Seryl-tRNA synthetase-mediated essential amino acids regulate β-casein synthesis via cell proliferation and mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary epithelial cells

Abstract

Essential amino acids (EAA) play an important role in promoting milk protein synthesis in primary bovine mammary epithelial cells (BMEC). However, the regulatory mechanisms involved in the relationship between EAA and milk protein synthesis have not been fully explored. This study examined the effects of seryl-tRNA synthetase (SARS) on EAA-stimulated β-casein synthesis, cell proliferation, and the mammalian target of rapamycin (mTOR) system in BMEC. First, BMEC were cultured in medium either lacking all EAA (-EAA) or that included all EAA (+EAA) for 12 h. The BMEC were then supplemented with the opposing treatments (-EAA supplemented with +EAA and vice versa) for 0 h, 10 min, 0.5 h, 1 h, 6 h, or 12 h, respectively. After the treatment-specific time allotment, proteins were collected for Western blotting. Subsequently, a 2 × 2 factorial design was used to evaluate the interactive of SARS inhibition (control or SARS inhibited) and EAA supply (+EAA or -EAA) on gene and protein abundance, cell viability, and cell cycle in BMEC. Based on the data obtained in the first experiment, the changes in protein abundance of β-casein and SARS depended on EAA treatment time in similar patterns. The protein abundance of β-casein, SARS, and mammalian target of rapamycin (mTOR)-related proteins, cell viability, cell cycle progression, and the mRNA abundance of cyclin D1 (CCND1, cell cycle progression marker) and marker of proliferation Ki-67 (MKI67, cell proliferation marker) were stimulated by the presence of EAA. Correspondingly, when cells were deprived of EAA, cell proliferation and abundance of these proteins and genes were reduced overall. Moreover, the decreases in these aspects were further exacerbated by inhibiting SARS, suggesting that an interaction between EAA and SARS is important for regulating protein synthesis. The results indicated that SARS stimulated the mTOR signaling pathway when EAA were present, enhanced EAA-stimulated cell proliferation, and contributed to increased β-casein production in BMEC.

Seryl-tRNA synthetase is involved in methionine stimulation of β-casein synthesis in bovine mammary epithelial cells

“…Indeed, we showed that SARS is involved in Met stimulation of cell viability, cell cycle progression, β-casein and total protein synthesis as well as in Met inhibition of protein degradation because SARS knockdown altered the effects of Met. These results were in line with our previous finding that the knockdown of SARS highly reduced cell proliferation, β-casein and protein turnover in BMEC without EAA supplementation (31) . Additionally, the reduction of β-casein under SARS knockdown in the present study was in agreement with the finding of leucyl-tRNA synthetase (23) , whose knockdown depressed β-casein protein production in BMEC, especially under leucine and Met deprivation.…”

“…Here in order to acquire the prominent effects of each measured item, we selected the various lengths of incubation times of BMEC. Because, our previous study (31) and this work in the following Fig. 1 found that 6 h treatment of EAA/Met deprivation and sufficiency in BMEC both demonstrated the most prominent effects on the protein expression and of SARS and casein.…”

“…Extensive studies demonstrated that AA play a crucial role in the regulation of milk protein synthesis through cell proliferation and the mTOR signalling pathway in vitro (2,17,18,37,38) , but the mechanisms how AA regulate milk protein production in BMEC require further studies. Our recent work demonstrated a potential regulatory role of SARS in milk protein production in the MG of dairy cows (26,27,31) . In the present study, our data revealed that SARS may be involved in affecting the mTOR and GCN2 routes by Met, thereby regulating milk protein synthesis in BMEC.…”

“…As protein turnover undergoes a dynamic process, the cellular balance between protein synthesis and degradation needs two and more days (32) . Based on our previous studies (31,33) , we usually treated bovine MG for 2 d to determine their protein turnover rates. Taking consideration of the theoretical and practical knowledge, here we chose 2 d for protein turnover assay.…”

Activation of G Protein-coupled Receptor 30 Promotes Proliferation of Goat Mammary Epithelial Cells via MEK/Erk&PI3K/Akt Signaling Pathway

“…Normal cell-cycle progression is a crucial event for every multicellular organism, due to it deciding body size and shape, tissue renewal and senescence, and is also important for reproduction [16], which is closely related to cell proliferation. In bovine mammary epithelial cells, an abundance of S and G2 phases [17,18] and up-regulated expression of cyclin D1 mRNA [17] were accompanied in the promotion of cell proliferation. In addition, G1 promoted the proliferation of GPR30-positive human thyroid cancer cells and upregulated the expression of cyclin A and D1 [19].…”