Pten Regulates Lactation In Dairy Cow Mammary Epithelial Cells

Pten gene is a tumor suppressor gene regulating many cellular processes, including growth, adhesion, and apoptosis. So far there was little research about the function of Pten gene in the development of dairy cow mammary gland. In the aim of detectting the role of Pten gene in the mammary gland of dairy cow, dairy cows mammary epithelial cells (DCMECs) in mid-lactation period were used as models to investigate the relationship of Pten expression and mammary glands development and lactation, which provides basic data for the study of ruminant mammary gland development and lactation mechanisms, and the theoretical support for milk production and milk quality of the artificial regulation at the same time.In this research, Holstein dairy cows were used as experimental animals, applying to qRT-PCR, Western blotting, and immunofiuorescence triple staining technology, Pten mRNA and protein expression at different development stages and various milk qualities of dairy cows mammary gland tissue were detected. Furthermore, DCMECs as research objects in vitro were used to study the function of Pten gene. Recombinant plasmid pGCMV-Pten-IRES-EGFP was constructed and transient transfected into cells to prosue the Pten gene overexpression experiment. Meanwhile, RNAi method was used to transfect Pten siRNA in the Pten gene inhibition experiment. We determined concentrations of β-casein, triglyceride, and lactose following Pten gene overexpression and inhibition by specific kits. To determine whether Pten gene affected DCMEC viability and proliferation, cells were analyzed by CASY-TT and flow cytometry. Genes involved in lactation-related signaling pathways were detected by qRT-PCR and Western blotting. After prolactin and glucose were added to the cell cultures, concentrations of β-casein, triglyceride, and lactose were detected, and Pten gene expression was also assessed. Thus investigating the role of Pten gene in the process of glucose transform into lactose induced by prolactin.The results revealed that the expression of Pten gene in the lactation period decreased significantly compared with dry period. The Pten mRNA and protein expression levels were decreased0.3-and0.4-fold in mammary glands of lactating cows producing high quality milk (milk protein>3.0%, milk fat>3.5%), compared with those cows producing low quality milk (milk protein<3.0%, milk fat<3.5%). When Pten was overexpressed, proliferation of DCMECs and concentrations for β-casein, triglyceride, and lactose were significantly decreased (P<0.05). Overexpression of the PTEN protein down-regulated expression of MAPK, Cyclin D1, AKT, mTOR, S6K1, STAT5, SREBP1, PPARy, PRLR, and GLUT1, but up-regulated4EBP1in DCMECs (P<0.05). The Pten gene inhibition experiment showed results that opposed to those of the gene overexpression experiment, while the expression of ELF5revealed no significant difference both in the Pten gene overexpression and inhibition experiments (P>0.05). Introduction of prolactin increased secretion of P-casein, triglyceride, and lactose, but decreased Pten gene expression levels (P<0.05). Addition of glucose increased both P-casein and lactose concentrations (P<0.05), but did not significantly alter triglyceride concentration and Pten gene expression levels (P>0.05).In summary, we showed that Pten gene is specifically involved in lactation of dairy cow mammary epithelial cells, and down-regulates DCMEC viability, proliferation ability, and the cell cycle along with β-casein, triglyceride, and lactose secretion. Pten gene targets and regulates the PI3K/AKT pathway, which in turn regulates other lactation-related signaling genes. Moreover, the expression of Pten gene can be down-regulated by prolactin, but the introduction of glucose to culture medium revealed no significant difference in Pten gene expression level in DCMECs.