Human Choriocarcinoma Research Articles

Activation of G protein-coupled estrogen receptor stimulates placental human chorionic gonadotropin expression through PKA-CREB signaling

The placenta-secreted human chorionic gonadotropin (hCG) is a hormone that plays a critical role in inducing ovarian progesterone production, which is required for maintaining normal pregnancy. The bioavailability of hCG depends on the expression of the beta-subunit of hCG (hCG-β) which is encoded by the chorionic gonadotropin beta (CGB) gene. G protein-coupled estrogen receptor (GPER) is a membrane estrogen receptor involved in non-genomic estrogen signaling. Estradiol (E2) has been shown to stimulate hCG production. However, the role of the GPER in regulating CGB expression remains unknown. In the present study, our results revealed that treatment with G1 upregulated CGB expression in two human choriocarcinoma cell lines, BeWo and JEG-3, and primary human cytotrophoblast cells. In addition, G1 treatment activated the cAMP-response element binding protein (CREB). Using a pharmacological inhibitor and siRNA-mediated knockdown approach, we showed that the stimulatory effect of G1 on CGB expression is mediated by the protein kinase A (PKA)-CREB signaling pathway. This study increases the understanding of the role of GPER in the human placenta. In addition, our results provide important insights into the molecular mechanisms that mediate hCG expression, which may lead to the development of alternative therapeutic approaches for treating placental diseases.

GDF-11 downregulates placental human chorionic gonadotropin expression by activating SMAD2/3 signaling

BackgroundThe production of human chorionic gonadotropin (hCG) by the placental trophoblast cells is essential for maintaining a normal pregnancy. Aberrant hCG levels are associated with reproductive disorders. The protein of hCG is a dimer consisting of an α subunit and a β subunit. The β subunit is encoded by the CGB gene and is unique to hCG. Growth differentiation factor-11 (GDF-11), a member of the transforming growth factor-β (TGF-β) superfamily, is expressed in the human placenta and can stimulate trophoblast cell invasion. However, whether the expression of CGB and the production of hCG are regulated by GDF-11 remains undetermined.MethodsTwo human choriocarcinoma cell lines, BeWo and JEG-3, and primary cultures of human cytotrophoblast (CTB) cells were used as experimental models. The effects of GDF-11 on CGB expression and hCG production, as well as the underlying mechanisms, were explored by a series of in vitro experiments.ResultsOur results show that treatment of GDF-11 downregulates the expression of CGB and the production of hCG in both BeWo and JEG-3 cells as well as in primary CTB cells. Using a pharmacological inhibitor and siRNA-mediated approach, we reveal that both ALK4 and ALK5 are required for the GDF-11-induced downregulation of CGB expression. In addition, treatment of GDF-11 activates SMAD2/3 but not SMAD1/5/8 signaling pathways. Moreover, both SMAD2 and SMAD3 are involved in the GDF-11-downregulated CGB expression. ELISA results show that the GDF-11-suppressed hCG production requires the ALK4/5-mediated activation of SMAD2/3 signaling pathways.ConclusionsThis study not only discovers the biological function of GDF-11 in the human placenta but also provides important insights into the regulation of the expression of hCG.EYYrMgGK1oNKot-fDMBUfiVideo

