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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 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 ?-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.

SUBMITTER: Liu Y

PROVIDER: S-EPMC6805561 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Liu Ying Y Wang Xuemei X Zhen Zhen Z Yu Yanbo Y Qiu Youwen Y Xiang Wensheng W

Cellular & molecular biology letters 20191022

<h4>Background</h4>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.<h4>Methods</h4>The expressions of GRP78 in BMECs stimulated with methionine, leucine, e ...[more]

PMID: 31660059

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Project description:ObjectiveTCP1 is one of the eight subunits of the TCP1 ring complex (TRiC) or the multi-protein mammalian cytosolic chaperone complex. TRiC participates in protein folding and regulates the expression of multiple signaling proteins and cytoskeletal components in cells. Although the clinical importance of its subunits has been clarified in various carcinomas, the function of TCP1 in ovarian cancer (OC) remains unclear. We aimed to identify the association between the expression of TCP1 and the development of epithelial OC (EOC) and patient prognosis, and explore the underlying mechanisms of TCP1 on the tumor progression of OC cells.MethodsTCP1 protein expression was tested in various ovarian tissues by immunohistochemistry, and the correlation between TCP1 expression and clinical physiologic or pathologic parameters of patients with EOC was analyzed. The relationship between TCP1 expression and the prognosis of patients with OC was investigated and analyzed using the Kaplan-Meier (KM) plotter online database. The expression level of TCP1 was then tested in different OC cell lines by Western blotting. Further, a model using OC cell line A2780 was constructed to study the functions of TCP1 in growth, migration, and invasion of human EOC cells. Finally, the possible regulating signaling pathways were discussed.ResultsTCP1 protein expression in OC or borderline tissues was significantly higher than that in benign ovarian tumors and normal ovarian tissue. The upregulated expression of TCP1 in OC was positively associated with the differentiation grade and FIGO stage of tumors and predicted poor clinical outcomes. Compared with IOSE-80 cells, TCP1 protein was overexpressed in A2780 cells. TCP1 knockdown using shRNA lentivirus inhibited the viability of A2780 cells. Western blotting showed that the phosphatidylinositol-3 kinase (PI3K) signaling pathway was activated in the tumor invasion in EOC driven by TCP1.ConclusionUpregulated TCP1 is correlated with the poor prognosis of patients with OC. The mechanism of cancer progression promoted by TCP1 upregulation may be linked to the activation of the PI3K signaling pathway, and TCP1 may serve as a novel target for the treatment of OC.

Project description:Primordial follicle pool established perinatally is a non-renewable resource which determines the female fecundity in mammals. While the majority of primordial follicles in the primordial follicle pool maintain dormant state, only a few of them are activated into growing follicles in adults in each cycle. Excessive activation of the primordial follicles accelerates follicle pool consumption and leads to premature ovarian failure. Although previous studies including ours have emphasized the importance of keeping the balance between primordial follicle activation and dormancy via molecules within the primordial follicles, such as TGF-β, E-Cadherin, mTOR, and AKT through different mechanisms, the homeostasis regulatory mechanisms of primordial follicle activation remain unclear. Here, we reported that HDAC6 acts as a key negative regulator of mTOR in dormant primordial follicles. In the cytoplasm of both oocytes and granulosa cells of primordial follicles, HDAC6 expressed strong, however in those activated primordial follicles, its expression level is relatively weaker. Inhibition or knockdown of HDAC6 significantly promoted the activation of limited primordial follicles while the size of follicle pool was not affected profoundly in vitro. Importantly, the expression level of mTOR in the follicle and the activity of PI3K in the oocyte of the follicle were simultaneously up-regulated after inhibiting of HDAC6. The up-regulated mTOR leads to not only the growth and differentiation of primordial follicles granulosa cells (pfGCs) into granulosa cells (GCs), but the increased secretion of KITL in these somatic cells. As a result, inhibition of HDAC6 awaked the dormant primordial follicles of mice in vitro. In conclusion, HDAC6 may play an indispensable role in balancing the maintenance and activation of primordial follicles through mTOR signaling in mice. These findings shed new lights on uncovering the epigenetic factors involved physiology of sustaining female reproduction.

Project description:BackgroundUbiquitin-conjugating enzyme E2C (UBE2C) has been shown to be associated with the occurrence of various cancers and involved in many tumorigenic processes. This study aimed to investigate the specific molecular mechanism through which UBE2C affects breast cancer (BC) proliferation.MethodsBC-related datasets were screened according to filter criteria in the Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA) database. Then differentially expressed genes (DEGs) were identified using Venn diagram analysis. By using DEGs, we conducted the following analyses including Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI), and survival analysis, and then validated the function of the hub gene UBE2C using quantitative reverse transcription-polymerase chain reaction (RT-qPCR), cell counting kit-8 (CCK-8) assay, transwell assay, and Western blot assay.ResultsIn total, 151 DEGs were identified from the GEO and TCGA databases. The results of GO analysis demonstrated that the DEGs were significantly enriched with mitotic nuclear division, lipid droplet, and organic acid-binding. KEGG analysis showed that the peroxisome proliferators-activated receptor (PPAR) signaling pathway, regulation of lipolysis in adipocytes, and proximal tubule bicarbonate reclamation were significantly enriched in the signal transduction pathway category. The top three hub genes that resulted from the PPI network were FOXM1, UBE2C, and CDKN3. The results of survival analysis showed a close relationship between UBE2C and BC. The results of CCK-8 and transwell assays suggested that the proliferation and invasion of UBE2C knockdown cells were significantly inhibited (P < 0.050). The results of Western blot assay showed that the level of phosphorylated phosphatase and tensin homology deleted on chromosome 10 (p-PTEN) was obviously increased (P < 0.050), whereas the levels of phosphorylated protein kinase B (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR), and hypoxia-inducible factor-1 alpha (HIF-1α) were dramatically decreased (P < 0.050) in the UBE2C knockdown cell.ConclusionUBE2C can promote BC proliferation by activating the AKT/mTOR signaling pathway.

Dataset Information

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

Publications

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

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