Cooperative Effects Of Activin A And Follistatin On Migration Of Trophoblasts

Trophoblasts are the basic units that perform placental functions,which have capacity of invading deeply into the decidualized maternal uterine tissue to remodel placental blood vessels and the uterine spiral arteries so as to favor embryo development.Activin A is one of transforming growth factorβ（TGF-β）superfamily members,which is a sex hormone regulatory protein to promote the secretion of follicle-stimulating hormone（FSH）by the anterior pituitary gland cells.Follistatin（FST）was initially discovered as a sex hormone regulatory protein,which has the opposite effect to activin A,inhibits the secretion of FSH by anterior pituitary gland cells.A large number of studies have shown that FST has a high affinity with activin A and antagonize the biological effects of activin A.Moreover,the studies found the levels of activin A and FST in the peripheral blood of normal pregnant women were significantly higher than those of non-pregnant women.It has been reported that activin A increased human trophoblast migration and invasion.However,it is still unclear whether the relationship between activin A and FST is an antagonistic or synergistic effect which is similar to that of estrogen and progesterone in the induction of decidualization during placental development.Therefore,this study focuses on trophoblasts to explore the regulatory role of FST on trophoblasts and whether activin A and FST jointly regulate trophoblast proliferation,migration,and invasion in synergy or antagonistic to each other,in order to reveal the biological significance and mechanism of the high expression of activin A and FST in pregnant women during the reproductive process.I.METHODS1.Detection of activities in Human trophoblast cell line HTR-8/SVneo cellsHTR-8/SVneo cell viability was detected by CCK-8 assay,cell proliferation and adhesion were detected by cell colony formation method and real-time cell-based assay（RTCA）,and the secretory levels of IL-6 and IL-1βwere detected by ELISA and NO level were detected by Griess method,the cell morphology was detected by Giemsa staining;Scratch test was used to detect the wound healing ability of cells,microfluidic technology was used to detect cell migration,and matrigel coated transwell method was used to detect cell invasion.2.Detection of activities in primary cultured trophoblasts of miceprimary cultured trophoblasts of E8.5 mice were cultured by enzyme digestion and identified by immunocytochemical staining;The detection of cell proliferation,adhesion,migration and invasion was the same as that of human HTR-8/SVneo cells mentioned above.3.Migration mechanism of activin A and FST regulating trophoblastsFlow cytometry was used to detect the Ca²⁺influx of trophoblasts;RT-RCR and Western blotting methods were used to detect the expression of cell migration related proteins,Act R,Smads and MAPKs signal proteins;immunocytochemical fluorescence staining was used to detect the Ezrin polarization of trophoblasts;the effect of JNK inhibitor and the effect of anti-Act RIIA and anti-Act RIIB antibody treatment on trophoblasts migration was detected.4.Expression and distribution of activin A and FST in uterus and embryo of miceTo establish normal pregnant mice and LPS induced abortion mice models,immunohistochemical staining was used to detect the expression and distribution of activin A and FST in uterine and embryonic tissues.II.RESULTS1.Effects of activin A and FST on the activities of HTR-8/SVneo cells（1）FST and Activin A+FST groups both promoted the secretion of IL-6、IL-1βand NO levels by HTR-8/SVneo cells,and both promoted the viability and proliferation of HTR-8/SVneo cells.（2）FST and Activin A+FST groups both inhibited adhesion of HTR-8/SVneo cells and induced migration and invasion of HTR-8/SVneo cells.2.Effects of activin A and FST on the activities of primary cultured trophoblasts of miceFST and Activin A+FST groups both promoted the viability and cell proliferation of mice primary trophoblasts,both inhibited the adhesion of mice primary trophoblasts,and both induced the migration and cell invasion of mice primary trophoblasts.3.Mechanism of activin A and FST regulating migration in trophoblasts（1）FST and Activin A+FST groups both up-regulated the expression of Ezrin（a polarizing molecular）and promoted the polarization distribution of Ezrin in human trophoblasts;FST and Activin A+FST groups both jointly promoted the expression of N-cadherin,vimentin,MMP2 and MMP9,and inhibited the expression of E-cadherin;The combined action increased the intracellular Ca²⁺influx level.（2）Activin A increased the phosphorylation level of Smad3 in human trophoblasts,while FST alone has no effect on expression of Smad3;In Smad3 knockdown group,activin A induced trophoblast migration significantly decreased,while in Smad3overexpression group,activin A induced trophoblast migration significantly increased.（3）FST and activin A+FST groups both induced the increase of phosphorylated JNK in human trophoblasts,while activin A alone has no effect on expression of JNK;After pretreating by JNK inhibitor（AS601245）,the migration of trophoblasts induced by FST and activin A+FST groups decreased significantly,but had no effect on the action of activin A.（4）Activin A could up-regulate the expression of Act RIIA in trophoblasts;FST and Activin A+FST groups both induced the expression of Act RIIB in human trophoblasts;Blocking the effect of Act RIIA on trophoblasts significantly reduced the migrated distance induced by activin A,but had no effect on the effect of FST alone;Blocking the effect of Act RIIB on trophoblasts significantly reduced the cell migrated distance induced by activin A+FST group.（5）FST and Activin A+FST groups both promoted the polarization distribution of Ezrin in primary cultured trophoblasts of mice;FST and Activin A+FST groups both jointly promoted the expression of N-cadherin,vimentin and MMP2 and increased the intracellular Ca²⁺influx level.（6）FST and activin A+FST groups both induced the increase of phosphorylated JNK in primary cultured trophoblasts of mice,while activin A alone has no effect on expression of JNK;After pretreating by JNK inhibitor（AS601245）,the migration and invasion of trophoblasts induced by FST group decreased significantly.4.Expression and distribution of activin A and FST in uterus and embryo of miceActivin A was mainly expressed in the trophoblasts of the embryonic side in E6.5pregnant mice,while activin A was widely distributed and increased in the local decidual tissue in E8.5 pregnant mice;FST was mainly distributed in the yolk sac of the embryo and the maternal decidualized endometrium outside the trophoblast.The expression of FST in E8.5 was higher than that in E6.5.Compared with normal pregnant mice,the uterine tissue of aborted mice showed embryo loss,placental structure disorder,and the decreased expression of activin A and FST.5.The effect of activin A and FST on the migration of other cellsIn order to eliminate false positive results caused by experimental error factors,mouse decidual cells and microglial cells（BV2 cells）identified as activin A-induced cell migration were selected for the experiment to further observe the combined effect of activin A and FST.The results showed that FST combined with activin A had an antagonistic effect on cell migration,consistent with previous research reports.Ⅲ.CONCLUSIONTo sum up,FST is a novel chemoattractant for migration and invasion of placental trophoblasts,the combined action of FST and activin A in trophoblasts is synergetic rather than antagonistic as previously reported.Therefore,the cooperative effect of FST and activin A jointly promoted the viability,migration and invasion of trophoblast cells related to the Act RIIB-JNK signal pathway which may be more conducive to the maintenance of fetal placental development.However,the biological effect of FST and activin A cooperating on trophoblasts may also be related to the complex factors of pregnancy and the regulatory network of placental function,and the FST binding receptor alone and deep mechanism of effect is not clear to be further explored.In addition,this study also suggests that the combination of FST and activin A may be more effective than the single treatment for some patients with pregnancy failure.