MALAT1 modulates trophoblast phenotype via miR-101–3p/VEGFA axis

Preeclampsia is a potentially life-threatening condition that can arise due to poor placentation and consequent abnormal uterine spiral artery remodeling. Abnormal placentation, in turn, is associated with aberrant trophoblast cell proliferation and apoptosis. Here, we investigated the lncRNA MALAT1 in trophoblast proliferation during early-onset preeclampsia (ePE). MALAT1 levels were examined in placental tissue samples from ePE patients and control patients. The effects and underlying mechanism of MALAT1 on proliferation, migration, invasion and apoptosis were investigated in the first-trimester extravillous trophoblast HTR-8/SVneo cells and the human choriocarcinoma JAR cells. MALAT1 levels were decreased in the placental tissue samples of ePE patients compared with those of control patients, and the levels of MALAT1 were positively correlated with the neonate birth-weight. Knockdown of MALAT1 attenuated the cell viability, proliferation, migration, invasion and the cell cycle progression of trophoblasts, but promoted the apoptosis of trophoblasts. The MALAT1 knockdown promoted miR-101–3p upregulation and VEGFA downregulation. Inhibitor of miR-101–3p increased vascular endothelial growth factor A (VEGFA) expression, and miR-101–3p mimic inhibited VEGFA expression. Luciferase assays showed that miR-101–3p could bind to both MALAT1 and VEGFA. The MALAT1 knockdown-induced induction in the cell vitality and proliferation were attenuated by miR-101–3p inhibitor. We conclude that endogenous MALAT1 promotes proliferation, migration and invasion of trophoblasts by inhibiting the miR-101–3p expression and the subsequent VEGFAupregulation. The reduced MALAT1 level in placental tissue may be involved in the pathogenesis of the ePE.

Rolipram suppresses migration and invasion of human choriocarcinoma cells by inhibiting phosphodiesterase 4-mediated epithelial-mesenchymal transition.

Rolipram is a selective phosphodiesterase-4 (PDE4) inhibitor. The effect of rolipram on the metastasis of choriocarcinoma is barely known. Here, we evaluated the role of rolipram in the migration and invasion of human choriocarcinoma cells in vitro.Human choriocarcinoma cells lines JEG3 and JAR were used in this study. The expression profile of PDE4 subfamily members in choriocarcinoma cells was evaluated using real-time PCR. The migration and invasion properties of choriocarcinoma cells before and after inhibition of PDE4 by rolipram or RNAi-directed knockdown were evaluated in vitro. Expression levels of MMP9, TIMP1, E-cadherin, vimentin, TGFβ1, SMAD1, and SMAD4 of choriocarcinoma cells were compared before and after rolipram treatment, RNAi-directed knockdown of PDE4D, and overexpression of PDE4D. We found PDE4D was the most commonly expressed isoform of PDE4 both in JEG3 and JAR cells. Rolipram and knockdown of PDE4D were efficient to inhibit the migration and invasion of choriocarcinoma cells in vitro, accompanied by decreased expression of MMP9 and TIMP1. Furthermore, rolipram and knockdown of PDE4D promoted the expression of E-cadherin but reduced the expression of vimentin in choriocarcinoma cells, and overexpression of PDE4D decreased the expression of E-cadherin but promoted the expression of vimentin. Rolipram suppressed migration and invasion of human choriocarcinoma cells in vitro, possibly by inhibiting epithelial-mesenchymal transition through PDE4 inhibition.

Stiff Extracellular Matrix Promotes Invasive Behaviors of Trophoblast Cells.

The effect of extracellular matrix (ECM) stiffness on embryonic trophoblast cells invasion during mammalian embryo implantation remains largely unknown. In this study, we investigated the effects of ECM stiffness on various aspects of human trophoblast cell behaviors during cell-ECM interactions. The mechanical microenvironment of the uterus was simulated by fabricating polyacrylamide (PA) hydrogels with different levels of stiffness. The human choriocarcinoma (JAR) cell lineage was used as the trophoblast model. We found that the spreading area of JAR cells, the formation of focal adhesions, and the polymerization of the F-actin cytoskeleton were all facilitated with increased ECM stiffness. Significantly, JAR cells also exhibited durotactic behavior on ECM with a gradient stiffness. Meanwhile, stiffness of the ECM affects the invasion of multicellular JAR spheroids. These results demonstrated that human trophoblast cells are mechanically sensitive, while the mechanical properties of the uterine microenvironment could play an important role in the implantation process.

