3-Dimensional JEG-3 choriocarcinoma cell organoids as a model for trophoblast expansion and differentiation.

JEG-3 choriocarcinoma cells have been widely used as a model for placental trophoblast. Herein, 3-dimensional (3D) JEG-3 organoids (JEG-3-ORGs) were established using a protocol that we recently developed for primary cytotrophoblast organoids (CTB-ORGs). 3D JEG-3-ORGs, cultivated in basic culture medium, rapidly divide and spontaneously undergo differentiation. Under stem cell culture conditions (activation of WNT/EGF signalling and inhibition of TGF-ß signalling) smaller organoids with reduced proliferative capacity were generated specifically abolishing expression of extravillous trophoblast (EVT)-specific genes. Similar to CTB-ORGs, removal of the WNT activator CHIR99021 induced re-expression of these genes in JEG-3-ORGs. Hence, JEG-3-ORGs could be used as a model for directed EVT differentiation.

Expression pattern and phosphorylation status of Smad2/3 in different subtypes of human first trimester trophoblast.

INTRODUCTION: TGF-ß superfamily members are thought to play a pivotal role in placental development and differentiation. However, their downstream effectors, the Smad transcription factors, have been poorly investigated in human trophoblasts. METHODS: Expression and localisation of the canonical TGF-ß targets Smad2/3 and their regulators (Smad4 and Smad7) were investigated in first trimester placenta and purified cytotrophoblast (CTB) subtypes using immunofluorescence, western blotting and qPCR. Canonical and non-canonical activation was analysed in nuclear/cytoplasmic extracts of trophoblast subtypes as well as in tissue sections using antibodies against Smad2/3, phosphorylated either at the C-terminus (pSmad2C/3C) or in their linker regions (pSmad2L/3L). Smad phosphorylation was also examined in differentiating extravillous trophoblasts (EVTs) in the absence or presence of decidual stromal cell (DSC)-conditioned medium. RESULTS: Smad2, Smad4 and Smad7 protein were uniformly expressed between 6th and 12th week placentae and the different isolated CTB subtypes. Activated pSmad2L was mainly detected in nuclei and cytoplasm of villous CTBs, whereas pSmad2C was absent from these cells. In contrast, pSmad2C could be detected in the cytoplasm of cell column trophoblasts and in the cytoplasm/nuclei of EVTs. Smad3 and its phosphorylated forms pSmad3C and pSmad3L specifically localised to EVT nuclei. During EVT differentiation autocrine activation of pSmad2C/3C and pSmad3L was observed. DSC-conditioned medium further increased Smad2/3 phosphorylation in EVTs. DISCUSSION: The lack of pSmad2C in villous CTBs suggests that other mitogens than TGF-ß could promote Smad2 linker phosphorylation under homeostatic conditions. Whereas autocrine signalling activates Smad2/3 in differentiating EVTs, paracrine factors contribute to Smad phosphorylation in these cells.

Notch signalling in placental development and gestational diseases.

Activation of Notch signalling upon cell-cell contact of neighbouring cells controls a plethora of cellular processes such as stem cell maintenance, cell lineage determination, cell proliferation, and survival. Accumulating evidence suggests that the pathway also critically regulates these events during placental development and differentiation. Herein, we summarize our present knowledge about Notch signalling in murine and human placentation and discuss its potential role in the pathophysiology of gestational disorders. Studies in mice suggest that Notch controls trophectoderm formation, decidualization, placental branching morphogenesis and endovascular trophoblast invasion. In humans, the particular signalling cascade promotes formation of the extravillous trophoblast lineage and regulates trophoblast proliferation, survival and differentiation. Expression patterns as well as functional analyses indicate distinct roles of Notch receptors in different trophoblast subtypes. Altered effects of Notch signalling have been detected in choriocarcinoma cells, consistent with its role in cancer development and progression. Moreover, deregulation of Notch signalling components were observed in pregnancy disorders such as preeclampsia and fetal growth restriction. In summary, Notch plays fundamental roles in different developmental processes of the placenta. Abnormal signalling through this pathway could contribute to the pathogenesis of gestational diseases with aberrant placentation and trophoblast function.

ERBB2 gene amplification increases during the transition of proximal EGFR(+) to distal HLA-G(+) first trimester cell column trophoblasts.

