Regulation of milk synthesis in the bovine mammary gland: The role of MRCK-alfa in prolactin-and amino acid-evoked lactogenesis

The mechanism of lactogenesis is one of the important fundamental question in life science. Understanding the mechanism of nutrient-mediated regulation of lactogenesis in mammary gland contributes to improve feed utilization efficiency and precision feeding to achieve higher milk yield and milk protein production and less pollution to the environment. Both in vitro and in vivo studies have shown the importance of supply of amino acids (AA), ratio of AA supplied, and the amount of energy on milk synthesis. However, knowledge on the interaction and its regulation mechanism between AA ratio and energy is largely lacking. Besides, it is well known that mTOR and JAK2/STAT5 signaling pathways are involved in regulating milk synthesis in response to the stimulation of nutrients and hormones. However, what other important signaling molecules are involved in transcriptional and translational regulation of milk protein synthesis in mammary cells? How nutrients (e.g., AA) and hormones (e.g., prolactin (PRL)) mediate the regulation of mTOR remain to be addressed. In this thesis, I investigated the effects of different lysine (Lys)/methionine (Met) ratios and glucose levels and their interaction on casein synthesis related gene and protein expression in bovine mammary epithelial cells (BMEC). The results showed that no interactions between Lys/Met ratio and glucose level on casein synthesis were found. Casein synthesis was regulated by Lys/Met ratio via JAK2/ELF5, mTOR and its downstream RPS6KB1 and EIF4EBP1 signaling, while glucose regulated casein synthesis through promoting cell proliferation, accelerating cell cycle progression, and via activating of the ELF5 and AMPK/mTOR signaling pathways. Furthermore, with a central focus on the role of MRCKα in milk synthesis, the regulatory mechanisms of nutrients (Met and leucine (Leu)) and PRL mediating the regulation of mTOR was investigated in BMEC. More than this, the mechanisms of MRCKα in regulating milk protein synthesis, in particular the physical interaction between MRCKα and 14-3-3γ, and their binding role in milk protein synthesis in BMEC were investigated. By silencing and overexpression, MRCKα was shown to be a novel regulator of milk synthesis and cell proliferation in BMEC. The data demonstrate that MRCKα is a critical mediator of prolactin-induced lactogenesis via stimulation of the mTOR/SREBP1/Cyclin D1 signaling pathway. Furthermore, the results indicate that MRCKα is a key mediator of the Met- and Leu-induced signaling cascade, acting downstream of PI3K and upstream of AKT to control β-casein synthesis in BMEC. The co-immunoprecipitation and Western blot analyses verified that 14-3-3γ physically interacts with MRCKα in BMEC. Using silencing and overexpression methods, the results further indicated that 14-3-3γ plays a regulatory role in Met and Leu-induced β-casein synthesis, probably by facilitating the MRCKα-AKT-mTOR signaling pathway. Together, I improved an integrated network of signaling pathways, which advances understanding of regulation of milk synthesis in bovine mammary epithelial cells under stimulation by AA, PRL or glucose.