ISAC-3-6 TNFα suppresses the constitutive expression of protease-activated receptor(PAR)-2 in placenta-derived immortalized human trophoblast cell lines (TCL-1 and HTR-8/SVneo)(Group 3 Perinatology,IS Award Candidate,International Session)
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L-type amino acid transporter 1 (LAT1) is a neutral amino acid transporter expressed in trophoblast giant cells onembryonic day 8 in mice. LAT1 is responsible for metabolism in blastocysts and cancer cells. Despite research concerning the aberrant high expression and indispensable function of LAT1 in various cancers, little is known about the role of LAT1 in regulating the behaviors of human trophoblast cells under different physiological and pathological conditions. The HTR8-SVneo human trophoblast cell line and JEG-3 and JAR choriocarcinoma cell lines are used as models for trophoblast cell biological research. The proliferation and apoptosis of these cells were assayed using the CCK-8 assay and flow cytometry, respectively. Transwell-chambers were used to observed migration and invasion of the cells. Immunofluorescent staining, western blot, and RT-PCR assays were used to determine the possible mechanism of LAT1 on human trophoblast cell behaviors with small interfering RNA or signal agonists and antagonist treatments. LAT1 was expressed in the trophoblast and choriocarcinoma cells. LAT1 was involved in regulating behaviors of these cells, such as cell proliferation, apoptosis, migration, and invasion. Detailed results suggested that LAT1 modulated trophoblast cell functions by mediation of mTORC1 signaling pathways. Our results implicate LAT1 as a very important regulator in human trophoblast cell behaviors at the maternal-fetal interface.
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In the present study five mouse trophoblastic cell lines were used, SM9-1, SM9-2 and SM10 from outbred (Swiss) mice, and HLA-B1 and HLA-B3 from inbred transgenic (HLA-B27) mice placentas generated in the laboratory of Dr. Joan S Hunt (K U Medical Center, Kansas City, KS). All of the cell lines demonstrated a basic set of characteristics that are strongly associated with and probably exclusive to trophoblast cells. Successful propagation of normal trophoblast cell lines with distinct phenotypes cultured in vitro provides an excellent model for the study of mechanisms regulating trophoblast invasion very similar to invasion by tumor cells. Application of these cells to in vitro invasion assay has uncovered some of the molecular mechanisms responsible for trophoblast invasiveness and its control. Using the in vitro invasion assay SM9-2 and SM-10 were identified as the most invasive and least invasive cell lines, respectively. By using RT-PCR we have shown that all lines express TNF-alpha mRNA and this level is high in HLA-B derived cell lines. Other experiments has revealed that only Swiss mice derived cell lines (SM9-1, SM9-2 and SM-10) express the TGF-beta 1 mRNA and among these lines SM9-2 has the highest level. In TGF-beta 1 activity assay, secreted conditioned medium of these cell lines further showed that SM9-2 line has the highest inhibitory activity on Mv-1-Lu cell line. Exogenous TGF-beta 1 down-regulates invasion as well as mRNA level in SM9-2 trophoblasts. However, neutralizing TGF-beta 1 antibody in this cell line up-regulates invasion minimally. Late gestational trophoblast cells show a major reduction of invasive ability which is an autocrine type negative regulation of trophoblast invasion, and is possibly mediated by TGF-beta 1 production by the trophoblasts. Thus trophoblast TGF-beta 1 could be implicated in the invasion of these cells and this invasive phenotype is retained with complete fidelity during their further propagation in culture.
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Human trophoblast invasion and differentiation are essential for a successful pregnancy outcome. Dysregulation of these processes can lead to placental pathologies such as pre-eclampsia. The molecular mechanisms; however, are poorly understood. Interleukin (IL)11--a cytokine that regulates endometrial epithelial cell adhesion, trophoblast motility and invasion during implantation--may be involved in some of these processes.The effect of IL11 on protein expression was investigated in trophoblastic HTR8/SVneo cells and primary extravillous trophoblasts (EVTs) purified from first- trimester placentas. Two-dimension (2D)-differential in-gel electrophoresis analyses revealed that 731 spots were significantly differentially regulated by IL11 in HTR8/SVneo cells: seven spots were analyzed by liquid chromatography-tandem mass spectrometry and 14 unique proteins identified. Protein disulfide isomerase family A, member 3 (PDIA3; endoplasmic reticulum p57) and glucose-regulated protein 78 (GRP78) were further validated to be regulated by IL11 in HTR8/SVneo and primary EVT. One dimension western blot analysis confirmed that PDIA3 was down-regulated in EVT. 2D western blot analysis revealed that GRP78 was post-translationally modified following IL11 treatment. Moreover, IL11 stimulated the secretion of GRP78 in EVT.Data suggest that IL11, possibly via signal transducers and activators of transcription 3 signaling pathway, regulates PDIA3 protein expression and modification/secretion of GRP78. This is the first study to identify PDIA3 and GRP78 as IL11 targets in invasive trophoblasts and identifies a possible mechanism by which IL11 regulates trophoblast function.