INTRODUCTION: Although, DNA copy-number alterations (CNAs) have been well documented in a number of adverse phenotypic conditions, accumulating data suggest that CNAs also occur during physiological processes. Interestingly, extravillous trophoblasts induce the expression of the transforming, proto-oncogene ERBB2, which is frequently amplified in human cancer. However, no data are available to address whether trophoblast-related ERBB2 expression might also be linked to genomic amplification. METHODS: Dual color silver as well as fluorescence in situ hybridization analyses were carried out to evaluate frequency and degree of ERBB2 gene and chromosome 17 copy numbers in first trimester placental cell columns and isolated trophoblasts. Proliferative EGFR(+) and differentiated HLA-G(+) trophoblasts were identified or separated by means of in situ immunofluorescence co-stainings and magnetic beads cell isolation, respectively. RESULTS: ERBB2 gene amplification is detected in approximately 40% of isolated HLA-G(+) trophoblasts. Although already detectable in EGFR(+) cells, the percentage and extent of ERBB2 amplification was markedly increased in HLA-G(+) trophoblasts in situ and after isolation. Accordingly, HLA-G(+) trophoblasts highly express ERBB2 on protein level. Finally, ERBB2 copy number variations occur independently of aneuploidy as the majority of ERBB2 amplifying cells were cytogenetically diploid for chromosome 17. DISCUSSION: ERBB2 gene amplification is a frequent event during EVT differentiation. This finding challenges the long standing paradigm, which associates gene amplification with pathological conditions and further supports recent evidences suggesting that CNAs are a normal feature of developmental processes.

Expression pattern and function of Notch2 in different subtypes of first trimester cytotrophoblast.

INTRODUCTION: Notch signalling has been shown to control cytotrophoblast (CTB) proliferation, differentiation and motility suggesting that the conserved signalling pathway could be critical for human placental development. Since individual Notch receptors have not been elucidated, we herein investigated expression pattern and function of Notch2 in different first trimester trophoblast subpopulations. METHODS: Localisation of Notch2 was analysed in first trimester placental and decidual tissues using immunofluorescence. Notch2 transcript and protein levels were studied by qRT-PCR and Western blotting in proliferative EGF receptor (EGFR)(+) and differentiated HLA-G(+) CTBs, respectively, isolated from early placentae by MACS. CTB migration through fibronectin-coated transwells as well as proliferation (EdU labelling) in floating villous explant cultures and primary CTBs were investigated in the presence of Notch2 siRNAs or specific antibodies blocking Notch2 cleavage. RESULTS: In tissue sections Notch2 expression was higher in HLA-G(+) distal cell column trophoblasts (dCCTs) compared to proximal CCTs. Accordingly, expression of Notch2 mRNA and protein were elevated in isolated HLA-G(+) CTBs compared to EGFR(+) CTBs. Notch2 was also detectable in interstitial CTBs as well as in intramural CTBs associated with maternal decidual vessels. Antibody-mediated inhibition of Notch2 signalling did not affect proliferation, but increased migration of SGHPL-5 cells and primary CTBs. Similarly, Notch2 siRNA treatment promoted trophoblast motility. DISCUSSION: Notch2 is present in differentiated cells of the extravillous trophoblast lineage, such as dCCTs, interstitial and intramural CTBs, suggesting diverse roles of the particular receptor. Notch2 signalling, activated by cell-cell contact of neighbouring dCCTs, could attenuate trophoblast migration.

Notch-dependent RBPJκ inhibits proliferation of human cytotrophoblasts and their differentiation into extravillous trophoblasts.

