Study On The Regulation Mechanism Of Serotonin On Proliferation Of Bovine Mammary Epithelial Cells (MAC-T)

Serotonin(5-HT) had been demonstrated to be an important local regulator of lactational homeostasis and involution. The previous research of our laboratory showed that 5-HT can bidirectional regulate the fate of mammary epithelial cells(MAC-T) by changing the value of Bax/Bcl-2. That was, short-time 5-HT treatment promoted MAC-T cell proliferation, inhibited apoptosis; whreas long-time 5-HT treatment inhibited MAC-T cells proliferation, promoted apoptosis. But the underlying cellular mechanism remains unclear.5-HT is the active substance produced in the breast, and serotonergic action may be related to its transport and metabolism, the receptors and the interaction with other factors. On the basis of previous studies in our laboratory, this paper further explored the mechanism of 5-HT regulating MAC-T cell proliferation from the above three aspects.In order to verify whether Serotonergic action in MAC-T cells related to the process of transport and metabolism,we detected the mRNA expression of MAO and SERT in bovine mammary tissue using RT-PCR technique. In MAC-T cells, we detected the expression of MAO, while we did not detect the expression of SERT. Therefore, serotonergic action in MAC-T cells is not related to the process of transport.Previous studies in our laboratory showed that MAC-T cells express multiple subtypes of5-HT receptors(5-HTR1 B, 2A, 2B, 4, 7, etc.), and that the promotion effect of 5-HT on MAC-T cell proliferation was achieved through 1B and 4 subtype receptors. Based on these findings, the involvements of 5-HTR1B/4 and its downstream signaling pathways were explored in this paper.The results showed that treatment MAC-T cells with 5-HT alone(20?M)for 24 h, the protein expression of mTOR?JAK2 and STAT5 were not affected. But when co-treatment MAC-T cells with 5-HT(20?M) and 1B(SB224289) or 4(SB204070) subtypes receptor antagonist for 24 h, the protein expression of mTOR, eEF2, JAK2, STAT5 significantly decreased.While the protein expression of 4EBP1?S6K1 are not significant. Thissuggests that 5-HT could bind with the 1B and 4 subtypes of receptors, activated mTOR-eEF2 and JAK2-STAT5 signaling pathways, and then promoted the proliferation of MAC-T cells.5-HT do not take effect alone but with other factors in the body. Studies have shown that the prolactin(PRL) increase the expression of TPH1(5-HT synthesis rate-limiting enzyme),and we found that when the cells were cultured in the medium with or not with PRL, the growth curve is totally different. We hypothesized that 5-HT may interact with PRL to regulate the growth of MAC-T cells. Therefore, we co-treated MAC-T cells with PRL and5-HT, and found that both the rate of cell proliferation and the expression levels of mTOR protein were significantly higher than those of 5-HT or PRL- treatment group. These results showed that 5-HT interacted with PRL, to promote the proliferation of MAC-T cells by activating mTOR signaling pathway.In summary, our results suggest that 1)5-HT bind with the 1B and 4 subtypes of receptors,activateding mTOR-eEF2 and JAK2-STAT5 signaling pathways, and then promote the proliferation of MAC-T cells; 2) 5-HT interact with PRL each other to regulate the proliferation of MAC-T cells by activating mTOR signaling pathway.