Study On The Mechanism Of Twist1-regulated Syncytialization Of Human Placental Trophoblast Cells Through GCM1

BackgroundThe breeding of a new life begins with the combination of egg and sperm. The fertilization of egg and sperm occurs in the ampulla of fallopian tube, fertilized egg in the fallopian tube moves to uterus. During the process of moving, fertilized egg through cleavage develops to morula. Four to five days after fertilization, fertilized egg develops into blastocyst and has been reached to uterine cavity at this time. Blastocyst consists of external trophectoderm and internal inner cell mass, and in the future trophectoderm develops into the placenta, the inner cell mass develops into a fetus.The placenta is a temporary organ; however it plays an important role during the whole process of pregnancy. Fetal normal development cannot be separated from the placenta. Placenta is the link between maternal and fetal, and is essential for the maternal-fetal health. The abnormal development of placenta leads to occurring of pregnancy related diseases, including pre-eclampsia, first trimester miscarriage, intrauterine growth retardation, which can result in fatal death in severe cases. Therefore, the normal growth of the placenta and the normal regulation of placental functions are very important for the normal development of fetus during pregnancy.Cell fusion is a common physiological process in mammals and involved in fertilization, human placental development, skeletal muscle and bone development and immune defense responses. During the process of fertilization, sperm through the zona pellucida and continue through the membrane of the egg. The formation of the zygote is due to the fusion of eggs and sperm. During the developmental process of human placental villi, the cell fusion event also exists. After the implanting of blastocyst into the uterus, the development of placenta began to form villi. In the process of the formation of placental villi, the differentiation of cytotrophoblast cells (CTBs) mainly along two pathways. Invasion migration pathways:cytotrophoblast cells differentiate into extravillous cytotrophoblast cells (EVTs) which have the invasion ability to migrate into the mother’s uterine. Fusion pathways:there is a kind of cytotrophoblast cells with proliferation and differentiation ability in placental villi. These cytotrophoblast cells can divide into two cells, one fuse into syncytiotrophoblast layer (STB) located the outside of villi, and the other cell continues to maintain the mitotic properties. During the developmental process of cytotrophoblast cells fuse to form syncytiotrophoblast, not only the nucleus of cytotrophoblast cells fuse into the syncytiotrophoblast layer and also the other organells in the cytoplasm of CTBs, including mitochondria, endoplasmic reticulum and golgi apparatus and so on. The characteristics of cytotrophoblast cells continually fuse into syncytiotrophoblast can expand the surface area of syncytiotrophoblast and at the same time updating existing substance in the syncytiotrophoblast, which lead to aging parts in syncytiotrophoblast discharge into the maternal blood circulation so as to complete own metabolism and realize the growth and maturation of the placenta. Aging and apoptosis of the material contents fit together to form syncytiotrophoblast nodules and such nodules will discharge from syncytiotrophoblast layer into maternal blood in order to maintain homeostasis in syncytiotrophoblast. This is crucial for metabolism of the placenta. If cell fusion is out of control and lead to a large number of apoptosis or necrotic material in syncytiotrophoblast discharge into the maternal blood, which is likely to cause excessive maternal pregnancy inflammatory reaction and then result in the occurrence of pregnancy related disease such as preeclampsia.The placenta is a place that involved the gas exchange, supplying nutrients and discharge of metabolic products between maternal and fetus. About the 3rd weeks after fertilization, the mesoderm inside the secondary villous differentiate into the capillaries and formed tertiary villi. At the moment, circulation system between fetal and maternal by placenta is established. Intervillous space is filled with blood provided by the mother’s uterine spiral artery; uterine spiral artery blood is rich in oxygen and nutrients necessary for fetal development. Syncytiotrophoblast located the outer layer of floating villi can be directly contact with the uterine spiral artery blood and for the exchange of nutrients, metabolites and gas. Syncytiotrophoblast can also block the