Abnormal development of invasive trophoblasts has been implicated in the pathogenesis of human pregnancy diseases such as pre-eclampsia. However, critical signalling pathways controlling formation and differentiation of these cells have been poorly elucidated. Here, we provide evidence that the canonical Notch pathway, operating through Notch-dependent activation of its key regulatory transcription factor RBPJκ, controls proliferation and differentiation in villous explant cultures and primary trophoblasts of early pregnancy. Immunofluorescence of first trimester placental tissue revealed expression of RBPJκ and its co-activators, the MAML proteins, in nuclei of proliferative cell column trophoblasts (CCT) and differentiated, extravillous trophoblasts (EVTs). However, RBPJκ expression, transcript levels of the Notch target gene HES1 and activity of a Notch/RBPJκ-dependent luciferase reporter decreased during in vitro differentiation of primary cytotrophoblasts on fibronectin. Silencing of RBPJκ using silencing RNAs (siRNAs) increased proliferation of CCTs in floating villous explant cultures analysed by outgrowth and BrdU labelling. Similarly, down-regulation of the transcription factor enhanced BrdU incorporation in isolated primary cultures. However, motility of these cells was not affected. In addition, gene silencing of RBPJκ increased cyclin D1 expression in the two trophoblast model systems as well as markers of the differentiated, EVT, i.e. integrin α1, ADAM12 and T-cell factor 4. In summary, the data suggest that Notch-dependent RBPJκ activity could be required for balanced rates of trophoblast proliferation and differentiation in human placental anchoring villi preventing exaggerated trophoblast overgrowth as well as premature formation of EVTs.

Review: the ADAM metalloproteinases - novel regulators of trophoblast invasion?

During pregnancy, the extravillous trophoblast (EVT) invades the maternal decidua and remodels spiral arteries reaching as far as the inner third of the myometrium. This process is mandatory to a successful pregnancy since EVTs regulate spiral artery remodeling to achieve maximal vasodilation and thus an adequate nutrient supply to the embryo or communicate with maternal leukocyte populations to guarantee acceptance of the allogeneic conceptus. To achieve this, EVTs undergo a remarkable and unique differentiation process, which yields different phenotypes such as proliferative cell column trophoblasts or growth-arrested, invasive interstitial or endovascular cytotrophoblasts. Matrix metalloproteinases have long been seen as imperative to trophoblast invasion because of their ability to degrade extracellular matrix and therefore allow cellular movement in foreign tissues. However, global gene expression analysis reveals that EVTs also express various members of distintegrin and metalloproteinases (ADAMs). These proteases are associated with the process of proteolytic shedding and activation of surface proteins including growth factors, cytokines, receptors and their ligands rather than extracellular matrix breakdown. While ADAM12 has been associated with chromosomal abnormalities as well as preeclampsia or intrauterine fetal growth restriction, the function of ADAMs in trophoblasts remains elusive. In this article, we review the diverse invasive trophoblast phenotypes, EVT-associated protease systems and related open questions. In addition, we examine recent information about relevant ADAM members and their putative implications for EVT biology.

The role of interleukin-1ß in human trophoblast motility.

The pleiotropic cytokine interleukin-1ß (IL-1ß) can promote physiological cell migration, as well as cancer cell invasion and metastasis. Its role in human trophoblast invasion, however, has not been satisfactorily answered since direct, indirect as well as no effects on trophoblast motility have been published. Therefore, the role of IL-1ß has been re-evaluated by exclusively using human primary trophoblast model systems. Immunofluorescence of first trimester placentae indicated IL-1 receptor 1 (IL-1R1) protein expression in first trimester villous cytotrophoblasts (vCTB) and extravillous trophoblasts (EVT). The latter expressed higher mRNA levels of the receptor as shown by comparative gene chip data of vCTB and EVT. Similarly, Western blot analyses and immunofluorescence revealed a time- and differentiation-dependent increase of IL-1R1 in primary EVT seeded on fibronectin. IL-1ß dose-dependently elevated migration of isolated first trimester EVT through fibronectin-coated transwells, which was inhibited in the presence of IL-1R antagonist (IL-1Ra), whereas proliferation of these cells was not affected. Similarly, the interleukin did not alter proliferation of vCTB and cell column trophoblasts in floating villi of early pregnancy, but promoted migration in villous explant cultures seeded on collagen I. Western blot analyses of supernatants of primary EVT and first trimester villous explant cultures revealed IL-1ß induced secretion of urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI)-1 and PAI-2, which was diminished upon combined IL-1ß/IL-1Ra treatment. In conclusion, these data suggest that IL-1ß directly promotes trophoblast motility of first trimester EVT involving the uPA/PAI system.

IFPA Award in Placentology lecture: molecular regulation of human trophoblast invasion.

