ERV3 human endogenous provirus mRNAs are expressed in normal and malignant tissues and cells, but not in choriocarcinoma tumor cells

Messenger RNA expression of a human endogenous provirus, ERV3, has been characterized in 170 specimens of normal and malignant human tissues and cells. In contrast to the high expression in first-trimester and full-term placental chorionic villi, most other human tissues expressed ERV3 mRNAs at a level of 2-30% of placenta. However, ERV3 mRNAs were not detected in choriocarcinoma tumor cell lines. These studies suggest that the ERV3 provirus may have been preempted for a biological function and disruption of its mRNA expression results in choriocarcinoma.     

Expression of the glycoprotein hormone alpha-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different cis-acting element mediates pituitary-specific expression.

The single-copy gene encoding the alpha subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoter-regulatory elements of the human and bovine alpha-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine alpha subunit genes in transgenic mice. From the results, we conclude that placental expression of the human alpha-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine alpha-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human alpha CRE and thereby rendered the bovine alpha-subunit promoter inactive in human choriocarcinoma cells. However, conversion of the bovine alpha CRE homolog to an authentic alpha CRE restored activity to the bovine alpha-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine alpha transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human alpha-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human alpha transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine alpha-subunit transgenes were expressed in mouse pituitary, indicating differences in the composition of the enhancers required for pituitary- and placenta-specific expression.     

Molecular and morphologic analyses of expression of ESX1L in different stages of human placental development

The mRNA expression of the ESX1L gene was analyzed by RT-PCR and in situ hybridization in human normal cytogenetically placentas, of different gestational ages. Our RT-PCR analysis showed that ESX1L mRNA is expressed from 5 weeks of gestation until term, suggesting a role not only in trophoblast differentiation but also in the maintenance of the villi and microvasculature. We also observed, by in situ hybridization, that ESX1L mRNA is expressed by cytotrophoblast from chorionic plate, syncytiotrophoblast and stromal cells of all terminal, intermediate and stem villi of term placentas. ESX1L mRNA expression was more pronounced in trophoblast cells of terminal villi than in intermediate and stem villi. In conclusion, ESX1L is expressed during all stages of placental development and is localized to sparse areas of trophoblast in terminal villi in association with cytotrophoblastic cells.     

Integrin-linked kinase (ILK) is highly expressed in first trimester human chorionic villi and regulates migration of a human cytotrophoblast-derived cell line

The placenta represents a critically important fetal-maternal interaction. Trophoblast migration and invasion into the uterine wall is a precisely controlled process and aberrations in these processes are implicated in diseases such as preeclampsia. Integrin-linked kinase (ILK) is a multifunctional, cytoplasmic, serine/threonine kinase that has been implicated in regulating processes such as cell proliferation, survival, migration, and invasion; yet the temporal and spatial pattern of expression of ILK in human chorionic villi and its role in early human placental development are completely unknown. We hypothesized that ILK would be expressed in trophoblast subtypes of human chorionic villi during early placental development and that it would regulate trophoblast migration. Immunoblot analysis revealed that ILK protein was highly detectable in placental tissue samples throughout gestation. In floating branches of chorionic villi, from 6 to 15 wk of gestation immunofluorescence analysis of ILK expression in placental tissue sections demonstrated that ILK was highly detectable in the cytoplasm and membranes of villous cytotrophoblast cells and in stromal mesenchyme, whereas it was barely detectable in the syncytiotrophoblast layer. In anchoring branches of villi, ILK was highly localized to plasma membranes of extravillous trophoblast cells. Transient expression of dominant negative E359K-ILK in the villous explant-derived trophoblast cell line HTR8-SVneo dramatically reduced migration into wounds compared to cells expressing wild-type ILK or empty vector. Therefore, our work has demonstrated that ILK is highly expressed in trophoblast subtypes of human chorionic villi during the first trimester of pregnancy and is a likely mediator of trophoblast migration during this period of development.     

Aberrant expression of imprinted lncRNA MEG8 causes trophoblast dysfunction and abortion

Long noncoding RNAs (lncRNAs) are a group of noncoding RNAs whose nucleotides are longer than 200 bp. Previous studies have shown that they play an important regulatory role in many developmental processes and biological pathways. However, the contributions of lncRNAs to placental development are largely unknown. Here, our study aimed to investigate the lncRNA expression signatures in placental development by performing a microarray lncRNA screen. Placental samples were obtained from pregnant C57BL/6 female mice at three key developmental time points (embryonic day E7.5, E13.5, and E19.5). Microarrays were used to analyze the differential expression of lncRNAs during placental development. In addition to the genomic imprinting region and the dynamic DNA methylation status during placental development, we screened imprinted lncRNAs whose expression was controlled by DNA methylation during placental development. We found that the imprinted lncRNA Rian may play an important role during placental development. Its homologous sequence lncRNA MEG8 (RIAN) was abnormally highly expressed in human spontaneous abortion villi. Upregulation of MEG8 expression in trophoblast cell lines decreased cell proliferation and invasion, whereas downregulation of MEG8 expression had the opposite effect. Furthermore, DNA methylation results showed that the methylation of the MEG8 promoter region was increased in spontaneous abortion villi. There was dynamic spatiotemporal expression of imprinted lncRNAs during placental development. The imprinted lncRNA MEG8 is involved in the regulation of early trophoblast cell function. Promoter methylation abnormalities can cause trophoblastic cell defects, which may be one of the factors that occurs in early unexplained spontaneous abortion.     

