Role Of PoFUT1 In Angiogenesis Of Uterine Decidua

Ⅰ.Background and Objective:Differentiation of HESCs(human endometrial stromal cells)and formation of new blood vessels during the process of embryo implantation and development are critical for the establishment and maintenance of gestation.Protein O-fucosyltransferase 1(po FUT1)is a endoplasmic reticulum(ER)localized enzyme which transfers O-Fucose in O-linkage from GDP-β-L-Fucose to serine or threonine by recognizing EGF-like repeats on the protein.Fucosylation of proteins plays a significant role in the process of uterine receptivity establishment and embryo implantation.In this study,we focus on the effect of po FUT1 on angiogenesis of uterine decidua and its underlying glycobiological mechanism,which further adds to our knowledge of pregnancy physiological and pathological process and provides a novel idea and theoretical base for the pregnancy complications therapies.Ⅱ.Method: 1.Immuofluorenscence was performed to determine the expression level of po FUT1 and vascular endothelial cells(VECs)markers in uterine biopsy tissue from healthy non-pregnant and pregnant women and female mouses.2.Human endometrial stromal cells(HESCs)were induced by decidualisation and VEGF,and then real-time PCR,western blot and immunocytofluorescense were performed to analyze the expression of po FUT1 and VECs markers on gene and protein level;The angiogenic ability was assessed by tube formation assay.3.The recombinant vector or si RNA were transfected into HESCs after decidulisation(human decidual cells,HDSCs),real-time PCR,western blot and immunocytofluorescense were performed to analyze the expression of VECs markers;Cytoskeleton staining,wound healing and tube formation experiments were performed to detect the effect of po FUT1 on cytoskeleton and the ability of migration and angiogenesis.4.The expression of O-Fucose on u PA was detected by co-immunoprecipitation(IP)and O-Fucose kits.5.The expression of Rho A signaling pathway related molecules was measured by western blot.6.For further research of the role that po FUT1 plays on regulation of angiogenesis in vivo,we performed po FUT1-si RNA injection through uterine horn in healthy pregnant mouses.Ⅲ.Results: 1.Immuofluorenscence data showed that the number of blood vessels was larger,the diameter was wider and the expression of po FUT1 and CD31 was higher in uterine endometrium of healthy non-pregnant women in secretory phase,compared with those in proliferative phase;Healthy pregnant women showed further remarkable increase.The expression of po FUT1 and CD31 in healthy pregnant mouse increased as gestation processed.2.Induction of decidualisation and VEGF promoted po FUT1 expression and enhanced the ability of angiogenesis.The result of real-time PCR,western blot and immunocytofluorescense showed that HESCs treated with decidualisation induction expressed much more po FUT1 and CD31 compared to control cells,and then after co-treated with VEGF the increase even more obvious.Tube formation assay result showed that HESCs treated with decidualisation induction expressed stronger ability of angiogenesis than the control group,and then after co-treated with VEGF,the ability became much more stronger.3.po FUT1 promoted the angiogenic ability on HDSCs.The results of real-time PCR,western blot and immunocytofluorescense showed that HDSCs transfected with silenced po FUT1 by specific si RNA exhibited decreased VECs markers.These results were confirmed after co-transfection of po FUT1-si RNA and po FUT1-c DNA,which significantly restored proteins level of VECs markers.The results of cytoskeleton staining,wound healing and tube formation experiments showed that HDSCs expressing lower po FUT1 showed fewer number of F-actin and weaker ability of migration and angiogenesis,whereas HDSCs co-transfected with po FUT1-c DNA showed increased number of F-actin and stronger ability of migration and angiogenesis.4.po FUT1 activated Rho A signaling pathway by upregulating the level of u PA modified with O-Fucose.Western blot results showed that po FUT1-si RNA could significantly downregulated O-Fucose expression on total proteins of HDSCs while po FUT1-c DNA induced O-Fucose upregulation.To confirm which protein exactly was O-fucosylated and effected angiogenesis in decidua,we used bioinformatics prediction approach(Uni Prot)and found out that u PA contained O-Fucosylated site.Co-immunoprecipitation(IP)results showed that,po FUT-si RNA could significantly downregulated O-Fucose expression on u PA while restored O-Fucose expression on u PA by induction of co-transfected with po FUT1-c DNA.HDSCs transfected with po FUT1-c DNA and u PA-c DNA expressed more u PA modified by O-fucosylation compared to control cells,whereas HDSCs transfected with po FUT1-c DNA and u PA-mutant-c DNA that without O-linked fucosylated site,the level of u PA with O-Fucose was unchanged compared to control cells.In order to verify the role of O-Fucose on u PA in the Rho A signaling pathway,we did a series of western blot,when HDSCs were co-transfected with po FUT1-c DNA and u PA-c DNA,the expression of Rho A signaling pathway related molecules(Rho A-GTP、p-LIMK、p-cofilin)were upregulated compared to control ones,whereas when co-transfeccted with po FUT1-c DNA and u PA-mutant-c DNA,the condition of signaling pathway was as the same as control group.And also,in order to demonstrate the role of po FUT1 in this procsss,we did the following work,upregulation of po FUT1 could activate Rho A signaling pathway,whereas cells transfected with po FUT1-c DNA using u PA antibody and Rho A inhibitor(C3 transferase)at the same time,the Rho A signaling pathway was inactivated.5.po FUT1 activated Rho A signaling pathway by upregulating the level of u PA with O-Fucose,which therefore promoted angiogenesis function of HDSCs.Cytoskeleton staining,wound healing and tube formation experiments showed consistent functional results as above.HDSCs transfected with po FUT1-c DNA and u PA-c DNA expressed increased number of F-actin and enhanced ability of migration and angiogenesis compared to control cells,whereas after transfected with po FUT1-c DNA and u PA mutant-c DNA,these changes disappeared.And also,upregulation of po FUT1 could promote the ability of migration and angiogenesis whereas HDSCs transfected with po FUT1-c DNA using u PA antibody and Rho A inhibitor at the same time,the number of F-actin was decreased and the ability of migration and angiogenesis was inhibited.6.The mouse horn injection model in vivo also showed that po FUT1-si RNA significantly reduced the amount and the diameter of blood vessel and the embryo implantation rate significantly compared to opposite control horn.Ⅳ.Conclusion: 1.po FUT1 and CD31 can be tested in human and mouse uterine.In human,the healthy non-pregnancy women in secretory phase express more po FUT1 and CD31 than those in proliferative phase,healthy early-pregnant women expressed further remarkable increased po FUT1 and CD31;In mouse,the expressions of po FUT1 and CD31 increase as gestation processes.2.Induction of decidualisation and VEGF promotes po FUT1 expression and enhances the ability of tube formation.Conversely,po FUT1 promotes angiogenic ability on HESCs after decidulisation.3.po FUT1 activates Rho A signaling pathway by upregulating the level of u PA with O-Fucose,which therefore promotes angiogenic ability on HDSCs.