Invasion of extravillous trophoblast cell types into maternal uterine tissues is essential for successful human placental development and progression of pregnancy. Whereas endovascular trophoblasts migrate into the maternal spiral arteries, interstitial trophoblasts invade the decidual stroma, colonize the vessels from outside and communicate with diverse uterine cell types such as decidual stromal cells, macrophages and uterine NK cells. For example, interstitial trophoblasts expressing polymorphic human leukocyte antigen-C interact with uterine NK cells through binding to their killer immunoglobulin-like receptors which likely plays a role in trophoblast invasion and reproductive success of pregnancy. Both extravillous trophoblast subtypes are critically involved in the vascular transformation of the spiral arteries into dilated conduits ensuring appropriate blood flow into the intervillous space. Failures in this remodeling process are thought to be associated with severe forms of fetal growth restriction, preeclampsia and other pregnancy complications warranting studies on the molecular regulation of extravillous trophoblast differentiation. Moreover, interstitial trophoblast-derived hormones may regulate diverse biological functions in the decidua. In particular, human chorionic gonadotrophin has been shown to promote angiogenesis and to suppress apoptosis of endometrial stromal cells. In return, decidual cells produce a plethora of soluble factors controlling trophoblast invasion in a time- and distance-dependent manner. However, the underlying mechanisms have not been fully elucidated. Here, we will summarize autocrine as well as paracrine factors regulating invasion of extravillous trophoblasts and discuss critical signaling cascades involved. In addition, we will focus on key regulatory transcription factors controlling cell column proliferation and differentiation of the human extravillous trophoblast.

Trophoblast invasion: assessment of cellular models using gene expression signatures.

Invasive, extravillous trophoblasts (EVT) of the human placenta are critically involved in successful pregnancy outcome since they remodel the uterine spiral arteries to increase blood flow and oxygen delivery to the placenta and the developing fetus. To gain more insights into their biological role different primary cell culture models are commonly utilised. However, access to early placental tissue may be limited and primary trophoblasts rapidly cease proliferation in vitro impairing genetic manipulation. Hence, trophoblastic cell lines have been widely used as surrogates to study EVT function. Although the cell lines share some molecular markers with their primary counterpart, it is unknown to what extent they recapitulate the invasive phenotype of EVT. Therefore, we here report the first thorough GeneChip analyses of SGHPL-5, HTR-8/SVneo, BeWo, JEG-3 and the novel ACH-3P trophoblast cells in comparison to previously analysed primary villous cytotrophoblasts (CTBs) and extravillous trophoblasts (EVTs). Analyses of approximately 14,000 commonly expressed genes revealed that EVTs most closely resemble CTBs with considerable differences to the group of choriocarcinoma cells (JEG-3, BeWo, ACH-3P) and the group of SV40 Large T Antigen-selected cell types (SGHPL-5, HTR-8/SVneo). Similarly, analyses of 912 genes discriminating EVT from CTB, or 370 EVT-specific genes did not unravel a particular cell line with close similarity to any of the primary cell types, although molecular signatures common to EVT and each group of cell lines could be identified. Considering the diversity of mRNA expression patterns it is suggested that molecular studies in trophoblast cell lines require verification of the critical steps in an appropriate primary model system.

Wnt signalling in implantation, decidualisation and placental differentiation--review.

The family of secreted Wingless ligands plays major roles in embryonic development, stem cell maintenance, differentiation and tissue homeostasis. Accumulating evidence suggests that the canonical Wnt pathway involving nuclear recruitment of ß-catenin and activation of Wnt-dependent transcription factors is also critically involved in development and differentiation of the diverse reproductive tissues. Here, we summarise our present knowledge about expression, regulation and function of Wnt ligands and their frizzled receptors in murine and human endometrial and placental cell types. In mice, Wnt signalling promotes early trophoblast lineage development, blastocyst activation, implantation and chorion-allantois fusion. Moreover, different Wnt ligands play essential roles in the development of the murine uterine tract, in cycling endometrial cells and during decidualisation. In humans, estrogen-dependent endometrial cell proliferation, decidualisation, trophoblast attachment and invasion were shown to be controlled by the particular signalling pathway. Failures in Wnt signalling are associated with infertility, endometriosis, endometrial cancer and gestational diseases such as complete mole placentae and choriocarcinomas. However, our present knowledge is still scarce due to the complexity of the Wnt network involving numerous ligands, receptors and non-canonical pathways. Hence, much remains to be learned about the role of different Wnt signalling cascades in reproductive cell types and their changes under pathological conditions.