poly(dA:dT)促进体外培养的人绒毛组织AIM2炎性体活化

目的:在妊娠过程中,胎盘可能暴露于多种病原微生物,威胁胎儿正常生长发育。为探讨人胎盘绒毛组织是否表达AIM2炎性体成员基因以及人胎盘组织的AIM2炎性体的活化形式。方法:以THP-1细胞来源的RNA和蛋白作为阳性对照,分别应用RT-PCR和Western blot方法检测人早孕期胎盘绒毛组织中AIM2炎性体两个相关基因AIM2和ASC的表达。分离和体外培养人胎盘绒毛膜组织,并用不同浓度的poly(d A:d T)进行转染,处理24小时后,分别收集组织培养上清和蛋白裂解液,Western blot检测蛋白裂解液中caspase-1的活化,ELISA检测培养上清中IL-1β的分泌。结果:RT-PCR和Western blot结果均显示人早孕期胎盘绒毛组织组成性表达AIM2炎性体相关基因AIM2和ASC。同时,体外培养的人胎盘绒毛组织在转染5μg/m L poly(d A:d T)后,caspase-1剪切片段p10显著增多,培养上清中IL-1β分泌也显著增多(P0.01)。结论:人胎盘绒毛组织存在功能性的AIM2炎性体,能够被胞内双链DNA活化。     

Role of MDR1 and MRP1 in trophoblast cells,elucidated using retroviral gene transfer

Natural differences in expression and retroviral transduction techniques were used to test the hypothesis that MDR1 P-glycoprotein (P-gp) and MRP1 (multidrug resistance-related protein) contribute to xenobiotic handling by placental trophoblast. RT-PCR and Western blotting in placenta, primary cytotrophoblast cell cultures, and BeWo, JAr, and JEG choriocarcinoma cell lines showed that MRP1 was ubiquitously expressed, whereas MDR1 was absent or minimally expressed in BeWo and JEG cell lines. In syncytiotrophoblast, P-gp was localized predominantly to the microvillous, maternal facing plasma membrane, and MRP1 to the basal, fetal facing plasma membrane. Functional studies showed that cyclosporin A-sensitive accumulation of [³H]vinblastine by cells containing both transport proteins was significantly different from those expressing predominantly MRP1. Retroviral gene transfer of MDR1 to BeWo cells confirmed that this difference was due to the relative expression of MDR1. Therefore, both P-gp and MRP1 contribute to xenobiotic handling by the trophoblast. Localization of P-gp to the microvillous membrane suggests an essential role in preventing xenobiotic accumulation by the syncytiotrophoblast and, therefore, in protecting the fetus. placenta; multidrug resistance; xenobiotic     

Choriocarcinoma cells increase the number of differentiating human cytotrophoblasts through an in vitro interaction

The human placenta arises from the zygote through single cell intermediates called cytotrophoblasts that in turn give rise to a syncytium. In culture, mononucleated cytotrophoblasts exhibit little, if any, cell division but are converted to multinucleated cells. Choriocarcinoma, the malignant tumor of placenta trophoblast, comprises a mixed population of dividing cellular intermediates that resemble cytotrophoblasts but are less differentiated. Because the choriocarcinoma intermediates arise from dividing cells, the tumor may contain one or more cell types in abundance not present in the population of isolated placental cells. To study placental differentiation through cell-cell interaction, choriocarcinoma cell lines were co-cultured with placenta-derived cytotrophoblasts, and placental hormone biosynthesis, as a marker of differentiation was examined. We reasoned that intermediates formed by the tumor might interact with and complement those intermediates in the placenta-derived cytotrophoblast population. Co-culturing either the JAr or JEG choriocarcinoma cell lines with cytotrophoblasts elevated the synthesis of the chorionic gonadotropin alpha and beta subunits 10-20 fold, and human placental lactogen 5-fold. The effect was specific for these trophoblast-derived cells, since comparable quantities of Chinese hamster ovary or HeLa cells did not affect the placental cytotrophoblast culture. Further experiments suggested that the source of enhanced synthesis was the cytotrophoblasts. We propose that an interaction between cytotrophoblasts and choriocarcinoma cells occurs, which results in an increased number of differentiating cytotrophoblasts. Such co-cultures may represent a model system for examining choriocarcinoma cell interaction with normal cells, a process known to occur in vivo. The data are also consistent with the hypothesis that the regulated chorionic gonadotropin production in the placenta is determined by interaction among trophoblast cells at different stages of differentiation.     

Cytological distribution of chorionic gonadotropin subunit and placental lactogen messenger RNA in neoplasms derived from human placenta

Normal trophoblast of the human placenta elaborates at least two major protein hormones, chorionic gonadotropin (hCG), and placental lactogen (hPL). There are several gestational trophoblastic diseases of the placenta called hydatidiform mole, invasive mole, and choriocarcinoma. Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and small quantities of hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their messenger RNA (mRNA) in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha and - beta mRNA can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and -beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and -beta in the syncytial- like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villous formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues that contain cytotrophoblastic elements.