Cell Line BeWo Research Articles

Rottlerin-mediated inhibition of Toxoplasma gondii growth in BeWo trophoblast-like cells

Autophagy is a crucial and physiological process for cell survival from yeast to mammals, including protozoan parasites. Toxoplasma gondii, an intracellular parasite, typically exploits autophagic machinery of host cell; however host cell upregulates autophagy to combat the infection. Herein we tested the efficacy of Rottlerin, a natural polyphenol with autophagic promoting properties, against Toxoplasma infection on the chorioncarcinoma-derived cell line BeWo. We found that Rottlerin, at sub-toxic doses, induced morphological and biochemical alterations associated with autophagy and decreased Toxoplasma growth in infected cells. Although autophagy was synergically promoted by Toxoplasma infection in combination with Rottlerin treatment, the use of the autophagy inhibitor chloroquine revealed that Rottlerin anti-parasitic effect was largely autophagy-independent and likely mediated by the converging inhibitory effect of Rottlerin and Toxoplasma in host protein translation, mediated by mTOR inhibition and eIF2α phosphorylation. Both events, which on one hand could explain the additive effect on autophagy induction, on the other hand led to inhibition of protein synthesis, thereby depriving Toxoplasma of metabolically essential components for multiplication. We suggest that modulation of the competition between pathogen requirement and host cell defense might be an attractive, novel therapeutic approach against Toxoplasma infection and encourage the development of Rottlerin-based new therapeutic formulations.

Role of microRNA-130b in placental PGC-1α/TFAM mitochondrial biogenesis pathway

Diabetes during pregnancy is associated with abnormal placenta mitochondrial function and increased oxidative stress, which affect fetal development and offspring long-term health. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master regulator of mitochondrial biogenesis and energy metabolism. The molecular mechanisms underlying the regulation of PGC-1α in placenta in the context of diabetes remain unclear. The present study examined the role of microRNA 130b (miR-130b-3p) in regulating PGC-1α expression and oxidative stress in a placental trophoblastic cell line (BeWo). Prolonged exposure of BeWo cells to high glucose mimicking hyperglycemia resulted in decreased protein abundance of PGC-1α and its downstream factor, mitochondrial transcription factor A (TFAM). High glucose treatment increased the expression of miR-130b-3p in BeWo cells, as well as exosomal secretion of miR-130b-3p. Transfection of BeWo cells with miR-130b-3p mimic reduced the abundance of PGC-1α, whereas inhibition of miR-130b-3p increased PGC-1α expression in response to high glucose, suggesting a role for miR-130b-3p in mediating high glucose-induced down regulation of PGC-1α expression. In addition, miR-130b-3p anti-sense inhibitor increased TFAM expression and reduced 4-hydroxynonenal (4-HNE)-induced production of reactive oxygen species (ROS). Taken together, these findings reveal that miR-130b-3p down-regulates PGC-1α expression in placental trophoblasts, and inhibition of miR-130b-3p appears to improve mitochondrial biogenesis signaling and protect placental trophoblast cells from oxidative stress.

Molecular analysis of sequence and splice variants of the human SLC13A4 sulfate transporter.

