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

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.