The expression and post-transcriptional regulation of FSTL1 transcripts in placental trophoblasts.
ABSTRACT: Follistatin-like-1 (FSTL1) is a widely expressed secreted protein with diverse but poorly understood functions. Originally described as a pro-inflammatory molecule, it has recently been reported to play a role in signaling pathways that regulate development and homeostasis. Distinctively, FSTL1 harbors within its 3'-UTR the sequence encoding microRNA-198 (miR-198), shown to be inversely regulated relative to FSTL1 expression and to exhibit opposite actions on cellular processes such as cell migration. We sought to investigate the expression of FSTL1 and to assess its interplay with miR-198 in human trophoblasts.We used a combination of northern blot analyses, quantitative PCR, small RNA sequencing, western blot and immunohistochemistry to characterize FSTL1 and miR-198 expression in placental trophoblasts. We also used reporter assays to examine the post-transcriptional regulation of FSTL1 and assess its putative regulation by miR-198.We detected the expression of FSTL1 transcript in both the human extravillous trophoblast line HTR-8/SVneo and in primary term human villous trophoblasts. We also found that the expression of FSTL1 was largely restricted to extravillous trophoblasts. Hypoxia enhanced the expression of FSTL1 protein in cultured primary villous trophoblasts. Interestingly, we did not detect any evidence for expression or function of mature miR-198 in human trophoblasts.Our data indicate that placental FSTL1 is expressed particularly in extravillous trophoblasts. We also found no evidence for placental expression of miR-198, or for its regulation of FSTL1, implying that the post-transcriptional regulation of FSTL1 by miR-198 is tissue specific.
Project description:Epithelial carcinomas are well known to activate a prolonged wound-healing program that promotes malignant transformation. Wound closure requires the activation of keratinocyte migration via a dual-state molecular switch. This switch involves production of either the anti-migratory microRNA miR-198 or the pro-migratory follistatin-like 1 (FSTL1) protein from a single transcript; miR-198 expression in healthy skin is down-regulated in favor of FSTL1 upon wounding, which enhances keratinocyte migration and promotes re-epithelialization. Here, we reveal a defective molecular switch in head and neck squamous cell carcinoma (HNSCC). This defect shuts off miR-198 expression in favor of sustained FSTL1 translation, driving metastasis through dual parallel pathways involving DIAPH1 and FSTL1. DIAPH1, a miR-198 target, enhances directional migration through sequestration of Arpin, a competitive inhibitor of Arp2/3 complex. FSTL1 blocks Wnt7a-mediated repression of extracellular signal-regulated kinase phosphorylation, enabling production of MMP9, which degrades the extracellular matrix and facilitates metastasis. The prognostic significance of the FSTL1-DIAPH1 gene pair makes it an attractive target for therapeutic intervention.
Project description:Human cytomegalovirus (CMV) is the leading non-genetic cause of fetal malformation in developed countries. CMV placental infection is a pre-requisite for materno-fetal transmission of virus, and fetal infection. We investigated the roles of the viral pentameric complex gH/gL/pUL128-pUL131A, and cellular platelet-derived growth factor receptor-? (PDGFR?) for CMV infection in first trimester extravillous-derived (SGHPL-4) and villous-derived (HTR-8/SVneo) trophoblast cells. Infection with four CMV clinical and laboratory strains (Merlin, TB40E, Towne, AD169), and Merlin deletion mutants of UL128-, UL130-, and UL131A-genes, showed a cell type-dependent requirement of the viral pentameric complex for infection of trophoblast cells. The viral pentameric complex was essential for infection of villous trophoblasts, but non-essential for extravillous trophoblasts. Blocking of PDGFR? in extravillous trophoblasts, which naturally express PDGFR?, inhibited entry of pentameric complex-deficient CMV strains, but not the entry of pentameric positive CMV strains. Transient expression of PDGFR? in villous trophoblasts, which are naturally deficient in PDGFR?, promoted the entry of CMV strains lacking gH/gL/pUL128-pUL131A, but had no effect on entry of pentameric positive CMV strains. These results suggest PDGFR? is an important cell receptor for entry of CMV mutant strains lacking gH/gL/pUL128-pUL131A complexes in some placental cells, suggesting these entry pathways could be potential antiviral targets.