Dl-THP recovered the decreased NKp44 expression level on CD56dim CD16+ natural killer cells partially in choriocarcinoma microenvironment

Natural killer cell-based immunotherapy has become a leading-edge tool against cancer, but still faces a variety of challenges, such as phenotype shift and dysfunction of NK cells in tumor microenvironment. Thus, finding potent agents that could inhibit the phenotype shift and incapacity of NK cells in the tumor microenvironment is essential for improving antitumor effects. dl-tetrahydropalmatine (dl-THP), one of the active alkaloids of Chinese herb Corydalis Rhizoma, has been proven to possess antitumor activity. However, whether dl-THP acts on NK cells to enhance antitumor activity remains unknown. In this study, we found that the proportion of blood CD56dimCD16+ NK cells was decreased while the proportion of CD56brightCD16- NK cells was increased when the cells were cultured in conditional medium (CM, medium from the human choriocarcinoma cell lines JEG-3). dl-THP could alter the varied proportion of CD56dimCD16+ NK cells and CD56brightCD16- NK cells in CM respectively. Importantly, the expression level of NKp44 on CD56dimCD16+ NK cells was dramatically reduced when the cells were cultured in CM, which could also be reversed by dl-THP. Furthermore, dl-THP increased the decreased NK-cell cytotoxicity when cells were cultured in CM. In summary, our study demonstrated that dl-THP could recover the decreased NKp44 expression level on CD56dimCD16+ NK cells and restore the cytotoxicity of NK cells in tumor microenvironment.

Curcuminoids inhibit human and rat placental 3β-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis

Ethnopharmacological relevanceIn traditional Chinese medicine, curcuma longa L has been applied to treat pain and tumour-related symptoms for over thousands of years. Curcuminoids, polyphenolic compounds, are the main pharmacological component from the rhizome of Curcuma longa L. Pharmacological investigations have found that curcuminoids have many pharmacological activities of anti-inflammatory, anti-tumour, and anti-metastasis. Aim of the study3β-Hydroxysteroid dehydrogenase (3β-HSD1) catalyses the production of steroid precursors for androgens and estrogens, which play an essential role in cancer metastasis. We explored the potency and mode of action of curcuminoids and their metabolites of inhibiting 3β-HSD1 activity and compared the species difference between human and rat. Materials and methodsIn this study, we investigated the direct inhibition of 6 curcuminoids on human placental 3β-HSD1 activity and compared the species-dependent difference in human 3β-HSD1 and rat placental homolog 3β-HSD4. Results: The inhibitory potency of curcuminoids on human 3β-HSD1 was demethoxycurcumin (IC50, 0.18 μM) > bisdemethoxycurcumin (0.21 μM)>curcumin (2.41 μM)> dihydrocurcumin (4.13 μM)>tetrahydrocurcumin (15.78 μM)>octahydrocurcumin (ineffective at 100 μM). The inhibitory potency of curcuminoids on rat 3β-HSD4 was bisdemethoxycurcumin (3.34 μM)>dihydrocurcumin (5.12 μM)>tetrahydrocurcumin (41.82 μM)>demethoxycurcumin (88.10 μM)>curcumin (137.06 μM)> octahydrocurcumin (ineffective at 100 μM). Human choriocarcinoma JAr cells with curcuminoid treatment showed that these chemicals had similar potency to inhibit progesterone secretion under basal and 8bromo-cAMP stimulated conditions. Docking analysis showed that all chemicals bind pregnenolone-binding site with mixed/competitive mode for 3β-HSD. ConclusionSome curcuminoids are potent human placental 3β-HSD1 inhibitors, possibly being potential drugs to treat prostate cancer and breast cancer.