Functional expression of the human neonatal Fc-receptor, hFcRn, in isolated cultured human syncytiotrophoblasts.

Materno-fetal IgG transfer in the mature human placenta involves transport across the syncytiotrophoblast (STB) and fetal endothelial cell layer. The MHC class I-related Fc gamma-receptor (hFcRn) localized in STB as well as in endothelial cells is involved in overall IgG transfer from the maternal into the fetal circulation. Functional hFcRn is a heterodimer of a transmembrane alpha-chain and beta2-microglobulin. To establish the basis for future studies to unravel the mechanism of IgG transport in STB, we investigated hFcRn alpha-chain and beta2-microglobulin expression in cytotrophoblasts (CTB) isolated from human term placentae and cultured in vitro under conditions where differentiation into multinuclear STB takes place (>or=48 h). Northern blot analysis demonstrated up-regulation of alpha-chain mRNA after 48 h of in vitro cultivation. Likewise, hFcRn alpha-chain and beta2-microglobulin were at the limit of detection by immunofluorescence microscopy in CTB immediately after isolation, but their expression increased upon STB formation. hFcRn alpha-chain co-localized with beta2-microglobulin in multinuclear STB and formed a functional, i.e. low pH IgG binding, receptor as shown by affinity isolation. The in vitro differentiated STB exhibited specific, low pH-dependent IgG binding to the plasma membrane. In conclusion, these cultures can now be applied to study the role of hFcRn in IgG transport and trafficking in STB cultures in vitro.

Expression, regulation and functional characterization of matrix metalloproteinase-3 of human trophoblast.

MMP-3 has been detected in human placenta and reduced expression of the enzyme was observed in invasive trophoblasts of patients with severe preeclampsia. However, detailed expression pattern, regulation and biological properties of the placental protease have not been elucidated so far. RT-PCR analyses, Western blotting and enzyme activity assays revealed that pro- and active form of MMP-3 were predominantly expressed in purified first trimester villous trophoblasts, in invasive cytotrophoblasts of differentiating explant cultures and in trophoblastic SGHPL-4 cells. Accordingly, immunofluorescene of first trimester placental tissues detected MMP-3 mainly in villous and extravillous cytotrophoblasts. IL-1beta, an inducer of MMP-3 in decidual cells, increased secretion and activity of the protease in trophoblast supernatants in a dose- and time-dependent manner. IL-1beta-stimulated production of the enzyme was suppressed in the presence of inhibitors of MAPK and AKT signalling. Similar to recombinant MMP-3, MMP-3 in supernatants of IL-1beta-stimulated decidual stromal or SGHPL-4 cells degraded IGFBP-1 in vitro resulting in the appearance of cleavage products at approximately 25, 22, 17, 14 and 11kD. However, cleavage assays using recombinant MMP-2 suggested that the gelatinase may contribute to IGFBP-1 degradation in trophoblast supernatants. Despite its effects on MMP-3 expression IL-1beta failed to significantly alter invasion of SGHPL-4 cells through Matrigel-coated transwells. In conclusion, the data suggest that invasive trophoblast cell models secrete bioactive MMP-3. Inducible expression of the protease involves MAPK and AKT signalling. In addition to the decidua, MMP-3 of trophoblasts may contribute to the regulation of the IGF system by degrading IGFBP-1.

Human tumour necrosis factor: physiological and pathological roles in placenta and endometrium.