entry of pathogens from the mother to fetus, such as resistance to Listeria bacteria and other pathogens. If syncytiotrophoblast layer structure destroyed, pathogens from the mother’s blood may invade and affect fetal growth and development. The placenta is also an organ with metabolic and endocrine functions, which can synthesize a variety of hormones, including human chorionic gonadotropin (hCG), human placental lactogen (hPL), estrogen, progesterone and placental growth factor etc. Normal synthesis and secretion of these hormones plays a vital role for the normal development and functions of placenta as well as maintaining pregnancy. Normal development of syncytiotrophoblast layer structure and then formed the well growth of placental barrier structure, which is the presupposition for gases and nutrients exchange between mother and fetus, resistance to pathogens form maternal blood and normal secretion of hormones and fully guaranteed the normal fetal development.Epithelial-mesenchymal transformation (EMT) is a converted process of epithelial cells to mesenchymal cells which has an active capacity and then able to move freely between the cell matrix. The typical characteristic of EMT is the reducing and missing of E-cadherin. Studies have found that some of the major transcription factors can promote epithelial to mesenchymal transition by inhibiting the expression of E-cadherin, including Snail, Slug, ZEB1, ZEB2 and Twistl. These transcriptional factors were known as EMT-related factors. Our research team has reported the Cullin7 can promote the invasion and migration of trophoblastic cells in the placental villi and epithelial to mesenchymal transition of cytotrophoblast cells through regulating the expression of Slug and ZEB1. Other studies have also found that some EMT-related factors play an important role in the process of invasion and migration differentiation of trophoblastic cells in placental villi, including Twistl, Snail and Slug. Above mentioned studies have shown that some EMT-related factor (ZEB1, Twistl, Snail and Slug) play an important role in the process of invasion and migration of the trophoblastic cells. However, there is little research reported that whether these EMT related factors also play an equally important role in the fusion differentiation pathway of cytotrophoblast cells during human placental development. Therefore, the main research purpose of this experiment is to detect the roles of EMT related factors in the fusion differentiation pathway of cytotrophoblast cells during the placental villous development. Through experimental study we confirmed that transcription factor Twistl is involved and play an important role in the fusion differentiation process of cytotrophoblast cells. And for the first time study found that Twistl promote the fusion of cytotrophoblast cells into syncytiotrophoblast by regulating the transcriptional activity of GCM1 and then increase the GCM1 expression. So EMT related factor Twist 1 not only play a role of epithelial-mesenchymal transformation in maternal-fetal interface of placenta and also play an important physiological function in another fusion differentiation pathway of cytotrophoblast cells. These findings will help us further understand the molecular mechanisms of formation of syncytiotrophoblast fused by cytotrophoblast cells and placental development. Study on the physiological mechanisms of placental formation provides molecular theory foundation of pregnancy-related diseases for clinical prevention caused by abnormal development of the placenta. In-depth understanding of the pathological mechanism of pregnancy related diseases in order to provide effective treatments in the future for the treatment of pregnancy-related disease.Part I the role of Twist 1 in the human placental trophoblast syncytialization[Objective] By detecting the expression change of EMT related factors during the spontaneously syncytialization of primary cytotrophoblast cells isolated from human term placenta and then study the roles of EMT related factors in the fusion process of trophoblast cells, which help us further understand the syncytialization differentiation pathway of cytotrophoblast cells during the process of placental development.