Metformin regulates autophagy via LGMN to inhibit choriocarcinoma

Choriocarcinoma has the problem of chemotherapy insensitivity and recurrence. Metformin may be a promising candidate to restrict choriocarcinoma progress because of its indirect and direct beneficial role on inhabitations of cancer cells without severe adverse side effects. In this study, metformin pressed the proliferation and invasion of choriocarcinoma JAR cells in vitro and the growth of the JAR subcutaneous xenografts in vivo. The high throughput sequencing and bioinformatics technology identified the low expression of legumain (LGMN) in lysosomal pathway caused by metformin, which was upregulated in human choriocarcinoma tissues compared with the early pregnancy tissues. As elevating metformin concentration and treatment time, the mRNA and protein expression of LGMN both depressed in two choriocarcinoma cell lines (JAR and JEG-3). LGMN was involved in metformin-mediated inhibition of cell proliferation and invasion. Furthermore, metformin induced autophagy via inhibiting LGMN through AKT/mTOR/LC3II signaling pathway of choriocarcinoma. Autophagy inhibitor could depress metformin-induced autophagy and improve cell proliferation and invasion ability dropped by metformin, while autophagy inducer could partially reverse the change of cell proliferation and invasion modulated by combination of metformin and LGMN overexpression. These results indicated that metformin inhibited cell proliferation and invasion ability by inducing autophagy in a LGMN-dependent manner so as to play a role in the treatment of choriocarcinoma.

Chemicals of environmental concern as inhibitors of human placental 3β-hydroxysteroid dehydrogenase 1 and aromatase: Screening and docking analysis

Many environmental pollutants act as endocrine-disrupting compounds by inhibiting human placental 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase type 1 (HSD3B1) and aromatase (CYP19A1) activities. In this study, we screened 13 chemicals of environmental concern for their ability to inhibit human HSD3B1 and CYP19A1 by measuring the conversion of pregnenolone to progesterone for HSD3B1 activity and the conversion of testosterone to 17β-estradiol for CYP19A1 activity in human JEG-3 choriocarcinoma cell microsomes. HSD3B1 had an apparent Km of 0.323 μM and an apparent Vmax of 0.111 nmol/mg/min and CYP19A1 had an apparent Km of 56 nM and an apparent Vmax of 0.177 nmol/mg protein/min. 17β-Estradiol, bisphenol A, and bisphenol AF competitively inhibited HSD3B1 with Ki values of 0.8, 284.1, and 141.2 μM, respectively, while diethylstilbestrol had a mixed inhibition on human HSD3B1 with the Ki of 8.0 μM. Ketoconazole, bisphenol A, and bisphenol AF noncompetitively inhibited CYP19A1 with Ki values of 10.3, 54.4, and 45.7 μM, respectively, while diethylstilbestrol and zearalenone competitively suppressed CYP19A1 with Ki values of 63.0 and 16.6 μM, respectively. Docking analysis showed that 17β-estradiol, diethylstilbestrol, bisphenol A, and bisphenol AF bound the steroid binding pocket facing the catalytic residues Y155 and K159 of HSD3B1, and that ketoconazole, bisphenol A, and bisphenol AF bound heme binding pocket while diethylstilbestrol and zearalenone bound the steroid binding site of CYP19A1. In conclusion, 17β-estradiol, diethylstilbestrol, bisphenol A, and bisphenol AF are human HSD3B1 inhibitors, and ketoconazole, zearalenone, diethylstilbestrol, bisphenol A, and bisphenol AF are human CYP19A1 inhibitors.

The miR-182-5p/NDRG1 Axis Controls Endometrial Receptivity through the NF-κB/ZEB1/E-Cadherin Pathway.

Endometrial receptivity is essential for successful pregnancy, and its impairment is a major cause of embryo-implantation failure. MicroRNAs (miRNAs) that regulate epigenetic modifications have been associated with endometrial receptivity. However, the molecular mechanisms whereby miRNAs regulate endometrial receptivity remain unclear. Therefore, we investigated whether miR-182 and its potential targets influence trophoblast cell attachment. miR-182 was expressed at lower levels in the secretory phase than in the proliferative phase of endometrium tissues from fertile donors. However, miR-182 expression was upregulated during the secretory phase in infertile women. Transfecting a synthetic miR-182-5p mimic decreased spheroid attachment of human JAr choriocarcinoma cells and E-cadherin expression (which is important for endometrial receptivity). miR-182-5p also downregulated N-Myc downstream regulated 1 (NDRG1), which was studied further. NDRG1 was upregulated in the secretory phase of the endometrium tissues and induced E-cadherin expression through the nuclear factor-κΒ (NF-κΒ)/zinc finger E-box binding homeobox 1 (ZEB1) signaling pathway. NDRG1-overexpressing or -depleted cells showed altered attachment rates of JAr spheroids. Collectively, our findings indicate that miR-182-5p-mediated NDRG1 downregulation impaired embryo implantation by upregulating the NF-κΒ/ZEB1/E-cadherin pathway. Hence, miR-182-5p is a potential biomarker for negative selection in endometrial receptivity and a therapeutic target for successful embryo implantation.