The cytokine tumour necrosis factor alpha (TNF) is a well known member of the TNF superfamily consisting of at least 18 ligands and 29 different receptors involved in numerous cellular processes. TNF signals through two distinct receptors TNFR1 and TNFR2 thereby controlling expression of cytokines, immune receptors, proteases, growth factors and cell cycle genes which in turn regulate inflammation, survival, apoptosis, cell migration, proliferation and differentiation. Since expression of TNF was discovered in amnion and placenta many studies demonstrated the presence of the cytokine and its receptors in the diverse human reproductive tissues. Whereas TNF has been implicated in ovulation, corpus luteum formation and luteolysis, this review focuses on the functions of TNF in human placental, endometrial and decidual cell types of normal tissues and also discusses its role in endometrial and gestational diseases. Physiological levels of the cytokine could be important for balancing cell fusion and apoptotic shedding of villous trophoblasts and to limit trophoblast invasion into maternal decidua. Regulation of the TNF/TNFR system by steroid hormones also suggests a role in uterine function including menstrual cycle-dependent destruction and regeneration of endometrial tissue. Aberrant levels of TNF, however, are associated with diverse reproductive diseases such as amniotic infections, recurrent spontaneous abortions, preeclampsia, preterm labour or endometriosis. Hence, concentrations, receptor distribution and length of stimulation determine whether TNF has beneficial or adverse effects on female reproduction and pregnancy.

Regulation of trophoblast invasion - a workshop report.

Trophoblast invasion during placental development helps to establish efficient physiological exchange between maternal and fetal circulatory systems. Trophoblast stem cells differentiate into multiple subtypes, including some that are highly invasive. Signalling to the trophoblast from decidua, uterine natural killer cells and vascular smooth muscle can regulate extravillous trophoblast differentiation. Important questions remain about how these cellular interactions promote trophoblast invasion and the signalling pathways that are involved. New and established biological models are being used to experimentally examine these interactions and the underlying molecular mechanisms.

Complex expression pattern of Wnt ligands and frizzled receptors in human placenta and its trophoblast subtypes.

Canonical Wingless (Wnt) signalling provoked by exogenous and endogenous Wnt ligands was recently shown to play a crucial role in the invasive differentiation of human trophoblasts. To gain insights into the expression pattern of the developmental regulators, we analysed all human Wnt ligands and their frizzled (FZD) receptors in the human placenta and different trophoblast model systems using semi-quantitative PCR. Fourteen out of 19 Wnt ligands and 8 out of 10 FZD receptors were detectable in placental tissues, however, expression patterns varied with gestational age and between different trophoblast subtypes suggesting cell-specific functions. Besides Wnt ligands acting through the canonical pathway, non-canonical ligands such as Wnt-5a, which may also activate alternative Wnt signalling pathways or inhibit canonical Wnt signalling, could be identified. Western blot analyses revealed secretion of Wnt-5a from primary trophoblast cultures and trophoblastic cell lines. To evaluate the potential role of Wnt-5a, SGHPL-5 trophoblast cells were transfected with luciferase reporter plasmids harbouring eight T-cell factor (TCF) DNA-recognition sequences which are exclusively activated through the canonical Wnt signalling pathway. Luciferase assays revealed that Wnt-3a-induced reporter activity was repressed by recombinant Wnt-5a indicating an antagonistic role in trophoblasts. The data suggest that a complex network of Wnt ligands and FZD receptors may regulate developmental processes of the human placenta.

Effects of different human chorionic gonadotrophin preparations on trophoblast differentiation.

Recent evidence from the literature suggested that hCG preparations purified from urine of pregnant women, which are widely used in in vitro studies and IVF programs, may contain contaminants such as EGF. To determine the putative biological effects of the contaminating growth factor, we here investigated distinct trophoblast differentiation processes in the presence of various hCG compounds. Western blot analyses indicated that treatment of trophoblastic SGHPL-5 cells and purified term trophoblasts with potentially EGF-contaminated hCG (hCG-A) resulted in auto-phosphorylation of the EGF receptor at tyrosine 1173 whereas supplementation of another urine-purified hCG preparation (hCG-B), recombinant holo-hCG or recombinant alphahCG had no effects. Phosphorylation was specifically blocked by the EGF receptor inhibitor PD153035. Urinary hCG-A was most effective in promoting invasion of SGHPL-5 cells through Matrigel-coated transwells, but increased invasiveness was also observed in the presence of hCG-B or recombinant holo-hCG. Similarly, the extent of syncytialisation of term trophoblasts, quantitated by nuclei in desmoplakin-negative areas, was highest upon addition of hCG-A or recombinant EGF as a control. PD153035 reduced invasion and fusion of trophoblasts supplemented with hCG-A, but did not diminish the effects provoked by hCG-B. In conclusion, the data suggest that the EGF contamination of hCG considerably affects trophoblast function. Experiments using EGF-free hCG preparations demonstrate that the hormone increases trophoblast invasion and syncytialisation.

