Study Of EnJSRV Envelope Protein Promotes Fusion Of Sheep Trophoblast Cells And Its Mechanism

Endogenous retrovirus(ERVs)are proviruses which present in all vertebrates.They are derived from the ancient infections of the host germ line by exogenous retroviruses(XRVs).The ovine genome contains approximately 27 copies of endogenous retroviruses(enJSRVs)that are related to the exogenous Jaagsiekte sheep retroviruses(JSRVs),an oncogenic retrovirus tropic to the lung.The envelope protein(Env)of enJSRV could induce the differentiation of mononuclear trophoblast cells into trophoblast giant binucleate cells,fusing with endometrial luminal epithelial cells(LECs)to create nonproliferative multinucleated syncytiotrophoblasts,which ultimately form the cotyledons of the placenta.Therefore,enJSRV-Env plays a critical role in sheep conceptus development and placental morphogenesis.Our group has demonstrated that enJSRV-Env can promote the fusion of sheep chorionic trophoblast cells(STCs),but it was appeared to focus only STCs,ignoring the important role of LECs in placenta formation.Therefore,based on the co-culture of STCs and LECs,this study investigated the effect of enJSRV-Env on the fusion of STCs.Furthermore,the signaling pathway activated by enJSRV-Env which induced cell fusion was explored.The specific research contents and results are as follows:(1)The primary LECs were isolated and cultured by pronase digestion,purified and identified.Then STC_S was fixed at 1×10~4,and LEC_Sand STC_S were added at the ratio of0:1(control),1:1,1:2,1:3,1:4,and 1:5,cultured for 24 h,48 h,72 h respectively.Giemsa staining was performed and 10 fields of view were randomly selected under an inverted microscope to count binucleate and multinucleated cells.The results showed that after co-culturing LEC_S and STC_S at a ratio of 1:1 for 48 h and 72 h,the number of binucleated giant cells and multinucleated cells in the chorionic trophoblast was relatively the highest,and no significant difference in these two time gradients.Considering the need for transfection and protein extraction in the later stage,the optimal co-culture conditions were determined by co-culturing the two cells at a ratio of 1:1 for 48 hours.(2)LECS and STCS were counted and labeled with Carboxyfluorescein succinimidyl aminoester(CFSE)and 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate(Di I)respectively,then they were co-cultured at a ratio of 1:1 for 48 h.Nuclei were stained with DAPI after cell fixation.Under the laser confocal microscope,LEC_S showed green fluorescence,STC_S showed orange fluorescence,nuclei showed blue fluorescence,and fusion cells showed yellow-green fluorescence.This indicated that LEC_S participated in the formation of chorionic trophoblast trinuclear cells.(3)The fusion efficiency of STC_S assessed after transfected into the co-cultured cells with the eukaryotic expression plasmids pEGFP-C1/enJSRV-env,pEGFP-C1/Hyal 2 and their corresponding interference plasmids enJSRV-env-sh RNA-3 and Hyal 2-sh RNA-3respectively.The results showed that the sheep chorionic trophoblast binucleate and multinucleate cells were increased significantly,the fusion efficiency of trophoblast cells was remarkable improved while the co-culture cells transfected with recombinant eukaryotic expression plasmid pEGFP-C1/enJSRV-env and co-transfected with pEGFP-C1/enJSRV-env and pEGFP-C1/Hyal 2.But there was no significant change in the binuclear and multinucleated cells of the sheep chorionic trophoblast while the co-cultured cells transfected with eukaryotic expression plasmids pEGFP-C1/Hyal 2 and Hyal 2-sh RNA-3.This result indicated that enJSRV-Env can promote the fusion of STC_S,while Hyal 2 only plays an auxiliary role of the receptor.(4)In this experiment,CoIP-MS technology was used to screen proteins or protein molecular groups that may interact with enJSRV-Env,and 508 differential expression proteins were found in the experimental group.PKA has come to our attention by bioinformatics analysis.Then,the authenticity of the combination of the two was verified in reverse through the Co-IP experiment.(5)Western blot and RT-qPCR were used to study the signal pathway activated by enJSRV-Env that induced cell fusion.The results showed that the expressions of PKA,MEK and ERK1/2 were significantly increased while the recombinant expression plasmid pEGFP-C1/enJSRV-env was transfected into co-cultured cells.This demonstrating that enJSRV-Env had a positive correlation with PKA,and had a certain activation effect on MEK/ERK1/2 pathway in cells.Then treatment of cells with PKA-specific inhibitor H89resulted in a significant decrease in MEK and ERK1/2 expression,indicating that enJSRV-Env activation of MEK/ERK1/2 signaling pathway could be mediated by PKA.Finally,after the cells were treated with PD98059,a specific inhibitor of ERK1/2,the number of binucleated and multinucleated cells in the sheep chorionic trophoblast was significantly reduced,indicating that the PKA/MEK/ERK1/2 pathway can mediate the process of enJSRV-Env-promoting STC_S fusion.(6)Hela cells were transfected with pEGFP-C1/enJSRV-env,and then the fusion efficiency of He La cells was detected,and the proliferation of He La cells was detected by MTT method;RNA-seq sequencing was used to screen the differentially expressed genes.Functional annotation and enrichment analysis were performed using GO and KEGG databases;RNA-seq results were verified by RT-qPCR and Western Blot,and related regulatory mechanisms were analyzed.The results showed that enJSRV-Env inhibited the proliferation of Hela cells,probably via DUSP6 and ERK1/2 signaling pathway.In this study,the optimal co-culture system of LEC_S and STC_S was established,that is,the optimal co-culture condition was co-cultured at a ratio of 1:1 for 48 h.Then,the fusion phenomenon of STCs and LECs was observed by fluorescent staining.It was determined that enJSRV-Env could promote the fusion of STCS,and the fusion process was mediated by the PKA/MEK/ERK1/2 pathway.In addition,this study also determined that enJSRV-Env inhibited the proliferation of Hela cells through DUSP6 and ERK1/2 signaling pathways.