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Objective
To explore the influence of heparanase (HPSE) on the expression of cytokines and predict its underlying molecular mechanism in trophoblast cells.
Methods
The human first-trimester extravillous trophoblast cell line HTR8/SVneo cells were selected as research subjects of this study. And a HPSE over-expression stably transfected HTR8/SVneo cell line and a HPSE normal-expression stably transfected HTR8/SVneo cell line were constructed. They were enrolled into test group and control group, respectively. And then 343 cytokines were semi-quantitatively detected using human cytokine antibody array kit and analyzed by Image J software to discover differential cytokine proteins in trophoblast cell lines of two groups. Western blotting was performed to validate the semi-quantitative results of cytokine antibody array by detecting AXL receptor tyrosine kinase, which had the most significant difference between trophoblast cell lines of two groups. The protein-protein interaction network was constructed through STRING protein online database, and the gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed by The Database for Annotation, Visualization and Integrated Discovery (DAVID) online bioinformatics resources.
Results
①The semi-quantitative results of cytokine microarray showed that levels of 15 cytokines, including AXL, intercellular adhesion molecule (ICAM)1, TEK receptor tyrosine kinase, urokinase-type plasminogen activator receptor (PLAUR), tissue inhibitor of metalloproteinase (TIMP)2, matrix metalloprotein (MMP)9, lymphocyte-activation gene (LAG)3, tumor necrosis factor receptor superfamily (TNFRSF)1B, TNFRSF1A, cytotoxic and regulatory T cell molecule (CRTAM), tissue inhibitor of metalloproteinase (TIMP)4, human platelet reactive protein (THBS)1, interleukin 17 receptor B (IL17RB), junctional adhesion molecule-like protein (AMICA), and C-C chemotactic factor 16 (CCL16) were decreased in HPSE-overexpressed trophoblast cell line of test group, compared with the control trophoblast cell line of control group. And the ratio of levels of differential cytokines in trophoblast cell line between test group and control group was less than 0.666 7.②The result of Western blotting showed that the level of AXL in HPSE-overexpressed trophoblast cell line of test group was obviously lower than that in trophoblast cell line of control group, and the difference was statistically significant (t=-6.931, P=0.020), which was consistent with the semi-quantitative results of cytokines microarray. ③HPSE was associated with MMP9 by vascular endothelial growth factor (VEGF)A, tumor protein p53 (TP53) and CD44 by STRING protein online database. And MMP9 was directly or indirectly associated with other 12 types of differential cytokines except LAG3, CCL16 and IL17RB to establish an interaction network. ④GO enrichment analysis of cytokines showed that the differential cytokines were mainly involved in 41 biological processes, including negative regulation of apoptosis and extracellular matrix (ECM) disassembly. Results of KEGG signaling pathway analysis showed that the differential cytokines were mainly involved in 6 signaling pathways, including proteoglycan in cancer signaling pathway and tumor necrosis factor signaling pathway.
Conclusions
Overexpression of HPSE may regulate the expression of cytokines in trophoblast cells. And it may affect the biological functions of trophoblast cells through the proteoglycan in cancer signaling pathway, which MMP9 is involved in. This study can provide a research direction and basis for further study of the influences of HPSE in trophoblast cells associated diseases.
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Heparanase; Cytokines; Signaling pathway; Protein interaction; Trophoblast cell
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Abstract Trophoblast giant cells of the rat placenta express cytochrome P450 17α-hydroxylase (P450c17) and synthesize androgens. The purpose of this study was to investigate androgen production and expression of P450c17 in the Rcho-1 trophoblast cell line. These cells are capable of differentiating along the trophoblast giant cell lineage. Androstenedione production increased approximately 70-fold as Rcho-1 trophoblast cells progressed from the proliferation to the differentiation state. P450c17 enzyme activity and mRNA also showed significant increases associated with trophoblast giant cell differentiation. To study the transcriptional regulation of the P450c17 gene, the activities of a series of P450c17 promoter–luciferase reporter constructs were evaluated following transient transfection into Rcho-1 trophoblast cells. A DNA region located – 98 bp upstream of the P450c17 gene transcriptional start site was the shortest promoter DNA construct consistently possessing activity in Rcho-1 trophoblast cells. Activities of longer constructs (−156 to −1560 bp) in this population of cells were significantly greater than the −98 bp promoter–reporter construct. The − 476 bp P450c17 construct showed maximal promoter activity in transiently transfected Rcho-1 trophoblast cells and was developmentally activated in stably transfected Rcho-1 trophoblast cells. Activation of the cyclic AMP/protein kinase A pathway did not significantly affect P450c17 promoter activity in Rcho-1 trophoblast cells, in contrast to its effects in mouse MA-10 Leydig cells. In summary, Rcho-1 trophoblast cells are capable of endocrine differentiation and are a useful in vitro system for studying the regulation of trophoblast androgen production and P450c17 gene expression. Journal of Endocrinology (1996) 150, 161–168