Tumour necrosis factor-alpha impairs chorionic gonadotrophin beta-subunit expression and cell fusion of human villous cytotrophoblast.

Growth factors expressed at the fetal-maternal interface modulate hormone expression of placental trophoblasts. The aim of this study was to investigate the effects of different cytokines on hCG subunit mRNA expression in differentiating villous cytotrophoblasts. Quantitative real-time PCR revealed a 1.8- and 6.9-fold increase of hCG-alpha and hCG-beta mRNA levels, respectively, between 36 and 60 h of term trophoblast syncytialization. Compared with controls, neither interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-10, IL-13 and IL-15 nor tumour necrosis factor (TNF)-alpha significantly altered hCG-alpha mRNA expression. Similarly, the ILs did not affect hCG-beta transcript levels. In contrast, TNF-alpha suppressed hCG-beta mRNA 3.8- and 1.8-fold at 36 and 60 h of term trophoblast differentiation. Accordingly, hCG secretion was impaired by TNF-alpha but not by the different ILs. Moreover, TNF-alpha reduced luciferase expression of reporter plasmids harbouring the proximal hCG-beta5 promoter to 35 and 77%, respectively, in primary term trophoblasts and trophoblastic SHGPL-5 cells. In addition, counting of nuclei in syncytialized, desmoplakin-negative areas revealed a 1.9-fold reduction of term trophoblast fusion in the presence of TNF-alpha. Similarly, floating explant cultures prepared from first trimester-denuded villi recovered the syncytium 2.8-fold less efficiently during 72 h of cytokine treatment. Concomitantly, TNF-alpha impaired induction of endogenous and secreted hCG-beta protein levels in these cultures. The data suggest that TNF-alpha decreases hCG-beta mRNA and protein expression by reducing gene transcription and trophoblast cell fusion. Suppression of these processes by TNF-alpha could partly explain the adverse effects of the cytokine on placental function and pregnancy outcome.

Trophoblast differentiation: progenitor cells, fusion and migration -- a workshop report.

Challenge lies ahead in unravelling the role played by trophoblast and its repertoire of expressed genes in normal human placental development, growth and pathology. Specific technical advances will clearly be required for characterisation of function. In particular, improvements in our repertoire of in vitro models are needed before many of the key questions can be answered. Recent advances in the study of human trophoblast differentiation are discussed.

TNFalpha-mediated induction of PAI-1 restricts invasion of HTR-8/SVneo trophoblast cells.

The pro-inflammatory cytokine TNFalpha has numerous effects on placental trophoblasts. Here, we investigated the effects of the cytokine on gene expression and function of the extravillous trophoblast cell line HTR-8/SVneo. Wound healing and Matrigel invasion assays demonstrate that TNFalpha impairs motility and invasiveness. In contrast, counting of cumulative cell numbers and FACS analyses revealed that the cytokine did neither affect proliferation nor distribution of cell cycle phases. Immunocytochemistry of the cytokeratin 18 neo-epitope suggests that TNFalpha did not induce apoptosis in HTR-8/SVneo cells. Gelatine zymography and enzyme activity assays of supernatants of TNFalpha-treated cells demonstrate elevation of the pro- and active form of MMP-9 suggesting that increased expression of the protease cannot overcome the TNFalpha-inhibitory effect on cell invasion. Semi-quantitative RT-PCR analyses suggest that the cytokine may not alter mRNA levels of uPA and tPA. However, elevated expression of PAI-1 was detected by RT-PCR, as well as by Northern and Western blot analyses. Supplementation of PAI-1-blocking antibodies restored invasion of TNF-alpha-incubated HTR-8/SVneo cells through Matrigel-coated transwells. In addition, immunocytochemistry revealed nuclear accumulation of the p65 subunit of NFkappaB in the presence of the cytokine. EMSA indicated TNFalpha-induced binding of the inflammatory transcription factor to an NFkappaB consensus sequence and to the NFkappaB recognition site located in the PAI-1 promoter. The data suggest that TNFalpha restricts trophoblast invasion mainly by increasing the expression of PAI-1. Induction of the inhibitor may involve TNFalpha-stimulated activation of NFkappaB.