Evaluation of the Estrogenic Action Potential of Royal Jelly by Genomic Signaling Pathway in Vitro and in Vivo.

Royal jelly (RJ) has beneficial effects on human health, and some of these effects are reported to be the result of its estrogenic activity; however, chemicals with estrogenic activities may disrupt physiological estrogen signaling leading to adverse effects on human health. Thus, clarification of the mode of action of RJ is needed. Here, we investigated whether the estrogen-like actions of RJ are induced via estrogen receptors (ERs)-mediated genomic actions by using an in vitro reporter assay in human choriocarcinoma JEG3 cells and an estrogen-responsive reporter (E-Rep) mouse line that can be used to sensitively detect transactivation of ERs in multiple organs simultaneously. In the in vitro reporter assay, ERs-dependent transcriptional activity was significantly increased by 17β-estradiol (E2) treatment at concentrations of 1 nM and above, confirming that the assay was highly responsive to estrogen; however, RJ did not exhibit any agonist activity via either the α or β form of ER. Similarly, in E-Rep mice, E2 showed significant ERs-dependent genomic action in 17 tissue types including uterus and mammary gland, whereas RJ did not. Thus, unlike endocrine-disrupting chemicals, the estrogen-like activity of RJ is unlikely to be due to genomic actions via ERs.

LRSAM1 E3 Ubiquitin Ligase Promotes Choriocarcinoma Progression and Metastasis via p53/p21 Signaling Impediment.

Objective The E3 ubiquitin ligase LRSAM1 (LRSAM1) was involved in many cancers, but whether it exerts anti- or protumor efficacies on choriocarcinoma cellular structures remains unknown. We wanted to explore the efficacies of aberrant LRSAM1 expression on human choriocarcinoma cellular structures and the underlying mechanisms. Methods LRSAM1 mRNA expressions in choriocarcinoma lines of cells JEG-3 and JAR cellular structures, as well as HTR8/sev8 human trophoblastic cell line cellular structures, were assessed using assay analysis of quantitative real-time polymerase chain reactions. We compared cell proliferation, migratory flow, invasive force, adhesion, and apoptotic process between cellular structures infected with si-LRSAM1 plasmids versus negative controls using CCK-8, clone formation, Transwell, adhesion, and flow cytometry assays. Protein expressions of LRSAM1, E-cadherin, and N-cadherin (indicators of epithelial-mesenchymal transformation) and p53/p21 pathway components were quantitated using a Western blot assay. The morphology of tumor lesions was observed in xenografted nude mice using immunohistochemistry (IHC) analyses. Results LRSAM1 was markedly overexpressed within JEG-3 and JAR choriocarcinoma cellular structures compared to HTR8/sev8 trophoblast cellular structures. Compared to si-NC, LRSAM1 knockdown robustly restricted cell proliferating, migratory flow, invasive force, and adhesion and fueled apoptotic cell process in JEG-3 as well as JAR cellular structures and suppressed tumor growth, as evidenced by the reduction in tumor volume and weight in naked mice inoculated with transfected cellular structures. Compared to si-negative control (si-NC), si-LRSAM1 significantly decreased Ki67 (a proliferating indicator) and N-cadherin expressions but reduced E-cadherin expression in JEG-3 and JAR cellular structures. Blocking the p53/p21 pathway by pifithrin-a (a p53 restrictor) successfully reversed the anti-inhibitory effect of LRSAM1 depletion, resulting in enhanced proliferating and metastasis in JEG-3 and JAR cellular structures. Conclusion LRSAM1 exerts tumorigenic roles in choriocarcinoma. Via the activating of the p53/p21 pathway of signaling and impediment of choriocarcinoma cell proliferating, migratory flow, and invasive force, LRSAM1 knockdown slows the course of the disease. For choriocarcinoma diagnosis and treatment, it serves as a new therapeutic target.

