Caihong Wang1, Fengqi Zhao1,2, Jianxin Liu1, Hongyun Liu1. 1. College of Animal Sciences, Zhejiang University, Hangzhou, China. 2. Laboratory of Lactation and Metabolic Physiology, Department of Animal and Veterinary Sciences, University of Vermont, Burlington, New Jersey, USA.
Abstract
BACKGROUND/AIMS: The aim of this study was to investigate the transport properties and utilization of methionyl-methionine dipeptide (Met-Met) in β-casein (β-CN) synthesis in bovine mammary epithelial cells (BMECs). METHODS: The transport properties were studied for the effects of time, pH, concentration, temperature and inhibitors using Met-Met-FITC in BMECs. BMECs were treated with different concentrations of Met-Met (0, 20, 40, 80, 120 and 160 µg/ml). In several experiments, the cells were treated with Janus kinase 2 (JAK2) inhibitor (tyrphostin AG-490, 50 µM) and mammalian target of rapamycin (mTOR) inhibitor (rapamycin, 100 ng/ml). RESULTS: The uptake of Met-Met-FITC by BMECs was rapid during the first fifteen minutes and became saturated after 15 minutes. The transport of Met-Met-FITC in BMECs exhibited a Michaelis constant of 52.4 µM and maximum transport velocity of 14.8 pmol/min/mg protein. The uptake of Met-Met-FITC in BMECs was pH-dependent, peaked at pH 6.5 and was significantly inhibited by other peptides, including Met-Lys, Lys-Lys, Gly-Met, Gly-Leu and Met-Leu. Knocking down the peptide transporter 2 (PepT2) with small interference RNA markedly decreased Met-Met-FITC uptake. Met-Met concentration-dependently increased the PepT2 expression and β-CN synthesis in BMECs with an optimal concentration of 80 µg/ml. At 80 µg/ml, Met-Met also enhanced the cell viability and cyclin D1 expression and promoted cell cycle transition from G1 phase to S phase. In addition, 80 µg/ml Met-Met increased the mRNA abundance of JAK2 and signal transducer and activator of transcription 5 (STAT5) and enhanced the phosphorylation of JAK2, STAT5, mTOR, p70 ribosomal S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1. The inhibition of JAK2 and mTOR significantly decreased Met-Met-induced increase in cell viability and β-CN synthesis in BMECs. CONCLUSION: Our data elucidated the properties of peptide transporter and its effect on β-CN synthesis in BMECs. Met-Met, taken up by PepT2, enhances cell proliferation and promotes β-CN synthesis by activating JAK2-STAT5 and mTOR signaling pathways in BMECs.
BACKGROUND/AIMS: The aim of this study was to investigate the transport properties and utilization of methionyl-methionine dipeptide (Met-Met) in β-casein (β-CN) synthesis in bovine mammary epithelial cells (BMECs). METHODS: The transport properties were studied for the effects of time, pH, concentration, temperature and inhibitors using Met-Met-FITC in BMECs. BMECs were treated with different concentrations of Met-Met (0, 20, 40, 80, 120 and 160 µg/ml). In several experiments, the cells were treated with Janus kinase 2 (JAK2) inhibitor (tyrphostin AG-490, 50 µM) and mammalian target of rapamycin (mTOR) inhibitor (rapamycin, 100 ng/ml). RESULTS: The uptake of Met-Met-FITC by BMECs was rapid during the first fifteen minutes and became saturated after 15 minutes. The transport of Met-Met-FITC in BMECs exhibited a Michaelis constant of 52.4 µM and maximum transport velocity of 14.8 pmol/min/mg protein. The uptake of Met-Met-FITC in BMECs was pH-dependent, peaked at pH 6.5 and was significantly inhibited by other peptides, including Met-Lys, Lys-Lys, Gly-Met, Gly-Leu and Met-Leu. Knocking down the peptide transporter 2 (PepT2) with small interference RNA markedly decreased Met-Met-FITC uptake. Met-Met concentration-dependently increased the PepT2 expression and β-CN synthesis in BMECs with an optimal concentration of 80 µg/ml. At 80 µg/ml, Met-Met also enhanced the cell viability and cyclin D1 expression and promoted cell cycle transition from G1 phase to S phase. In addition, 80 µg/ml Met-Met increased the mRNA abundance of JAK2 and signal transducer and activator of transcription 5 (STAT5) and enhanced the phosphorylation of JAK2, STAT5, mTOR, p70 ribosomal S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1. The inhibition of JAK2 and mTOR significantly decreased Met-Met-induced increase in cell viability and β-CN synthesis in BMECs. CONCLUSION: Our data elucidated the properties of peptide transporter and its effect on β-CN synthesis in BMECs. Met-Met, taken up by PepT2, enhances cell proliferation and promotes β-CN synthesis by activating JAK2-STAT5 and mTOR signaling pathways in BMECs.