The solute linked carrier 13A4 gene (SLC13A4) is abundantly expressed in the human and mouse placenta where it is proposed to transport nutrient sulfate to the fetus. In mice, targeted disruption of placental Slc13a4 leads to severe and lethal fetal phenotypes, however the involvement of SLC13A4 in human development is unknown. A search of the NCBI and Ensembl gene databases identified two alternatively spliced SLC13A4 mRNA transcripts and 98 SLC13A4 gene variants, including 85 missense, 4 splice site, 5 frameshift and 2 nonsense variants, as well as 2 in-frame deletions. We examined the relative abundance of the two SLC13A4 mRNA transcripts and then compared the sulfate transport function and plasma membrane expression of both isoforms as well as 6 sequence variants that predict disrupted SLC13A4 protein structure and function. SLC13A4 mRNA variant 1 has three additional nucleotides CAG compared to SLC13A4 mRNA variant 2 as a result of alternative splicing at the 5'-end of exon 6. Using qRT-PCR, we show a 4-fold higher abundance of SLC13A4 mRNA variant 1 compared to variant 2 in term human placentas and cultured BeWo and JEG-3 cell lines. The corresponding SLC13A4 protein isoforms 1 and 2 were found to have similar sulfate uptake activity and apical membrane expression in cultured MDCK cells. In addition, sulfate uptake into MDCK cells was similar between SLC13A4 isoform 1 and four missense variants N300S, F310C, E360Q and I570V, whereas V513M and frameshift variant L72Sfs led to partial (≈75% decrease) and complete loss-of-function, respectively. Localisation of these variants in MDCK cells showed N300S, E360Q, V513M and I570V expression on the apical plasma membrane, L72Sfs intracellularly and F310C on both apical and basolateral membranes. Our finding of partial and complete loss-of-function variants warrants further studies of the potential involvement of SLC13A4 in fetal pathophysiology.

Cytotoxicity of Endocytosis and Efflux Inhibitors in the BeWo Cell Line.

AimsThe purpose of this study was to determine the cell viability and cytotoxicity of various endocytosis and efflux inhibitors which can be used to determine transport and uptake mechanisms in the BeWo (b30 clone) human placental trophoblast cell line. Ethanol and dimethylsulfoxide (DMSO) were also studied since they are often used as cosolvents for administration of these inhibitors.MethodologyThe water-soluble tetrazolium-1 (WST-1) assay was used to quantify cell viability and the lactate dehydrogenase (LDH) assay was used to determine cytotoxicity.ResultsBy the WST-1 assay, reduced cell viability was observed following 4 hours of exposure to chlorpromazine (10 μg/mL), colchicine (1 mM), filipin (3 μg/mL), gentamicin (2 mM), GF120918 (1 μM), methyl-β-cyclodextrin (5 mM), and verapamil (100 μM). By the LDH assay, however, no cytotoxicity was observed after 4 hours of exposure to the aforementioned compounds. Amiloride (500 μM), ethanol (up to 0.1% v/v), and DMSO (up to 0.1% v/v) did not reduce cell viability nor induce cytotoxicity.ConclusionThis information is valuable when selecting potential inhibitors of endocytosis and efflux and the selection of time points for mechanistic studies.

Netrin-4 Expression and Regulation by Hypoxia in Human Placenta

Netrin-4 is a well-known actor involved in axonal guidance. Recently we have reported the cellular localization of netrin-4 in human placenta. Furthermore we demonstrated its role in placental angiogenesis. Here we determined netrin-4 mRNA expression in first trimester placenta. First, we showed an increase in netrin-4 mRNA expression at 11-13 weeks of gestation (wg), suggesting a negative regulation of netrin-4 expression by hypoxia. In fact, the human placenta undergoes a transition from a low oxygenated to a highly oxygenated environment during the first trimester of pregnancy. This physiological switch in oxygen tension is a prerequisite for proper placental development and involves the hypoxia-inducible factor (HIF-1), a protein that is up-regulated under hypoxic conditions. Then, we used the trophoblast cell line BeWo to test this hypothesis. BeWo cells cultured under hypoxic conditions exhibited decreased levels of netrin-4 mRNA expression. Using siRNA strategy, hypoxia-inducible factor 1α (HIF-1α) knock-down induced a significant increase in netrin-4 mRNA expression. Altogether these results indicate that netrin-4 expression in the trophoblast cells is regulated by hypoxia via the HIF-1 pathway.

Heme-mediated apoptosis and fusion damage in BeWo trophoblast cells.

