pathway
Cellular & Molecular Biology Letters
Cyclin D1
GRP78
Bovine mammary epithelial cells
mTOR
SREBP-1c
title GRP78 regulates milk biosynthesis and the proliferation of bovinemammaryepithelial cells through the mTOR signaling pathway
title_full GRP78 regulates milk biosynthesis and the proliferation of bovinemammaryepithelial cells through the mTOR signaling pathway
title_fullStr GRP78 regulates milk biosynthesis and the proliferation of bovinemammaryepithelial cells through the mTOR signaling pathway
title_full_unstemmed GRP78 regulates milk biosynthesis and the proliferation of bovinemammaryepithelial cells through the mTOR signaling pathway
title_short GRP78 regulates milk biosynthesis and the proliferation of bovinemammaryepithelial cells through the mTOR signaling pathway
title_sort grp78 regulates milk biosynthesis and the proliferation of bovinemammaryepithelial cells through the mtor signaling pathway
topic Cyclin D1
GRP78
Bovine mammary epithelial cells
mTOR
SREBP-1c
url http://link.springer.com/article/10.1186/s11658-019-0181-x
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