Prolactin Regulates LAT1 Expression In Mammary Epithelial Cells Of Dairy Cows

Milk protein is an important component of milk,it can provide all necessary amino acids for human growth and development.Milk protein is an important criterion for ev aluating milk quality.The milk with high content of milk protein has become an inevitable trend in healthy food production,which is related to the health of people.In recent years,with the progress of science and technology,milk yield has increased significantly in China.However,compared with the world’s dairy industry in the developed countries,the milk protein content in China is still significantly lower,which seriously restricts China’s dairy industry development.How to increase the content of milk protein has become a hot topic in lactation research.The synthesis of milk protein is regulated by a variety of molecules during the lactation period of dairy cows.Studying the synthesis mechanism of milk protein could help us find a solution to in crease the content of milk protein in raw milk.Prolactin(PRL)is mainly synthesized and secreted from the pituitary gland and the placenta of the female mammal.It is transported through the blood circulation and acts on the mammary gland,which plays a vital role in lactation.During mammalian pregnancy,PRL regulates the growth and differentiation of mammary epithelial cells.During lactation,PRL induces the synthesis and secretion of milk.In the mammary gland,PRL binds to the PRL receptor on the pla sma of the mammary epithelial cells and initiates a series of corresponding intracellular signal transduction cascade effects.It is reported that PRL induced the synthesis of milk protein via the JAK2-STAT5 signaling pathway.L-type amino acid transporter 1(LAT1)is an amino acid transporter which expresses in mammary gland and other organs.It is responsible for transporting large neutral amino acids into cells and is related to intracellular protein synthesis.Although the e xpression of LAT1 is different in various animals,its structure and function are conservative.LAT1 contains multiple cysteine residue transmembrane domains.4F2 heavy chain(4F2hc)can combine with LAT1 by disulfide bond to form a dimer,which makes LAT1 stabilize on the membrane and uptake amino acids into cells.Preliminary studies in our laboratory have shown that LAT1 and 4F2 hc were expressed in mammary tissues.The expression levels of LAT1 and 4F2 hc were significantly higher in lactating cows than that in dry cows,suggesting that LAT1 plays a role in the regulation of mammary gland lactation.However,the precise mechanism of LAT1 on lactation regulation is still poorly understood.The relationship between PRL stimulation and LAT1 expression in mamm ary gland of dairy cow is still unclear.In this study,western blot,q RT-PCR,immunofluorescence,and gene interference were used to investigate the regulation of PRL on LAT1 expression and milk protein synthesis.First,the expression of LAT1 and milk protein in mammary epithelial cells of dairy cows with different PRL concentrations treatment were detected.Then with LAT1 gene interference or BCH inhibition,we detected whether PRL-induced milk protein synthesis is involved in LAT1.Next,we detected whether PRL regulates LAT1 expression via STAT5 signaling pathway.The results showed that PRL stimulation induced LAT1 expression and β-casein synthesis in mammary epithelial cells of dairy cows.5μg/m L PRL is the optimal concentration for inducing the expression of LAT1 and β-casein in vitro.The expression of β-casein decreased when mammary epithelial cells were treated with BCH or LAT1 si RNA in the presence of PRL,indicating that PRL induced milk protein synthesis by LAT1.When mammary epithelial cells were treated with PRLR si RNA or STAT5 si RNA,the expression of LAT1 decreased,which in turn decreased milk protein synthesis.This result indicated that PRL induced LAT1 expression via STAT5 signaling pathway.In this process,PRL also induced the transport of LAT1 to the plasma.This study enriches the basic theories of lactation biology.Studying the relationship among PRL,LAT1,and milk protein synthesis can help us better understand the regulatory mechanism of lactation in dairy cows,which supports the theoretical basis for improving milk quality through artificial means.