Placental malaria (PM) is a complication associated with malaria infection during pregnancy that often leads to abortion, premature delivery, intrauterine growth restriction and low birth weight. Increased levels of circulating free heme, a by-product of Plasmodium-damaged erythrocytes, is a major contributor to inflammation, tissue damage and loss of blood brain barrier integrity associated with fatal experimental cerebral malaria. However, the role of heme in PM remains unknown. Proliferation and apoptosis of trophoblasts and fusion of the mononucleated state to the syncytial state are of major importance to a successful pregnancy. In the present study, we examined the effects of heme on the viability and fusion of a trophoblast-derived cell line (BeWo). Results indicate that heme induces apoptosis in BeWo cells by activation of the STAT3/caspase-3/PARP signaling pathway. In the presence of forskolin, which triggers trophoblast fusion, heme inhibits BeWo cell fusion through activation of STAT3. Understanding the effects of free plasma heme in pregnant women either due to malaria, sickle cell disease or other hemolytic diseases, will enable identification of high-risk women and may lead to discovery of new drug targets against associated adverse pregnancy outcome.

Molecular hydrogen ameliorates several characteristics of preeclampsia in the Reduced Uterine Perfusion Pressure (RUPP) rat model

Oxidative stress plays an important role in the pathogenesis of preeclampsia. Recently, molecular hydrogen (H2) has been shown to have therapeutic potential in various oxidative stress-related diseases. The aim of this study is to investigate the effect of H2 on preeclampsia. We used the reduced utero-placental perfusion pressure (RUPP) rat model, which has been widely used as a model of preeclampsia. H2 water (HW) was administered orally ad libitum in RUPP rats from gestational day (GD) 12–19, starting 2 days before RUPP procedure. On GD19, mean arterial pressure (MAP) was measured, and samples were collected. Maternal administration of HW significantly decreased MAP, and increased fetal and placental weight in RUPP rats. The increased levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and diacron reactive oxygen metabolites as a biomarker of reactive oxygen species in maternal blood were decreased by HW administration. However, vascular endothelial growth factor level in maternal blood was increased by HW administration. Proteinuria, and histological findings in kidney were improved by HW administration. In addition, the effects of H2 on placental villi were examined by using a trophoblast cell line (BeWo) and villous explants from the placental tissue of women with or without preeclampsia. H2 significantly attenuated hydrogen peroxide-induced sFlt-1 expression, but could not reduce the expression induced by hypoxia in BeWo cells. H2 significantly attenuated sFlt-1 expression in villous explants from women with preeclampsia, but not affected them from normotensive pregnancy. The prophylactic administration of H2 attenuated placental ischemia-induced hypertension, angiogenic imbalance, and oxidative stress. These results support the theory that H2 has a potential benefit in the prevention of preeclampsia.

Effects of Phytoestrogen Extracts Isolated from Elder Flower on Hormone Production and Receptor Expression of Trophoblast Tumor Cells JEG-3 and BeWo, as well as MCF7 Breast Cancer Cells.

Herein we investigated the effect of elderflower extracts (EFE) and of enterolactone/enterodiol on hormone production and proliferation of trophoblast tumor cell lines JEG-3 and BeWo, as well as MCF7 breast cancer cells. The EFE was analyzed by mass spectrometry. Cells were incubated with various concentrations of EFE. Untreated cells served as controls. Supernatants were tested for estradiol production with an ELISA method. Furthermore, the effect of the EFE on ERα/ERβ/PR expression was assessed by immunocytochemistry. EFE contains a substantial amount of lignans. Estradiol production was inhibited in all cells in a concentration-dependent manner. EFE upregulated ERα in JEG-3 cell lines. In MCF7 cells, a significant ERα downregulation and PR upregulation were observed. The control substances enterolactone and enterodiol in contrast inhibited the expression of both ER and of PR in MCF7 cells. In addition, the production of estradiol was upregulated in BeWo and MCF7 cells in a concentration dependent manner. The downregulating effect of EFE on ERα expression and the upregulation of the PR expression in MFC-7 cells are promising results. Therefore, additional unknown substances might be responsible for ERα downregulation and PR upregulation. These findings suggest potential use of EFE in breast cancer prevention and/or treatment and warrant further investigation.

