Iodothyronine Deiodinase 2: Its Regulation Of Trophoblast Cell Fate Decisions And Association With Early Recurrent Miscarriage

Introduction:Trophoblast development and trophoblast-uterus interactions,which constitute human placental development,involve a complex interplay of several molecules and mechanistic events whose respective identification and characterization have not been fully achieved.Dysregulated expression of these molecules and the impairment of the mechanistic events often result in pregnancy complications,such as early miscarriage（spontaneous pregnancy loss in the first trimester）.Regardless of the high prevalence of early recurrent miscarriage and the associated consequences,its pathogenesis and pathophysiology have not been fully clarified.Due to this,there are no specific and highly reliable proactive therapeutic interventions against the occurrence of this pregnancy complication.Iodothyronine deiodinase 2（Di O2）is a selenium-containing enzyme that is generally known for eliciting cellular actions by first converting tetraiodothyronine（T4）to triiodothyronine（T3）.It has been reported that the expression of this enzyme is controlled by cyclic adenosine monophosphate（c AMP）,which is a critical regulator of placental development,yet its involvement in placentation and pregnancy complications remains unknown.Besides,it has not been investigated whether this enzyme can modulate cellular activities in a manner that is not mediated by thyroid hormone metabolism.Thus,in this study,the aim was to investigate the role of Di O2 in trophoblast cell fate decisions in the absence of thyroid hormones and to assess its placental villous expression in early recurrent miscarriage patients.The study also aimed at investigating the role of T4 and T3 in trophoblast cell fate decisions.Materials and Methods:The placental villous expression of Di O₂was determined with immunofluorescence.“Exaggeration of function”and“loss of function”experiments–via Di O₂ overexpression and Di O₂ knockdown–were used to investigate the role of Di O₂ in trophoblast cell cycle,proliferation,migration,invasion and apoptosis in the absence of thyroid hormones.RPMI media supplemented with fetal bovine serum（FBS）that had been deprived of thyroid hormones were used to culture trophoblast cell line and first trimester human placental villous explants.Cell proliferation was measured with the cell counting kit 8（CCK8）while cell cycle and apoptosis were studied with flow cytometry.Cell migration and invasion were measured with the wound healing assay and the transwell cell invasion assay,respectively.At the m RNA level,gene expression was assessed with real time quantitative polymerase chain reaction（RT-q PCR）;and at the protein level,gene expression was assessed with western blotting.Results:It was found that Di O₂is expressed in the cytotrophoblast（CTB）,proximal column trophoblast（PCT）,distal column trophoblast（DCT）and syncytiotrophoblast（STB）of the first trimester placental villi.Its overexpression arrested trophoblast cell line proliferation at the G1 phase of the cell cycle by downregulating cyclin D1 and PCNA,while promoting apoptosis via increased activity of caspase-3 and inhibition of the AKT and ERK1/2 signaling pathways.Also,it augmented trophoblast cell migration and invasion via the upregulation of N-cadherin,vimentin,fascin-1,twist-1,snail-1,snail 2,zeb-1,zeb-2,HIF1-αand MMP9.Di O₂ knockdown elicited the opposite effects.However,neither Di O₂ overexpression nor Di O₂knockdown affected the expression of cytokeratin-7 andβ-catenin.Interestingly,each of these effects of Di O₂ manipulation was not mediated by thyroid hormone metabolism.Inhibition of the AKT or ERK1/2 signaling pathways abrogated the proliferative effects and anti-apoptotic effects of Di O₂knockdown but did not have a significant effect on the anti-invasive effects of Di O₂knockdown.Silencing of WSB1 decreased the proliferation of the cells and increased their apoptosis via the upregulation of Di O₂.However,there was a decrease in the invasiveness of the cells via the downregulation of several epithelial mesenchymal-transition genes following the silencing of WSB1.Assessment of the early miscarriage placental villi revealed a decrease in the expression of Di O2,WSB1,N-cadherin,vimentin,fascin-1 and twist-1 but not E-cadherin whose expression remained unchanged.Treatment of the cells with T4 or T3 enhanced their proliferation,migration and invasion but inhibited their apoptosis by regulating the AKT and ERK1/2 signaling pathways and by targeting several transcription factors.Conclusions:These results indicate that during placental development,Di O2 may switch off the proliferative ability of the trophoblast cells while facilitating their differentiation into an invasive phenotype;and that its downregulation may contribute to the shallow trophoblast invasion that precedes early miscarriage.Hence,Di O2 is a potential therapeutic target against early recurrent miscarriage.This study has also revealed,for the first time,that Di O₂ does not always function through thyroid hormone signaling,but can directly target transcription factors and signaling pathways to regulate cell activities.Moreover,this study has indicated that WSB1 and T4 control the expression of Di O₂ to modulate trophoblast cell activities.Hence,the utilization of Di O₂and WSB1 as new markers for studying trophoblast development are highly recommended.