[Method] From May 2014 to May 2015, the human placental tissues used in this study were collected from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Placentas at different gestational stages (first trimester (6-8 weeks), second trimester (18-20 weeks) and full-term (38-40 weeks)) from patients who underwent legal abortion or cesarean section were included. The research model of spontaneously syncytialization of primary cytotrophoblast cells was established by the culture of CTBs isolated from human full-term placenta. Primary cytotrophoblast cells were harvested at different time points during the longer periods of culture. The mRNA and protein expression levels of β-hCG were detected by real-time quantitative PCR and western blotting methods, and immunofluorescence method was used to examine the syncytial formation of primary cytotrophoblast cells which to determine the successful establishment of the study model of spontaneously syncytialization. The expression levels of EMT related factors (Twist1, Snail, Slug, ZEB1 and ZEB2) during the spontaneously syncytialization of primary CTBs were examined using real-time quantitative PCR. The expression localization of Twistl protein in human placentas at different gestational stages was detected by immunohistochemistry. The expression level of Twistl protein during spontaneous syncytialization of primary CTBs and forskolin (FSK)-induced fusion process of BeWo cells was evaluated through western blotting method. When the expression of Twistl protein was decreased by targeting inhibition using specific siRNA in BeWo cells and then BeWo cells treated with FSK for next 48h were induced to fusion, we observed its impact on fusion capacity of BeWo cells treated with FSK included the syncytial formation and the expression level of β-hCG protein.[Results] (1) Immunofluorescence showed that primary cytotrophoblast cells spontaneously gathered to from multinuclear syncytium during the process of culture. With the extension of incubation time, the bigger the syncytium formed by CTBs. The number of nuclei in syncytium can reach more than 10 after 72 hours. The mRNA and protein expression levels of β-hCG increased in a time-dependent manner during spontaneous syncytialization of primary CTBs. (2) Real-time quantitative PCR showed that the mRNA expression of EMT related factor Twistl was markedly up-regulated during the spontaneous syncytialization of primary CTBs. In contrast, the mRNA levels of Slug, Snail, ZEB1 and ZEB2 decreased significantly during the in vitro spontaneous syncytialization process. (3) The expression of Twist1 protein in human placentas was detected by immunohistochemistry, which showed that positive immunostaining for Twist 1 protein was detected in the nucleus of CTB cells and the STB, as well as stromal and endothelial cells in human placental tissue from the first, second and third trimesters. (4) Western blotting showed that the level of Twist 1 protein was elevated significantly at 24 hours and continuously to 72 hours during spontaneous fusion of primary CTBs. Meanwhile, the Twist 1 protein level was significantly higher in the FSK-treated Be Wo cells compared to the dimethylsulfoxide (DMSO)-treated control group. (5) Twist1 protein expression was efficiently down-regulated in BeWo cells transfected with Twist1 siRNA compared to the scrambled siRNA-treated cells under FSK stimulation. Twist1 knockdown markedly decreased BeWo cells fusion in the presence of FSK, as demonstrated by the reduction in the β-hCG protein level detected by western blotting and a significant decrease in the formation of syncytia as measured by calculating the fusion index.[Conclusions] (1) During the culture process of primary cytotrophoblast cells isolated from human full-term placenta, CTBs spontaneously gathered and gradually formed a number of multinuclear syncytium which predicts that the model for spontaneous syncytialization of primary CTB cells was effective. (2) The mRNA and protein expression levels of EMT related factor Twistl was up-regulated with spontaneously syncytialization of primary CTB cells, and the expression of Twistl protein in human placentas at different stages of pregnancy has significant nuclear localization signals in the CTB cells and STB of placental villi. These results predict that Twistl was involved in human trophoblast syncytialization. (3) Function experiment of Twistl carried out using human chorionic carcinoma trophoblastic cell line BeWo cells showed that Twistl knockdown by siRNA markedly decreased FSK-treated fusion of BeWo cells, as demonstrated by the reduction in the P-hCG protein level and a significant decrease in fusion index. Above results further confirmed the important role of Twist1 in human trophoblast syncytialization.Part Ⅱ Twistl is involved in trophoblast syncytialization by regulating GCMl(Objective] First part has been preliminary confirmed that Twistl can promote trophoblast cells syncytialization during development process of human placental villi. So far, the researches about detailed molecular mechanism underlying trophoblast cells fusion regulated by Twistl are rarely reported. GCM1 is currently recognized as important molecules, which can promote CTBs fuse into STB in human placental villi. This part preliminarily investigates whether Twistl promote the trophoblast cells fusion through regulating GCM1 expression and the potential molecular mechanisms of fusion promoted by Twistl.