Project description:BACKGROUND: SERPINE2, one of the potent serpins belonging to the plasminogen activator (PA) system, is involved in the tissue remodeling. We previously demonstrated the expression patterns of Serpine2 in the mouse placenta and uterus, indicating that Serpine2 is a major PA inhibitor in the placenta and uterus during the estrous cycle, pregnancy, and lactation. In this study, we further investigated the expression pattern of SERPINE2 in the human placenta and explored possible functional roles of SERPINE2 in regulating trophoblast activity. METHODS: Placental tissues from various trimesters were collected for real-time reverse-transcription polymerase chain reaction quantification. Immunohistochemical staining was performed in placental tissues to assure localization of SERPINE2. SERPINE2 small interfering (si) RNA was applied to suppress its expression in villous explants and extravillous trophoblast-like 3A cells. Subsequent experiments to evaluate SERPINE2 levels, villous outgrowth, trophoblast invasion, and tube formation were performed. RESULTS: SERPINE2 messenger RNA was detected in the human placenta during pregnancy with the highest levels in the third trimester. The SERPINE2 protein was present in villous syncytiotrophoblasts and trophoblasts of chorionic villi for anti-SERPINE2 immunostaining. Extravillous trophoblasts in the chorionic plate and basal plate confronting the invasive face of anchoring villi were also positive. In most decidual cells, SERPINE2 was observed in the cytoplasm. In addition, fibrinoid deposit was weakly immunoreactive. Introduction of SERPINE2 siRNA into villous explants and trophoblast cells led to significantly reduced villous outgrowth, and trophoblastic migration and invasion. Moreover, capillary-like network formation of 3A cells in Matrigel was greatly attenuated by SERPINE2 siRNA and SERPINE2 antiserum. CONCLUSIONS: These data identify the temporal and spatial SERPINE2 distribution in the human placenta and suggest its possible role in modulating tissue remodeling of extravillous trophoblasts in the placenta during pregnancy.
Project description:Development of the human placenta and its different epithelial trophoblasts is crucial for a successful pregnancy. Besides fusing into a multinuclear syncytium, the exchange surface between mother and fetus, progenitors develop into extravillous trophoblasts invading the maternal uterus and its spiral arteries. Migration into these vessels promotes remodelling and, as a consequence, adaption of blood flow to the fetal-placental unit. Defects in remodelling and trophoblast differentiation are associated with severe gestational diseases, such as preeclampsia. However, mechanisms controlling human trophoblast development are largely unknown. Herein, we show that Notch1 is one such critical regulator, programming primary trophoblasts into progenitors of the invasive differentiation pathway. At the 12th wk of gestation, Notch1 is exclusively detected in precursors of the extravillous trophoblast lineage, forming cell columns anchored to the uterine stroma. At the 6th wk, Notch1 is additionally expressed in clusters of villous trophoblasts underlying the syncytium, suggesting that the receptor initiates the invasive differentiation program in distal regions of the developing placental epithelium. Manipulation of Notch1 in primary trophoblast models demonstrated that the receptor promotes proliferation and survival of extravillous trophoblast progenitors. Notch1 intracellular domain induced genes associated with stemness of cell columns, myc and VE-cadherin, in Notch1- fusogenic precursors, and bound to the myc promoter and enhancer region at RBPJ? cognate sequences. In contrast, Notch1 repressed syncytialization and expression of TEAD4 and p63, two regulators controlling self-renewal of villous cytotrophoblasts. Our results revealed Notch1 as a key factor promoting development of progenitors of the extravillous trophoblast lineage in the human placenta.
Project description:ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 repeats, member 13) is primarily synthesized in liver. The biosynthesis of ADAMTS13 and its physiological role in placenta are not known.We used real-time polymerase chain reaction, immunohistochemistry, and Western blotting analyses, as well as proteolytic cleavage of FRETS (fluorescent resonance energy transfers)-VWF73, to determine ADAMTS13 expression in placenta and trophoblasts obtained from individuals with normal pregnancy and patients with severe preeclampsia. We also determined the role of ADAMTS13 in extravillous trophoblasts using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound scratch assay, transwell migration assay, tube formation assay, and tissue outgrowth assays. We showed that full-length and proteolytically active ADAMTS13 was expressed in normal human placenta, primarily in the trophoblasts and villous core fetal vessel endothelium during pregnancy. Placental expression of ADAMTS13 mRNA, protein, and proteolytic activity was at the highest levels during the first trimester and significantly reduced at the term of gestation. Additionally, significantly reduced levels of placental ADAMTS13 expression was detected under hypoxic conditions and in patients with preeclampsia. In addition, recombinant ADAMTS13 protease stimulated proliferation, migration, invasion, and network formation of trophoblastic cells in culture. Finally, knockdown of ADAMTS13 expression attenuated the ability of tube formation in trophoblast (HTR-8/SVNEO) cells and the extravillous trophoblast outgrowth in placental explants.Our results demonstrate for the first time the expression of ADAMTS13 mRNA and protein in normal and abnormal placental tissues and its role in promoting angiogenesis and trophoblastic cell development. The findings support the potential role of the ADAMTS13-von Willebrand factor pathway in normal pregnancy and pathogenesis of preeclampsia.
Project description:Analysis of gene expression in extravillous trophoblasts and column cells isolated from matrigel embedded 1st trimester placental explants cultured in 1%, 5% or 20% oxygen. The aim of this study is to identify differentially expressed genes and pathways potentially altered by varying oxygen conditions within anchoring column trophoblasts and invasive extravillous trophoblasts. Overall design: Total RNA was isolated from extravillous trophoblasts and column cells propagated from Matrigel-embedded placental explants. All explants were derived from placental villous tissues from 5-8 week gestation placentas. Prior to RNA isolation, placental explants were cultured in either 1%, 5% or 20% oxygen for 48 hours, after which RNA was extracted from trophoblast cells imbedded within Matrigel using Trizol LS reagent. Following RNA extraction, RNA was convereted to cDNA libraries and hybridized onto Affymetrix Clariom S Assay reagents and arrays.