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Placentation requires the production of numerous growth factors, hormones and transcription factors. Many of them, like the adipose tissue‑derived leptin or adiponectin, have been identified in the placenta and their role has been established in the proliferation and subsequent development of the placenta. Apelin is another adipokine known for proliferative effects in different cell types. PCR, immunoblotting and immunocytochemistry were used to study mRNA and protein expression of apelin and its receptor (APJ) in syncytiotrophoblast (BeWo) and cytotrophoblast (JEG‑3) cells as well in immunohistochemistry in human normal placenta slides. The effect of apelin on cell proliferation study was investigated by alamarBlue® and Cell Counting Kit‑8 assays, the cell cycle by the flow cytometry method and the protein expression of cyclins and phosphorylation level of extracellular signal‑regulated kinases (ERK)1/2, phosphatidylinositol 3'‑kinase/protein kinase B (Akt), signal transducer and activator of transcription 3 (Stat3) and 5'‑monophosphate‑activated protein kinase (AMPKα) were studied by western blotting. Apelin was increased in JEG‑3 compared with in BeWo cells, while APJ was the same in both placenta cell lines. Immunocytochemical analyses revealed high cytoplasmic and/or membrane apelin localisation in JEG‑3, while BeWo cells exhibited markedly weaker apelin signal in the cytoplasm. Apelin increased cell proliferation as well as the percentage of cells in the G2/M phase of the cell cycle, cyclin proteins and the expression of all kinases mentioned above. In conclusion, apelin by promotion of trophoblast cell proliferation by APJ and ERK1/2, Stat3 and AMPKα signalling could be a new important adipokine in the regulation of early placental development.
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Human blastocysts that fail to implant following IVF secrete elevated levels of miR-661, which is taken up by primary human endometrial epithelial cells (HEECs) and impairs their adhesive capability. MicroRNA miR-661 downregulates mouse double minute homologue 2 (MDM2) and MDM4 in other epithelial cell types to activate p53; however, this has not been examined in the endometrium. In this study MDM2 protein was detected in the luminal epithelium of the endometrium, the site of blastocyst attachment, during the mid secretory receptive phase of the menstrual cycle. The effects of miR-661 on gene expression in and adhesion of endometrial cells was also examined. MiR-661 overexpression consistently downregulated MDM2 but not MDM4 or p53 gene expression in the Ishikawa endometrial epithelial cell line and primary HEEC. Adhesion assays were performed on the real-time monitoring xCELLigence system and by co-culture using Ishikawa cells and HEECs with HTR8/SVneo trophoblast spheroids. Targeted siRNA-mediated knockdown of MDM2 in endometrial epithelial cells reduced Ishikawa cell adhesion (P<0.001) and also reduced HTR8/SVneo trophoblast spheroid adhesion to Ishikawa cells (P<0.05) and HEECs (P<0.05). MDM2 overexpression using recombinant protein treatment resulted in enhanced HTR8/SVneo trophoblast spheroid adhesion to Ishikawa cells (P<0.01) and HEECs (P<0.05). This study highlights a potential new mechanism by which human blastocyst-secreted miR-661 reduces endometrial epithelial cell adhesion; via downregulation of MDM2. These findings suggest that MDM2 contributes to endometrial-blastocyst adhesion, implantation and infertility in women.
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Trophoblast giant cells are one of the primary endocrine cell types of the rodent placenta. Placental lactogen-I (PL-I) is the initial prolactin (PRL) family member expressed as trophoblast giant cells differentiate. In this report, we use the Rcho-1 trophoblast cell line as a model for studying the regulation of PL-I gene expression during trophoblast giant cell differentiation. Evidence is provided for trophoblast cell expression of epidermal growth factor receptor (EGFR), ErbB2, fibroblast growth factor receptor 1 (FGFR1), transforming growth factor-alpha, and heparin-binding EGF. EGF and FGF-2 stimulated PL-I mRNA and protein accumulation and PL-I promoter activity in a concentration-dependent manner. These latter growth factor actions on PL-I promoter activities were specifically inhibited by cotransfection with dominant negative constructs for EGFR and FGFRs respectively. Utilization of the mitogen-activated protein kinase (MAPK) pathway by EGF and FGF-2 in trophoblast cells was demonstrated by growth factor stimulation of a Gal4 DNA binding/Elk1 transactivational domain fusion construct, and more specifically by activation of extracellular signal regulated kinase and p38 MAPK. PL-I gene activation was also sensitive to disruption of MAPK and activation protein-1 (AP-1) signaling pathways. In conclusion, autocrine/paracrine pathways involving EGFR and FGFR1, MAPK and AP-1 are shown to participate in the regulation of the PL-I gene in differentiating trophoblast cells.
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