Effect And Mechanism Of Kisspeptin-10 On The CSN2 Synthesis Of Bovine Mammary Epithelial Cells

Milk protein is the main material basis of milk nutrition.Milk protein content is an important symbol of the core competitiveness of dairy industry.Proteins and peptides play important roles in the synthesis of milk protein.Kps(Kisspeptins)encoded by KISS-1 which is a tumor suppressor gene,is a peptide hormone which can pyrolysis to several small peptide in the body,such as Kp-10(Kisspeptin-10)and other small peptides,these peptides both works by binding with the specific G Protein Coupled Receptor 54(GPR-54).The effects of Kps are mainly involved in regulating the secretion of pituitary hormone,seasonal reproduction,energy balance and puberty initiation and some importment bioligical activites.Because of that Kps has become one of the current hot topics in the study of neuroendocrine physiology.Existing research shows that Kps in some cells can activate ERK1/2 and mTOR,both of them are the key signaling pathways in the process of milk protein synthesis,but about the effect on milk protein synthesis in bovine mammary epithelial has not been reported.Therefore,this experiment analyzed the relationship between Kps receptor GPR54 level and beta-casein(CSN2)content in high quality group and low quality group in mammary tissues of dairy cows,and defined the correlation between Kps receptor GPR54 and CSN2 content.Then,we isolated bMECs,and added Kp-10 and/or signal blockers to study the effect and mechanism of Kp-10 on CSN2 synthesis in bMECs.First of all,we detected the CSN2 and GPR54 genes and proteins expression of high protein and low protein group of bovine mammary gland organization,and speculated the correlation between Kps and milk protein synthesis,the results showed that CSN2 and GPR54 gene and protein expression of mammary gland tissue in high milk protein group were significantly higher than that of low protein group,the correlation analysis showed that GPR54 level and CSN2 content was significantly positively related,which can be speculated that Kps could promote CSN2 synthesis by its receptor GPR54.Then,we isolated and cultured bMECs from Holstein cows of lactation through the tissue block culture method,and used the CK-18 antibody to identify the type by immunofluorescence expriement,and used flow cytometry to test the purity of isolated bMECs,and then we detected CSN2 and GPR54 expression in bMECs from 1-4 generations by quantitative real-time PCR and Western blot.The results showed that the cultured cells were bMECs with a purity of 95.11%.CSN2 and GPR54 were stable expressed in bMECs from 1-4 generations.These results indicated that the purity of isolated bMECs could meet experimental requirements,and the cells could stable express the GPR54 and CSN2,providing a basis for promoting the synthesis of CSN2 by Kps.Further,we used a small peptide hormone Kp-10 to carry out subsequent experiments.The study found that different concentrations Kp-10(0-100 nM)will affect the CSN2 expression in bMECs,and Kp-10 in 100 nM can significantly promote CSN2 gene and protein level,Peptide-234 that a specificity blocker of GPR54 can inhibit the above effects.All of above data exemplified that Kp-10 promoted the synthesis of CSN2 in bMECs by activating GPR54;Further studies have found that Kp-10 can activate mTOR,AKT,STAT5 and ERK1/2 such the secritical signaling pathways were involved in the process of milk protein synthesis in bMECs,and both of them can inhibit the effect of Kp-10 on CSN2 synthesis after treatment with corresponding blockers.The above results show that Kp-10 has the effect of promoting CSN2 synthesis in bMECs,which is accomplished by activating GPR54 and its downstream signal mTOR,AKT,STAT5 and ERK1/2.