Exposure to manganese and lead disrupt the human placental serotonergic system

Objectives: Serotonin is crucial for placental function and fetal development. Maternal exposure to metals can interfere with the placenta functioning and fetal development. The aim of this study is to determine if low concentrations of manganese (Mn) and lead (Pb) alter placental serotonin system. Methods and results: Birth biological samples from a cohort of pregnant women were collected and Mn and Pb levels were measured in maternal and cord blood, and placenta. Expression of placental serotonin 5-HT2A receptor mRNA is lower in high-level groups of Pb and Mn in maternal blood compared to low-level groups. Placental expression of the serotonin transporter (SERT) protein is lower in high-level group of Pb in cord blood compared to low-level group. To better understand the effect of metals on the placental serotonin system, BeWo cell line (trophoblast model) was exposed to increasing concentrations of Mn and Pb. Exposure to 1 nM of Pb reduced SERT expression by 64% in BeWo cells. SERT activity in BeWo cells exposed to Mn (1000 nM) and Pb (0.01 nM) was decreased by 30% and 52%, respectively, and was accompanied by a decrease in serotonin cell level. Conclusion: This study shows that low concentrations of Mn and Pb affect placental serotonin system, which could disturb the proper course of pregnancy and fetal development.

Alterations in epithelial sodium channels (ENaC α, β, γ, δ) and its regulator pendrin in response to sodium chloride in BeWo and JEG3 cell lines

Alterations in epithelial sodium channels (ENaC α, β, γ, δ) and its regulator pendrin in response to sodium chloride in BeWo and JEG3 cell lines

Regulation of hypoxia-inducible factor-1α, regulated in development and DNA damage response-1 and mammalian target of rapamycin in human placental BeWo cells under hypoxia

IntroductionHypoxia-inducible factor-1 (HIF-1), regulated in development and DNA damage response-1 (REDD1) and mammalian target of rapamycin (mTOR) are crucial mediators of many metabolic processes in various cell types under hypoxia. The involvement and regulation of these three factors underlying trophoblasts' response to hypoxia remains to be determined. MethodsSpecific siRNAs were applied to inhibit the expression of the corresponding genes and to investigate the roles of HIF-1α in modulating REDD1/mTOR and REDD1 in regulating mTOR/HIF-1α in the human choriocarcinoma cell line BeWo under normoxia and hypoxia. ResultsExposure of BeWo cells to 1% oxygen (compared with 21% oxygen) led to a remarkable increase of both HIF-1α and REDD1 and an obvious decrease of mTOR at both the mRNA and protein levels. Interference of HIF-1α expression by siRNA resulted in an apparent reduction of REDD1 parallel with that of HIF-1α during normoxia and hypoxia. Additionally, the hypoxia-induced REDD1 increase was blocked through loss of HIF-1α, and the downregulation of mTOR in hypoxia could be partly obstructed by HIF-1α-siRNA transfection. Separately, the modulation effect of REDD1 was confirmed in an experiment demonstrating that the hypoxia-induced decrease of mTOR was inhibited by REDD1 knockdown, which was measured by changes in both the mRNA and protein levels. The disruption of REDD1 expression also led to increased accumulation of HIF-1α under both normoxia and hypoxia. DiscussionThe HIF-1α-REDD1-mTOR pathway was involved in the response to hypoxia in BeWo cells. Hypoxia-induced REDD1 upregulation is mediated by a HIF-1α-dependent pathway. Disruption of REDD1 blocked the effects of hypoxia on suppressing mTOR and resulted in additional accumulation of HIF-1α in BeWo cells.

