Study On Regulating Sheep Chorionic Trophoblast Cells Fusion By EnJSRV-Env Through Activating MAPK Signaling Pathway

The genomes of endogenous retrovirus(ERVs)have been widely found in the genomes of a variety of vertebratas.These endogenous retroviruses are closely related to the development of their host placenta.At least 27 endogenous retroviruses associated with exogenous sheep lung adenoma retroviruses have been found in the sheep genomes and called endogenous jaagsiekte sheep retrovirus(enJSRVs),the structures of these endogenous retroviruses correspond to exogenous retroviruses.EnJSRV plays an important role in the sheep placenta,and enJSRV envelope protein(Env)can promote the process of fusion of sheep chorionic trophoblast cells,but the mechanism is not clear yet.In this study,the expression of enJSRV envelope protein in sheep placenta was detected by immunohistochemistry,and then transiently transfected the recombinant plasmid pEGFP-C1/enJSRV-env into sheep chorionic trophoblast cells,treated with Erk inhibitor,p38 inhibitor and JNK inhibitor treatment.The phosphorylated levels of Erkl/2,p38 and JNK were detected by Western Blot,and analysis of whether enJSRV-Env activated MAPK signaling pathway in sheep chorionic trophoblast cells.The differences of cell-cell fusion among the groups with three inhibitors treatments and the groups without inhibitors were observed by laser confocal microscopy.Then we detected the phosphoiylated levels of Erkl/2,p38 and JNK in 293T cells transiently transfected with the recombinant plasmid pEGFP-C1/enJSRV-env,and compared with the results of sheep chorionic trophoblast cells,then the mechanism of enJSRV-Env in the different cells was analyzed preliminary.The results showed that enJSRV-Env was mainly expressed in sheep chorionic trophoblast cells.The phosphorylated levels of Erkl/2,p38 and JNK in sheep chorionic trophoblast cells transiently transfected with the recombinant plasmid pEGFP-C1/enJSRV-env were significant higher than the control group and the groups with inhibitors treatments(P<0.05).The phosphorylation of three protein kinases were significantly inhibited after adding three inhibitors.We observed 1.6 sheep binucleated chorionic trophoblast cells in average per field of the group transiently transfected with the recombinant plasmid pEGFP-C1/enJSRV-env,0.6 sheep binucleated chorionic trophoblast cells with Erk inhibitor treatment,0.8 sheep binucleated chorionic trophoblast cells with p38 inhibitor treatment and 0.8 sheep binucleated chorionic trophoblast cells with JNK inhibitor treatment.We detected the phosphoiylated level of p38 in 293T cells transiently transfected with the recombinant plasmid pEGFP-C1/enJSRV-env was significant higher than the control group(P<0.05),and the phosphorylated level of JNK in 293T cells transiently transfected with the recombinant plasmid pEGFP-Cl/enJSRV-env was extremely significant higher than the control group(P<0.01).The results showed that enJSRV-Env activated MAPK signaling pathway in sheep chorionic trophoblast cells and may regulate the process of fusion of sheep chorionic trophoblast cells through activating MAPK signaling pathway.The mechanism of enJSRV-Env may be different in the different kinds of cells.