Regulation Mechanism Of Serotonin On Proliferation And Apoptosis And Casein Synthesis In Bovine Mammary Epithelial Cells(MAC-T)

Serotonin(5-HT) has been demonstrated to be an important local regulator of lactational homeostasis and involution.Whereas, the effect and mechanisms of 5-HT on the proliferation and apoptosis of bovine mammary epithelial cell are poorly understood. The bovine mammary gland expresses multiple functional isoforms of serotonin receptors(HTR1B, 2A,2B, 4 and 7), and expression of milk protein levels were stimulated by blocking HTR1 B and HTR2 A with the selective antagonists. However its mechanism has not been reported at present. In this study, we use bovine mammary epithelial cells(MAC-T cells) as a model, to determine the effect of 5-HT on cell proliferation and apoptosis of MAC-T, ascertain its mechanism, and explore intracellular signal pathway of 5-HT regulating milk protein synthesis.First of all, MAC-T cells was subcultured and identified. The cells showed a high rate of replication and a cobblestone-like appearance at confluence, and growth curve presented an“S” shape. Cytoskeleton protein(cytokeratin-18) was detected in the cells. Treating with PRL and INS, casein was detected at mRNA and protein level. After continuous passage, freezing and thawing, little change in cell morphology and function was found. mRNA of 5-HT synthesis rate-limiting enzyme-TPH1, 5-HT1 B, 2A, 2B, 4,7,5A, 3A, 1F, 1D, 2C, 1E, 1A receptors subtype were detected by RT-PCR. These findings suggested that MAC-T cells could synthesize and secrete casein, 5-HT and express multiple functional isoforms of serotonin receptors, which showed that MAC-T cells could be used as a model in this study.In order to determine the effect of 5-HT on cell proliferation and apoptosis of MAC-T,MAC-T cells were treated cells with various concentrations of 5-HT for different times(12h,24 h, 48 h, 72h), then cell proliferation was evaluated by CCK-8 kit. It was found that short-time(12h, 24 h, 48h) 5-HT treatment promoted MAC-T cell proliferation, whreas sustained(72h) 5-HT treatment inhibited MAC-T cells proliferation. To probe the functional activities of individual receptors, MAC-T cells were treated with selective 5-HTR2B(SB204741), 5-HTR2A(Ritanserin), 5-HTR4(SB204070), 5-HTR7(Pimozide), 5-HTR1B(SB224289) antagonist for 24 h. The results showed that MAC-T proliferation induced by5-HT was inhibited significantly by SB224289 and SB204070, but not affected by Ritanserin,SB204741 or Pimozide. Treatment cells with 200μM 5-HT for 72 h and 20μM 5-HT for 24 h respectively, the relative expressions of cell proliferation and apoptosis-related factors such as caspase3, caspase8, Bax, Bcl-2 were determined by real-time quantitative PCR(RT-qPCR).The results demonstrated that treatment cells with 200μM 5-HT for 72 h significantly inhibited the mRNA expression of Bcl-2, increased the mRNA expression of caspase3 and caspase8,and heightened Bax / Bcl-2 values. While treatment cells with 200μM 5-HT for 24 h significantly reduced the mRNA expression of caspase3 and Bax / Bcl-2 values. These results suggested that 5-HT influenced MAC-T cell proliferation and apoptosis by changing expression level of caspase3 and the ratio of Bax / Bcl-2.In order to explore the signaling pathways of 5-HT regulating casein synthesis, the cells treated with 20μM 5-HT for 48 h, the key kinases and regulating factors participated in the milk protein synthesis such as JAK2, STAT5, mTOR, rpS6K1,4EBP1, eIF4 E were detected by RT-qPCR. We found STAT5 was reduced significantly(p<0.05), and the mRNA expression of JAK2 was also reduced, although difference is not significant. However, the relative expression of 4EBP1, eIF4 E, eEF2, mTOR, rpS6 Kl mRNA was not affected by 5-HT. These results indicated that JAK2-STAT5 signaling pathways involved 5-HT regulation of casein synthesis.In summary, at lower concentrations and earlier time points, 5-HT promoted MAC-T cell proliferation, whereas at higher concentrations and later time points, 5-HT inhibited MAC-T cell proliferation. Both Selective 5-HT1 B and 4 antagonists significantly inhibited proliferation of MAC-T induced by 5-HT. Thus, serotonin regulates MAC-T cells proliferation and apoptosis probably though 5-HTR1 B or 5-HTR4 subtype, thereby changing the expression caspase3 and Bax / Bcl-2 value. JAK2-STAT5 signaling pathways involved5-HT regulation of casein synthesis.