25 Low dose aspirin corrects in vitro the placental cell derived sFlt-1/PlGF imbalance characteristic of preeclampsia

Introduction Preeclampsia (PE) is a multi-system disorder typically involving pregnancy-induced maternal hypertension and proteinuria presenting in the second half of pregnancy. The placenta is the key organ responsible for the pathogenesis of PE, with altered release of placenta-derived anti- (eg sFlt-1) and pro- (eg PlGF) angiogenic proteins implicated in maternal PE pathophysiological events. Epidemiological data indicate that low-dose aspirin (LDA) commenced before 16 weeks gestation is clinically effective in preventing PE, but its mechanism of action is unclear. We hypothesized that the prophylactic efficacy of LDA treatment may be associated with a corrective effect of LDA on the altered release of placenta-derived angiogenic proteins seen in PE pregnancies. Objective To test this hypothesis, we examined the effects of pregnancy sera from normotensive and PE cases with and without LDA on the in vitro production and release of sFlt-1 and PlGF from a trophoblast-derived cell line (BeWo). Methods The effects of LDA (5 μg/mL) following treatment of BeWo cells in vitro with either PE or normotensive serum on the expression of angiogenic proteins sFlt-1 and PlGF were determined using real-time PCR. The secretion of these angiogenic proteins in the conditioned media was screened with a commercially designed protein array. Candidate protein validation was then performed using an ELISA. All experiments were performed in triplicate at least six independent occasions. A p-value Results Angiogenic profiling at the mRNA level identified significantly increased expression of Flt-1 [Normotensive: 1.04 ± 0.03 vs PE: 2.68 ± 0.07, p Conclusion Increased placental release of the anti-angiogenic protein sFlt-1 and reduced placental release of the pro-angiogenic protein PlGF into the maternal circulation are key contributors to the maternal vascular endothelial cell dysfunction that is central to the development of the maternal syndrome of PE. The ability of LDA to rebalance these key angiogenic proteins in an in vitro trophoblast cell-line model may offer an important mechanistic insight at a molecular level regarding the clinical utility of LDA in preventing PE.

Effect of bisphenol A on biological behavior of human trophoblastic BeWo cell line

目的 研究环境内分泌干扰物双酚A(bisphenol A，BPA)对人滋养细胞株BeWo增殖、凋亡、侵袭能力的影响，探讨BPA对不良妊娠结局的可能作用机制。 方法 不同浓度BPA处理24 h后，流式细胞术及细胞增殖实验检测BeWo细胞凋亡及增殖的改变。分离培养原代子宫内膜细胞，建立子宫内膜间质细胞与BeWo细胞体外共培养侵袭实验模型，Transwell法检测BPA对BeWo侵袭能力的影响。采取单因素方差分析及LSD法进行统计学分析。 结果 （1）1 000 μmol/L BPA引起BeWo细胞活力降低，凋亡率为（89.0±1.4）%，较对照组的（2.8±0.7）%明显增加（P<0.05）。10 μmol/L、1.0 μmol/L BPA对BeWo细胞增殖具有促进作用，与对照组相比相对存活率分别为（124.3±10.3）%和（115.7±7.7）%（P值均<0.05）。（2）在共培养模型中，BeWo细胞暴露于BPA后，侵袭力下降，呈浓度依赖性，侵袭指数在100 μmol/L组为0.50±0.02，1.0 μmol/L组为0.84±0.04，0.01 μmol/L组为0.96±0.01（P值均<0.05）。 结论 BPA能够抑制人胎盘滋养细胞株BeWo细胞的侵袭能力，影响细胞增殖能力，可能导致不良妊娠结局。

GATA3 inhibits GCM1 activity and trophoblast cell invasion.

