14-3-3γ Regulates Milk Protein Synthesis And Cell Proliferation In Dairy Cow Mammary Epithelial Cells

Milk protein content is an important indicator of milk quality which is the major benefits symbol of dairy industry. Milk protein content of Chinese cows are commonly low, therefore it is urgent to clarify the mechanism of milk protein synthesis in order to find effective technical ways to improve milk quality. Milk protein synthesis mechanism is one of the important topics in the field of molecular biology and lactation physiology. Domestic and international research have achieved some significant progress, and confirmed that JAK/Stat5 pathway and m TOR/S6K1 pathway to be essential for milk protein synthesis. In addition, mammary epithelial cell growth and proliferation directly affect the synthesis of lactose, milk protein and fat. 14-3-3 proteins are widely expressed in living tissues and cells, they involve in many biological processes including cell growth, cell cycle, proliferation, metabolism, signal transduction, gene transcription and protein synthesis. 14-3-3 proteins dictate their substrates’ subcellular localization to promote or inhibit signal transduction, such as mTOR pathway. 14-3-3 proteins interact with many substrate proteins and have different functions in vivo. Despite extensive research has been carried out, it is still necessary to performed in-depth exploration on the exact mechanisms of 14-3-3 proteins regulating protein synthesis and cell proliferation. In a previous research from our lab, 14-3-3γ was shown to associate with milk protein synthesis using two-dimensional gel electrophoresis and mass spectrometry analysis. This suggests that 14-3-3γ may be involved in regulation of milk protein synthesis. In this study, we analyzed the function of 14-3-3γ in lactation regulation and proliferation regulation, further identified proteins interact with 14-3-3γ. This study aims to reveal the molecular mechanism of 14-3-3γ regulating milk protein synthesis and cell proliferation via interacting proteins.Detections of cytokeratin 18 and β-casein were performed by imunofluorescence to identify the purity and ability to synthesize milk protein in dairy mammary epithelial cell(DCMECs). Cells were treated with Met(0.6mM) for 24 h, we detected the effects of methionine on 14-3-3γ and β-casein expression by western blotting analysis and detected the effects of methionine on 14-3-3γ expression by immunofluorescence analysis. We detected the effects of methionine on cell number and cell viability using Casy® Model DT cell counter and detected the effects of methionine on proliferation index and cell cycle by flow cytometric analysis. Further we constructed eukaryotic expression vector of 14-3-3γ to perform overexpression experiments and synthesized inhibition fragments of 14-3-3γ to perform inhibition experiments. We detected the protein levels of mTOR, p-m TOR, Stat5 a, p-Stat5 a, Cyclin D1 and β-casein after 14-3-3γ overexpression and inhibition by western blotting analysis. We detected the levels of p-m TOR and p-Stat5 a after 14-3-3γ overexpression and inhibition by immunofluorescence analysis. Further we detected the effects of 14-3-3γ overexpression and inhibition on cell number and cell viability using Casy® Model DT cell counter and detected the effects of methionine on proliferation index and cell cycle by flow cytometric analysis. Further we performed coprecipitation experiments and mass spectrometry analysis to find the proteins interact with 14-3-3γ and relates to milk protein synthesis. We further explore effects of 14-3-3γ overexpression and inhibition on interacting proteins, and whether interacting proteins have effects on milk protein synthesis and cell proliferation.This study determined the effects of methionine on 14-3-3γ expression and cell proliferation. The results showed that protein expression of 14-3-3γ and β-casein significantly increased after methionine stimulation. Met stimulation also promoted viability and increased cell number. In addition, the proliferation index and percentage of cells in S and G2-M phases all increased whereas percentage of cells in G0-G1 phase decreased after methionine stimulation, further demonstrating methionine stimulation promoted cell proliferation. This suggests that 14-3-3γ associates with milk protein synthesis and cell proliferation.The experiment further examined the effects of 14-3-3γ overexpression and inhibition on milk protein synthesis and cell proliferation. The results showed that 14-3-3γ overexpression upregulated the protein levels of mTOR, Stat5 a and β-casein, and promoted both m TOR and Stat5 a to be phosphorylated. Results of 14-3-3γ inhibition were just the opposite. This indicates 14-3-3γ can regulate milk protein synthesis through mTOR and Stat5 a. 14-3-3γ overexpression also increased cell number and promoted viability. In addition, the proliferation index and percentage of cells in S and G2-M phases all increased whereas percentage of cells in G0-G1 phase decreased after 14-3-3γ overexpression. Results of 14-3-3γ inhibition were just the opposite. In addition, overexpression and inhibition of 14-3-3γ showed that 14-3-3γ positively regulates Cyclin D1 expression. These results indicate 14-3-3γ positively regulates phosphorylation levels of m TOR and Stat5 a thus to promote milk protein synthesis and cell proliferation.This experiment identified proteins interact with 14-3-3γ, the results showed that 44 proteins immunoprecipitated with 14-3-3γ, belonging to 8 categories, proteins associate with transcription, translation, signal transduction, proliferation, cell cycle, transporter, cytoskeleton and enzymes. Protein translation is crucial for milk protein synthesis, so we chose e IF1 AX, RPS7 and e IF5 as research objects, and verified whether they really interact with 14-3-3γ. We utilized western blotting, immunofluorescence colocalization and fluorescence resonance energy transfer analysis to verify whether there is a direct interaction between these three proteins and 14-3-3γ. The results showed that e IF1 AX, RPS7 and e IF5 specifically bind to 14-3-3γ, the overlap coefficients of 14-3-3γ with e IF1 AX, RPS7 and e IF5 were all over 90%, and the mean FRET efficiency value of 14-3-3γ with the three proteins were 20%-35%, which were much higher than controls(<5%). These results clearly indicate that 14-3-3γ directly interacts with e IF1 AX, RPS7 and eIF5, and regulation of 14-3-3γ on lactation may be related to interaction between these three proteins.This study further examined the effects of methionine on e IF1 AX, RPS7 and e IF5 expression. The results showed that methionine treatment resulted in significantly higher levels of 14-3-3γ, e IF1 AX, RPS7, e IF5 and β-casein, which suggests that e IF1 AX, RPS7 and e IF5 are probably related to protein synthesis.We also examined the effects of 14-3-3γ overexpression and inhibition on e IF1 AX, RPS7 and e IF5 expression. The results showed that 14-3-3γ overexpression increased the expression of e IF1 AX, RPS7 and e IF5. Conversely, inhibition of 14-3-3γ downregulated the expression of e IF1 AX, RPS7 and e IF5. In addition, 14-3-3γ overexpression increased the expression of e IF2α but decreased the level of p-e IF2α. Results of 14-3-3γ inhibition were just the opposite. We then carried out e IF1 AX, RPS7 and e IF5 overexpression respectively to give further evidence. We found that after e IF1 AX, RPS7 and e IF5 overexpression, both e IF2α and β-casein were increased, whereas p-e IF2α significantly decreased. These data suggest that 14-3-3γ affects expression of e IF1 AX, RPS7 and e IF5 to regulate milk protein synthesis and cell proliferation in dairy cow mammary epithelial cells.