Project description:BACKGROUND: The trophoblast compartment of the placenta comprises various subpopulations with distinct functions. They interact among each other by secreted signals thus forming autocrine or paracrine regulatory loops. We established a first trimester trophoblast cell line (ACH-3P) by fusion of primary human first trimester trophoblasts (week 12 of gestation) with a human choriocarcinoma cell line (AC1-1). RESULTS: Expression of trophoblast markers (cytokeratin-7, integrins, matrix metalloproteinases), invasion abilities and transcriptome of ACH-3P closely resembled primary trophoblasts. Morphology, cytogenetics and doubling time was similar to the parental AC1-1 cells. The different subpopulations of trophoblasts e.g., villous and extravillous trophoblasts also exist in ACH-3P cells and can be immuno-separated by HLA-G surface expression. HLA-G positive ACH-3P display pseudopodia and a stronger expression of extravillous trophoblast markers. Higher expression of insulin-like growth factor II receptor and human chorionic gonadotropin represents the basis for the known autocrine stimulation of extravillous trophoblasts. CONCLUSION: We conclude that ACH-3P represent a tool to investigate interaction of syngeneic trophoblast subpopulations. These cells are particularly suited for studies into autocrine and paracrine regulation of various aspects of trophoblast function. As an example a novel effect of TNF-alpha on matrix metalloproteinase 15 in HLA-G positive ACH-3P and explants was found.
Project description:Early in pregnancy, trophoblast invasion into the decidua and inner myometrium is essential for establishment of proper implantation, maternal-fetal exchange, and immunological tolerance of the feto-placental allograft. Unlike villous trophoblasts (VTs), extravillous trophoblasts (EVTs) are unique in their capacity to invade the maternal decidua and myometrium. The largest human microRNA (miRNA) gene cluster, the chromosome 19 miRNA cluster (C19MC), is expressed almost exclusively in the placenta and, rarely, in certain tumors and undifferentiated cells. In the work reported here, we found that the expression of C19MC miRNAs is higher in VTs than in EVTs. Using a bacterial artificial chromosome (BAC)-mediated overexpression of C19MC miRNAs in an EVT-derived cell line, which does not naturally express these miRNAs, we found that C19MC miRNAs selectively attenuate cell migration without affecting cell proliferation or apoptosis. A microarray analysis revealed that C19MC miRNAs regulate target transcripts related to cellular movement. Our data also implicated a specific C19MC member, miR-519d, indirectly regulating the EVT invasive phenotype by targeting CXCL6, NR4A2 and FOXL2 transcripts through a 3'UTR miRNA-responsive element. Together, our data suggest a role for C19MC miRNAs in modulating the migration of EVTs.
Project description:Trophoblast differentiation during early placental development is critical for successful pregnancy and aberrant differentiation causes preeclampsia and early pregnancy loss. During the first trimester, cytotrophoblasts are exposed to low oxygen tension (equivalent to~2%-3% O2) and differentiation proceeds along an extravillous pathway (giving rise to invasive extravillous cytotrophoblasts) and a villous pathway (giving rise to multinucleated syncytiotrophoblast). Interstitial extravillous cytotrophoblasts invade the decidua, while endovascular extravillous cytotrophoblasts are involved in re-modelling uterine spiral arteries. We tested the idea that sodium butyrate (an epigenetic modulator) induces trophoblast differentiation in early gestation rhesus monkey trophoblasts through activation of the Wnt/?-catenin pathway. The results show that syncytiotrophoblast formation was increased by butyrate, accompanied by nuclear accumulation of ?-catenin, and increased expression of EnvV2 and galectin-1 (two factors thought to be involved in trophoblast fusion). Surprisingly, the expression of GCM1 and syncytin-2 was not affected by sodium butyrate. When trophoblasts were incubated with lithium chloride, a GSK3 inhibitor that mimics Wnt activation, nuclear accumulation of ?-catenin also occurred but differentiation into syncytiotrophoblast was not observed. Instead the cells differentiated to mononucleated spindle-shaped cells and showed molecular and behavioral characteristics of endovascular trophoblasts. Another highly specific inhibitor of GSK3, CHIR99021, failed to induce endovascular trophoblast characteristics. These observations suggest that activation of the Wnt/?-catenin pathway correlates with both trophoblast differentiation pathways, but that additional factors determine specific cell fate decisions. Other experiments suggested that the differential effects of sodium butyrate and lithium chloride might be explained by their effects on TNF? production. The results provide valuable tools to manipulate trophoblast differentiation in vitro and to better understand the differentiation pathways that occur during early gestation.
Project description:Seven patient paired primary human HLA-G+ extravillous trophoblasts (EVT) and Villous trophoblasts (VT) obtained from 1st trimester (7-9 weeks) villous tissue were obtained. RNA was isolated directly after isolation and purification using FACS sort for CD45-HLA-G+ (EVT) and CD45-HLA-G-EGFR1+ (VT) fractions. Expression profiles were compared to two samples of the choriocarcinoma cell line JEG-3 and four samples of decidual stromal cells (DSC) at passage 2 after cell culture.