GSK3β Regulates Lactation And Proliferation Through MTOR/S6K1Pathway In Dairy Cow Mammary Epithelial Cells

In recent years, with the development of the dairy research and healthy consumption needs, the needs of the people for dairy products gradually strengthened. However, high incidence of cow, low of the raw milk quality, have become the main factors which restrict the development of the dairy cattle breeding, also are important problems of the development of dairy industry. Therefore, to improve milk quality, and increase milk production now is the researchers focus on the important scientific problems. Milk protein and milk fat contents and compositions are the important indexes of milk quality, using advanced molecular biological technology, to explore the formation and regulation of milk protein and milk fat contents have gradually become the main research direction of this field. In recent years, the researchers found that downstream of PI3K/AKT, mTOR pathway can not noly regulate cell growth and proliferation, and nutrients and hormones can regulate milk protein synthesis through mTOR pathway; sterol regulatory element binding protein (SREBP) is fat synthesis genes which is an important transcriptional regulation factor. GSK3β gene, however, the same as an important substrates, downstream of AKT, Glycogen synthase kinase30(GSK3β) how to work with mTOR signaling pathways affecting cell proliferation and the synthesis of milk in the cow mammary gland epithelial cells research less, thus we need to do further research on the function and mechanism of GSK3β in mammary gland development and lactation.In this test we successfully build the cow mammary gland epithelial cells in vitro culture model, through GSK3β overexpression and inhibition experiment, using RT-PCR and Western blot techniques to detect GSK3β on lactation related protein expression through mTOR pathway; Application of immunofluorescence staining of cells we detected the expression of GSK3β,p-GSK3β, p-mTOR and SREBP within DCMECs; Using TG kits detected the cell secretion of triglycerides; We applicated of CASY cell dynamic analyzer to test cells vitality and proliferation ability of dairy cattle mammary gland epithelials in vitro; Using flow cytometry we detected the influence of GSK3β on cell cycle, finally we determined the influence of GSK3β on cow mammary gland proliferation of mammary epithelial cells. Through mTOR inhibition test and the treatment of methionine (Met), we also detected the relationship between GSK3β and mTOR/S6K1pathway to milk synthesis.Experimental results showed that GSK3P was distributed in the nucleus and cytoplasm in DCMECs, and mainly expressed in the cytoplasm and the round of nuclear membrane, whereas a few existed in the nucleus. At24h after GSK3β overexpression, lactation related genes mTOR, S6K1, SREBP1, CyclinDl in mRNA and protein expression level were decreased; Compared with the control group, β-casein expression in GSK3β transfection group was decreased, triglycerides secretion of DCMECs, cell viability and proliferation ability, compared with the control group were decreased significantly. Using LiCl, one of the GSK3β specificity protein inhibitor which inhibits the expression of GSK3P notably, we found that p-GSK3β expression were increased, related protein expressions were significantly increased,compared with control; β-casein expression and triglyceride were significantly increased than the control group, and the cell vitality and proliferation ability were promoted. Using mTOR specificity protein inhibitor rapamycin which significantly inhibits the expression of mTOR, we observed the inhibition of the expression of mTOR pathway related protein and synthesis of triglycerides, but no significant change for GSK3β; Inhibition of mTOR at the same time suppressed the activity of GSK3β, also the expression of lactation related proteins, rapamycin also suppressed the promoting effect of LiCl on cow mammary gland epithelial cells. Methionine (0.6mmol/L) significantly increased the expression lactation related protein at mRNA level in DCMECs, and increased triglyceride production significantly, furthermore significantly promoted cell vitality and proliferation, whereas decreased GSK3β expression; Compared with the Met group, in Met+LiCl group the p-GSK3P expression increased significantly, the cell vitality and proliferation were higher, mammary related protein expressions were increased significantly.Above all, GSK3β influences gene expression of lactation signals in mammary epithelial cells such as AKT1, mTOR, S6K1, β-casein, SREBP1and CyclinDl, also inhibites the secretion of triglyceride and cell proliferation. The effects of GSK3β inhibitor LiCl and mTOR inhibitor rapamycin to GSK3β activity have similar effects as the inhibition above. Met anhance mTOR/S6K1signaling pathway and promoted GSK3β phosphorylation inactivation. These results suggest that mTOR/S6K1signaling pathway can phosphorylated deactivation GSK3β, then remove the inhibition, thus promoting the lactation and proliferation of DCMECs.