[Method] The expression localization of Twistl and GCM1 in human placental villi of early pregnancy was detected by immunohistochemistry. Primary CTBs isolated from human full-term placentas were cultured under normoxia/hypoxia (20%/2% O2) conditions last for 72 hours, cells were collected at different time points during the longer periods of culture. Meanwhile, the expression levels of Twistl, GCM1 and β-hCG under different culture conditions were evaluated by real-time quantitative PCR and western blotting methods. Twistl protein was decreased by targeting inhibition using specific siRNA in BeWo cells and subsequently cells were stimulated with FSK to induce fusion for an additional 48 h. Through real-time quantitative PCR observed the effect of Twistl siRNA on the expression mRNA levels of GCM1 and its downstream molecules syntin1 and syncytin2 in the process of FSK-treated BeWo cells fusion.293 T cells were co-transfected with expression plasmids of Twist1 and reporter constructs containing intron 2 region of GCM1 gene, and tested the effect of different amount of expression vectors on the transcriptional activity of GCM1 by luciferase reporter assay after culturing for 48 hours. The relationship between Twist 1 protein and GCM1 target gene was further investigated by chromatin immunoprecipitation (ChIP).[Results] (1) Immunohistochemical results showed that the expression of Twistl and GCM1 proteins in first-trimester human placental tissues were similar and both localized in the nuclei of the cytotrophoblast cells and the syncytiotrophoblast. (2) Compared with normoxia (20% O2), the primary CTBs syncytialization was inhibited under the hypoxic environment (2% O2) as indicated by the decreased protein level of β-hCG. Under normoxic environment (20% O2), the elevated expression levels of Twistl and GCM1 of primary CTBs were both observed during the syncytialization process. However, the expression level of Twistl protein failed to increase significantly during the spontaneous syncytialization of human primary CTBs under hypoxic conditions (2% O2,), and a similar result was observed for GCM1 mRNA. (3) Knockdown of Twistl protein by specific siRNA transfection in FSK-stimulated BeWo cells resulted in a significant reduction of GCM1 mRNA expression compared with the control siRNA, and a similar result was observed for GCM1 downstream molecules, such as syncytinl and syncytin2. (4) Luciferase reporter assay (LRA) showed that the luciferase activity of the GCM1 was significantly increased by the induction of Twistl. Furthermore, the induced luciferase activity of the GCM1 by Twistl was dose-dependent, the higher amount of Twistl expression vector was cotransfected accompanied by the stronger luciferase activity was observed. (5) Chromatin-immunoprecipitation (ChIP) showed that Twist 1 protein was associated with a region containing several E-boxes in intron 2 of the GCM1 gene in human trophoblastic choriocarcinoma BeWo cells treated with forskolin for 48 hours.[Conclusions] (1) Twistl and GCM1 proteins showed a similar expression profile in human chorionic villi, and meanwhile the change trend of Twistl and GCM1 expression was also similar when the primary CTBs isolated from full-term placenta were cultured under the culture conditions with different oxygen concentration. These results speculated that whether there is a possible link between Twistl and GCM1. (2) Knockdown of Twistl by siRNA in BeWo cells treated with forskolin led to a significantly reduced expression of GCM1, syncytin 1 and syncytin 2 mRNA levels. Above results suggested that Twistl may through regulating the expression of GCM1 to promote the fusion of trophoblastic cells. (3) To further verify the hypothesis, luciferase report assay found that transcriptional activity of GCM1 was induced by Twist1, and the binding of Twistl to a region containing enriched E-boxes in intron 2 of the GCM1 gene in BeWo cells treated with forskolin was observed by ChIP. In conclusion, Twistl protein binds to a region containing enriched E-boxes in intron 2 of the GCM1 gene so as to regulate the expression of GCM1, and ultimately promote trophoblast syncytialization.