GSK3β Regulates Milk Synthesis in and Proliferation of Dairy Cow Mammary Epithelial Cells via the mTOR/S6K1 Signaling Pathway

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase, whose activity is inhibited by AKT phosphorylation. This inhibitory phosphorylation of GSK3 beta can in turn play a regulatory role through phosphorylation of several proteins (such as mTOR, elF2B) to promote protein synthesis. mTOR is a key regulator in protein synthesis and cell proliferation, and recent studies have shown that both GSK3 beta and mTORC1 can regulate SREBP1 to promote fat synthesis. Thus far, however, the cross talk between GSK3 beta and the mTOR pathway in the regulation of milk synthesis and associated cell proliferation is not well understood. In this study the interrelationship between GSK3 beta and the mTOR/S6K1 signaling pathway leading to milk synthesis and proliferation of dairy cow mammary epithelial cells (DCMECs) was analyzed using techniques including GSK3 beta overexpression by transfection, GSK3 beta inhibition, mTOR inhibition and methionine stimulation. The analyses revealed that GSK3 beta represses the mTOR/S6K1 pathway leading to milk synthesis and cell proliferation of DCMECs, whereas GSK3 beta phosphorylation enhances this pathway. Conversely, the activated mTOR/S6K1 signaling pathway downregulates GSK3 beta expression but enhances GSK3 beta phosphorylation to increase milk synthesis and cell proliferation, whereas inhibition of mTOR leads to upregulation of GSK3 beta and repression of GSK3 beta phosphorylation, which in turn decreases milk synthesis, and cell proliferation. These findings indicate that GSK3 beta and phosphorylated GSK3 beta regulate milk synthesis and proliferation of DCMECs via the mTOR/S6K1 signaling pathway. These findings provide new insight into the mechanisms of milk synthesis.