Inhibition of YAP/TAZ-TEAD activity induces cytotrophoblast differentiation into syncytiotrophoblast in human trophoblast.

During placentation, placental cytotrophoblast (CT) cells differentiate into syncytiotrophoblast (ST) cells and extravillous trophoblast (EVT) cells. In the placenta, the expression of various genes is regulated by the Hippo pathway through a transcription complex, Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ)-TEA domain transcription factor (TEAD) (YAP/TAZ-TEAD) activity. YAP/TAZ-TEAD activity is controlled by multiple factors and signaling, such as cAMP signaling. cAMP signaling is believed to be involved in the regulation of trophoblast function but is not yet fully understood. Here we showed that YAP/TAZ-TEAD expressions and their activities were altered by cAMP stimulation in BeWo cells, a human choriocarcinoma cell line. The repression of YAP/TAZ-TEAD activity induced the expression of ST-specific genes without cAMP stimulation, and transduction of constitutively active YAP, i.e. YAP-5SA, resulted in the repression of 8Br-cAMP-induced expressions of ST-specific genes in a TEAD-dependent manner. We also investigated the role of YAP/TAZ-TEAD in maintaining CT cells and their differentiation into ST and EVT cells using human trophoblast stem (TS) cells. YAP/TAZ-TEAD activity was involved in maintaining the stemness of TS cells. Induction or repression of YAP/TAZ-TEAD activity resulted in marked changes in the expression of ST-specific genes. Using primary CT cells, which spontaneously differentiate into ST-like cells, the effects of YAP-5SA transduction were investigated, and the expression of ST-specific genes was found to be repressed. These results indicate that the inhibition of YAP/TAZ-TEAD activity, with or without cAMP stimulation, is essential for the differentiation of CT cells into ST cells.

Calnexin Is Involved in Forskolin-Induced Syncytialization in Cytotrophoblast Model BeWo Cells.

Calnexin (CNX), a membrane-bound molecular chaperone, is involved in protein folding and quality control of nascent glycoproteins in the endoplasmic reticulum. We previously suggested critical roles of calreticulin, a functional paralogue of CNX, in placentation, including invasion of extravillous trophoblasts and syncytialization of cytotrophoblasts. However, the roles of CNX in placentation are unclear. In human choriocarcinoma BeWo cells, which serve as an experimental model of syncytialization, CNX knockdown suppressed forskolin-induced cell fusion and β-human chorionic gonadotropin (β-hCG) induction. Cell-surface luteinizing hormone/chorionic gonadotropin receptor, a β-hCG receptor, was significantly down-regulated in CNX-knockdown cells, which suggested the presence of a dysfunctional autocrine loop of β-hCG up-regulation. In this study, we also found abundant CNX expression in normal human placentas. Collectively, our results revealed the critical role of CNX in the syncytialization-related signaling in a villous trophoblast model and suggest a link between CNX expression and placenta development.

VEGF-B targeting by aryl hydrocarbon receptor mediates the migration and invasion of choriocarcinoma stem-like cells

Unlike other members of the VEGF family, the function of VEGF-B in tumor progression remains to be elucidated. Thus, the present study aimed to determine the function of VEGF-B in human choriocarcinoma cells by investigating its detailed effects and molecular mechanisms. VEGF-B and aryl hydrocarbon receptor (AhR) expression were evaluated by reverse transcription-quantitative PCR analysis and western blot analysis in JEG-3 cells and choriocarcinoma stem-like cells (CSLCs) and their proliferation, migration, and invasion after the transfection of short hairpin RNA VEGF-B, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; AhR agonist) treatment or StemRegenin 1 (SR1; AhR antagonist) treatment were examined by cell proliferation assay, wound healing assay and Transwell assay. In addition, luciferase reporter analysis and bioinformatics data mining were used to investigate the association between VEGF-B and AhR. Upregulation of VEGF-B and AhR expression was observed in CSLCs. Following VEGF-B knockdown or SR1 treatment, the proliferative, migratory, and invasive abilities of CSLCs were significantly decreased, contrary to the findings after TCDD treatment. It was also found that AhR enhanced VEGF-B transcriptional activity by binding to the relative promoter region. These observations indicated that VEGF-B may be an oncogene that promotes choriocarcinoma cell migration and invasion targeted by AhR. Therefore, targeting VEGF-B may provide a novel therapeutic opportunity for choriocarcinoma.