Development of human placenta involves the invasion of trophoblast cells from anchoring villi into the maternal decidua. Placental transcription factor GCM1 regulates trophoblast cell invasion via transcriptional activation of HtrA4 gene, which encodes a serine protease enzyme. The GATA3 transcription factor regulates trophoblast cell differentiation and is highly expressed in invasive murine trophoblast giant cells. The regulation of trophoblastic invasion by GCM1 may involve novel cellular factors. Here we show that GATA3 interacts with GCM1 and inhibits its activity to suppress trophoblastic invasion. Immunohistochemistry demonstrates that GATA3 and GCM1 are coexpressed in villous cytotrophoblast cells, syncytiotrophoblast layer, and extravillous trophoblast cells of human placenta. Interestingly, GATA3 interacts with GCM1, but not the GCM2 homologue, through the DNA-binding domain and first transcriptional activation domain in GCM1 and the transcriptional activation domains and zinc finger 1 domain in GATA3. While GATA3 did not affect DNA-binding activity of GCM1, it suppressed transcriptional activity of GCM1 and therefore HtrA4 promoter activity. Correspondingly, GATA3 knockdown elevated HtrA4 expression in BeWo and JEG-3 trophoblast cell lines and enhanced the invasion activities of both lines. This study uncovered a new GATA3 function in placenta as a negative regulator of GCM1 activity and trophoblastic invasion.

Macrophage Exosomes Induce Placental Inflammatory Cytokines: A Novel Mode of Maternal-Placental Messaging.

During pregnancy, the placenta forms the interface between mother and fetus. Highly controlled regulation of trans-placental trafficking is therefore essential for the healthy development of the growing fetus. Extracellular vesicle-mediated transfer of protein and nucleic acids from the human placenta into the maternal circulation is well documented; the possibility that this trafficking is bi-directional has not yet been explored but could affect placental function and impact on the fetus.We hypothesized that the ability of the placenta to respond to maternal inflammatory signals is mediated by the interaction of maternal immune cell exosomes with placental trophoblast. Utilizing the BeWo cell line and whole placental explants, we demonstrated that the human placenta internalizes macrophage-derived exosomes in a time- and dose-dependent manner. This uptake was via clathrin-dependent endocytosis. Furthermore, macrophage exosomes induced release of proinflammatory cytokines by the placenta. Taken together, our data demonstrates that exosomes are actively transported into the human placenta and that exosomes from activated immune cells modulate placental cytokine production. This represents a novel mechanism by which immune cells can signal to the placental unit, potentially facilitating responses to maternal inflammation and infection, and thereby preventing harm to the fetus.

150 IN VITRO MODEL FOR EFFECT OF ENDOCRINE-DISRUPTING CHEMICALS ON PLACENTAL CATION TRANSPORTER CHANNEL

Calcium, copper, iron, oxygen, and carbon dioxide are essential factors in fetal growth. These molecules are transferred by specific receptors located on the cell membrane or cytoplasm in placenta. Calcium, copper, and iron transfer genes are regulated by oestrogen, placental lactogen, and vitamin D. During pregnancy, expression of these receptors is controlled by the nutritional status of the maternal and fetal environment. Some synthetic plastics contain endocrine-disrupting chemicals (EDC), which have similar structures to steroid hormones or endogenous hormones related to reproduction. These substances disturb action of hormones (e.g. increasing oestrogen or progesterone) by interacting with their receptors or affecting the expression of transporting genes for cations. We used a BeWo cell line (human trophoblast cell line) to test the effect of EDC during pregnancy. The cells were cultured in phenol red-free DMEM supplemented with 5% charcoal dextran-stripped fetal bovine serum for 48 h to ensure the depletion of steroid hormones in the cells. Ethinyl oestradiol (EE), which activates oestrogen receptors, was used as a positive control. Then, EE (10–9, 10–8, and 10–7 M), octylpehnol (OP; 10–7, 10–6, and 10–5 M), nonylphenol (NP; 10–8, 10–7, and 10–6 M), and bisphenol A (BPA; 10–7, 10–6, and 10–5 M) were treated in BeWo cells for 48 h, and the cells were harvested. The mRNA and protein levels for calcium transporting genes (PMCA1 and TRPV6), copper transporting genes (CTR1 and ATP7A), and iron transporting genes (IREG1 and HEPH) were quantified by RT-qPCR, and Western blotting, respectively. Experiments were carried 3 times, and results were statistically analysed by GraphPad Prism6 (GraphPad Software, San Diego, CA, USA). We observed dose-dependent decreases in mRNA levels of PMCA1, TRPV6, ATP7A, and IREG1 compared with control group in OP-, NP-, or BPA-treated groups. Protein levels showed a similar pattern to mRNA levels. Based on our data, we confirmed that these EDC affect metal ion channels such as calcium, copper, and iron transporters during pregnancy.