Inefficient development of syncytiotrophoblasts in the Atp11a-deficient mouse placenta

The P4-ATPases ATP11A and ATP11C function as flippases at the plasma membrane to translocate phosphatidylserine from the outer to the inner leaflet. We herein demonstrated that Atp11a-deficient mouse embryos died at approximately E14.5 with thin-walled heart ventricles. However, the cardiomyocyte- or epiblast-specific Atp11a deletion did not affect mouse development or mortality. ATP11C may have compensated for the function of ATP11A in most of the cell types in the embryo. On the other hand, Atp11a, but not Atp11c, was expressed in the mouse placenta, and the Atp11a-null mutation caused poor development of the labyrinthine layer with an increased number of TUNEL-positive foci. Immunohistochemistry and electron microscopy revealed a disorganized labyrinthine layer with unfused trophoblasts in the Atp11a-null placenta. Human placenta-derived choriocarcinoma BeWo cells expressed the ATP11A and ATP11C genes. A lack of ATP11A and ATP11C eliminated the ability of BeWo cells to flip phosphatidylserine and fuse when treated with forskolin. These results indicate that flippases at the plasma membrane play an important role in the formation of syncytiotrophoblasts in placental development.

Uptake of antiepileptic drugs in forskolin-induced differentiated BeWo cells: alteration of gabapentin transport

Previous studies have indicated that the expression levels of several transporters are altered during placental trophoblast differentiation. However, changes in the transport activities of therapeutic agents during differentiation must be comprehensively characterised. Antiepileptic drugs, including gabapentin (GBP), lamotrigine (LTG), topiramate, and levetiracetam, are increasingly prescribed during pregnancy. The objective of this study was to elucidate differences in the uptake of antiepileptic drugs during the differentiation process. Human placental choriocarcinoma BeWo cells were used as trophoblast models. For differentiation into syncytiotrophoblast-like cells, cells were treated with forskolin. The uptake of GBP and LTG was lower in differentiated BeWo cells than in undifferentiated cells. In particular, the maximum uptake rate of GBP transport was decreased in differentiated BeWo cells. Furthermore, GBP transport was trans-stimulated by the amino acids His and Met. We investigated the profiles of amino acids in undifferentiated and differentiated BeWo cells. Supplementation with His and Met, which demonstrated trans-stimulatory effects on GBP uptake, restored GBP uptake in differentiated cells. The findings of this study suggest that drug transport in BeWo cells can be altered before and after differentiation, and that the altered GBP uptake could be mediated by the intracellular amino acid status.

Placental genomics mediates genetic associations with complex health traits and disease

As the master regulator in utero, the placenta is core to the Developmental Origins of Health and Disease (DOHaD) hypothesis but is historically understudied. To identify placental gene-trait associations (GTAs) across the life course, we perform distal mediator-enriched transcriptome-wide association studies (TWAS) for 40 traits, integrating placental multi-omics from the Extremely Low Gestational Age Newborn Study. At P ; < ; 2.5times {10}^{-6}, we detect 248 GTAs, mostly for neonatal and metabolic traits, across 176 genes, enriched for cell growth and immunological pathways. In aggregate, genetic effects mediated by placental expression significantly explain 4 early-life traits but no later-in-life traits. 89 GTAs show significant mediation through distal genetic variants, identifying hypotheses for distal regulation of GTAs. Investigation of one hypothesis in human placenta-derived choriocarcinoma cells reveal that knockdown of mediator gene EPS15 upregulates predicted targets SPATA13 and FAM214A, both associated with waist-hip ratio in TWAS, and multiple genes involved in metabolic pathways. These results suggest profound health impacts of placental genomic regulation in developmental programming across the life course.

Bilirubin Reduces the Uptake of Estrogen Precursors and the Followed Synthesis of Estradiol in Human Placental Syncytiotrophoblasts via Inhibition and Downregulation of Organic Anion Transporter 4.

Estrogen biosynthesis in human placental trophoblasts requires the human organic anion transporter 4 (hOAT4)-mediated uptake of fetal derived precursors such as dehydroepiandrosterone-3-sulfate (DHEAS) and 16α-hydroxy-DHEA-S (16α-OH-DHEAS). Scant information is available concerning the contribution of fetal metabolites on the impact of placental estrogen precursor transport and the followed estrogen synthesis. This study substantiated the roles of bilirubin as well as bile acids (taurochenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid, chenodeoxycholic acid) on the inhibition of hOAT4-mediated uptake of probe substrate 6-carboxylfluorescein and DHEAS in stably transfected hOAT4-Chinese hamster ovary cells, with the IC50 of 1.53 and 0.98 μM on 6-carboxylfluorescein and DHEAS, respectively, for bilirubin, and 90.2, 129, 16.4, and 12.3 μM on 6-CF for taurochenodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, and chenodeoxycholic acid. Bilirubin (2.5-10 μM) concentration-dependently inhibited the accumulation of estradiol precursor DHEAS in human choriocarcinoma JEG-3 cells (reduced by 60% at 10 μM) and primary human trophoblast cells (reduced by 80% at 10 μM). Further study confirmed that bilirubin (0.625-2.5 μM) concentration-dependently reduced the synthesis and secretion of estradiol in primary human trophoblast cells, among which 2.5 μM of bilirubin reduced the synthesis of estradiol by 30% and secretion by 35%. In addition, immunostaining and Western blot results revealed a distinct downregulation of hOAT4 protein expression in primary human trophoblast cells pretreated with 2.5 μM of bilirubin. In conclusion, this study demonstrated that bilirubin reduced the uptake of estrogen precursors and the followed synthesis of estradiol in human placenta via inhibition and downregulation of organic anion transporter 4. SIGNIFICANCE STATEMENT: Fetal metabolites, especially bilirubin, were first identified with significant inhibitory effects on the hOAT4-mediated uptake of estrogen precursor DHEAS in hOAT4-CHO, JEG-3 and PHTCs. Bilirubin concentration-dependently suppressed the estradiol synthesis and secretion in PHTCs treated with DHEAS, which was synchronized with the decline of hOAT4 protein expression. Additionally, those identified bile acids exhibited a weaker inhibitory effect on the secretion of estradiol.

Characterization of Extracellular Vesicles Labelled with a Lipophilic Dye Using Fluorescence Nanoparticle Tracking Analysis.

Research on extracellular vesicles (EVs) has intensified over the past decade, including fluorescent membrane labeling of EVs. An optimal fluorescent method requires the size of EVs to be preserved after labeling. Lipophilic fluorescent dyes, such as CellMask™ Green (CMG), have been widely used for this purpose. Here, we investigated conditions affecting the optimum CMG labeling of EVs derived from human choriocarcinoma cells (JAr) and different biological fluids using fluorescence NTA (fl-NTA). The effect of CMG labeling on the size, concentration and zeta potential (ZP) on JAr EVs purified with different methods were measured along with biological fluid-derived EVs. With the increase of CMG dye concentration, a significant decrease in the mean size of fluorescent nanoparticles (fl-NPs) was observed. The ZP of fl-NPs originating from JAr cells with the lowest and highest dye concentrations showed a significant shift towards more and less negative ZP values, respectively. Differences in the concentration of fl-NPs were observed for JAr EVs purified using size-exclusion chromatography (SEC) alone and SEC in combination with tangential flow filtration. The proportion of CMG labeling of NPs varied across different biological sources. CMG labeling may be a reliable technique for the detection of EVs using fl-NTA.