Effect of folic acid on human trophoblast health and function in vitro

IntroductionThe combined intake of folic acid (FA) from prenatal multivitamin supplements and fortified foods can result in FA intake values that exceed the tolerable upper intake level (UL). It is unclear what impact FA intake above the UL may have on the feto-placental unit. Our objective was to determine the effects of increasing concentrations of FA on trophoblast health and function in vitro. MethodsTwo human placental cell lines [HTR-8/SVneo (n = 5 experiments) and BeWo (n = 5 experiments)] and human placenta tissue explants (n = 6 experiments) were exposed to increasing concentrations of FA (2–2000 ng/mL) for 48-h. Intracellular total folate concentration, trophoblast proliferation, viability, apoptosis, placenta cell invasion and β-hCG hormone release were assessed. ResultsExposure to increasing FA concentrations resulted in higher intracellular total folate in placental cell lines and tissue explants (p < 0.05); yet, only minimal effects of excess folic acid were observed on the primary indicators of placental health and function studied. Specifically, treatment with excess folic acid (2000 ng/mL) resulted in reduced cellular viability in the villous trophoblast BeWo cell line and increased rates of proliferation in the HT8-8/SVneo extravillous trophoblast cell line (p < 0.05). Further, deficient concentrations of folic acid (2 ng/mL) resulted in decreased cell viability and invasive capabilities of the HTR-8/SVneo extravillous trophoblast cell line (p < 0.05). DiscussionOur results demonstrate that placental health and function may be compromised in conditions of folate deficiency, and not necessarily in conditions of excess FA. This finding supports the recommendation of prenatal folic acid supplementation in the North American population. Further work aimed at clarifying the therapeutic window of FA intake in the obstetrical population is warranted.

The expression of thyroid hormone receptors (THR) is regulated by the progesterone receptor system in first trimester placental tissue and in BeWo cells in vitro

BackgroundThyroid hormones are essential for the maintenance of pregnancy and a deficiency in maternal thyroid hormones has been associated with early pregnancy losses. The aim of this study was a systematic investigation of the influence of mifepristone (RU 486) on the expression of the thyroid hormone receptor (THR) isoforms THRα1, THRα2, THRβ1 and THRβ2 on protein and mRNA-level. MethodsSamples of placental tissue were obtained from patients with mifepristone induced termination of pregnancy (n=13) or mechanical induced termination of normal pregnancy (n=20), each from the 4th to 13th week of pregnancy. Expression of THRα1, THRα2, THRβ1 and THRβ2 was analysed on protein level by immunohistochemistry and on mRNA level by real time RT-PCR (TaqMan). The influence of progesterone on THR gene expression was analysed in the trophoblast tumour cell line BeWo by real time RT-PCR (TaqMan). ResultsNuclear expression of THRα1, THRα2 and THRβ1 is downregulated on protein level in mifepristone (RU 486) treated villous trophoblast tissue. In decidual tissue, we found a significant downregulation only for THRα1 in mifepristone treated tissue. On mRNA level, we also found a significantly reduced expression of THRA but no significant downregulation for THRB in placental tissue. The gene THRA encodes the isoform THRα and the gene THRB encodes the isoform THRβ. The majority of cells expressing the thyroid hormone receptors in the decidua are decidual stromal cells. In addition, in vitro experiments with trophoblast tumour cells showed that progesterone significantly induced THRA but not THRB expression. ConclusionsTermination of pregnancy with mifepristone (RU 486) leads to a downregulation of THRα1, THRα2 and THRβ1 in villous trophoblasts and in addition to a decreased expression of THRA in placental tissue. Decreased expression of THRα1 induced by RU486 could also be found in the decidua. Therefore inhibition of the progesterone receptor may be responsible for this downregulation. This assumption is supported by the finding, that stimulation of the progesterone receptor by progesterone itself up-regulated THRA in trophoblast cells in vitro.

Dexamethasone Downregulates SLC7A5 Expression and Promotes Cell Cycle Arrest, Autophagy and Apoptosis in BeWo Cells.

Synthetic glucocorticoids (GCs) such as dexamethasone (Dex) are widely given to pregnant women to induce maturation and improve viability of preterm infants. Despite the beneficial effects, synthetic GCs have adverse effects on placental growth and nutrient transport system. However, the molecular mechanisms involved in these events remain unknown. Here we use a human placental choriocarcinoma cell line (BeWo) as model to explore the pathway linking amino acids transport with cell viability under Dex challenge. BeWo cells treated with Dex (100 nM) for 24 h demonstrated G1/S cell cycle arrest together with enhanced autophagy and apoptosis. Concurrently, the amino acid carrier SLC7A5 was down-regulated in association with impaired cellular amino acids uptake and inhibition of mammalian target of rapamycin (mTOR) signaling. Similar cellular responses were observed in BeWo cells treated with BCH, a classical System L inhibitor which inactivates SLC7A5. The glucocorticoid receptor (GR) antagonist RU486 was able to diminish Dex-induced translocation of GR into nucleus and to abolish these effects. Furthermore, Dex treatment significantly promoted the binding of GR to the proximal promoter sequence of SLC7A5 gene. Taken together, our results show that Dex downregulates SLC7A5 expression via GR-mediated transrepression. The impaired amino acids uptake leads to inhibition of mTOR signaling which in turn causes inhibited proliferation and enhanced autophagy and apoptosis in BeWo cells. These findings indicate that SLC7A5 mediates the effect of Dex on cell viability, thus providing a novel molecular target for the prevention and treatment of Dex-induced cell cycle arrest and apoptosis in placental cells.

Toll-like receptor profiling of seven trophoblast cell lines warrants caution for translation to primary trophoblasts

IntroductionExcessive placental inflammation is associated with pregnancy complications. Toll-like receptors (TLRs) are sensors for danger signals from infections and damaged tissue and initiate inflammation. Trophoblasts in the placenta broadly express TLRs. Trophoblast cell lines are used as surrogates for primary trophoblasts for in vitro studies, but the inflammatory translatability of trophoblast cell lines warrants examination. We aimed to assess TLR1-10 gene expression and activation in seven trophoblast cell lines and compare this to primary trophoblasts. MethodsThe five choriocarcinoma trophoblast cell lines BeWo, JAR, JEG-3, AC1M-32 and ACH-3P, and the two SV40 transfected trophoblast cell lines HTR-8/SVneo and SGHPL-5 were included and compared to primary first trimester trophoblasts (n = 6). TLR1-10 gene expression was analyzed by RT-qPCR. Cells were stimulated by specific TLR1-9 ligands for 24 h and cytokine release was measured by a 10-plex immunoassay. ResultsAll choriocarcinoma cell lines demonstrated broad TLR gene expression, but lacked functional cytokine response to TLR ligand activation. In contrast, SV40 transfected cell lines showed restricted TLR gene expression, but SGHPL-5 cells displayed significantly increased levels of interleukin (IL)-6, IL-8, IL-12 and vascular endothelial growth factor A after TLR3 and/or TLR4 activation (P < 0.01), while TLR2 activation increased IL-6 and IL-8 levels (P < 0.05). HTR8/SVneo cells responded to TLR3 activation by increased IL-6 and interferon (IFN)-γ (P < 0.05). The SGHPL-5 TLR profile most closely resembled primary trophoblast. DiscussionThe characterized trophoblast cell line TLR profiles serve as a reference and warrant caution when selecting trophoblast cell lines as in vitro models for immune responses in primary trophoblasts.