Wnt5a inhibited human trophoblast cell line HTR8/SVneo invasion: implications for early placentation and preeclampsia

Objective: Wnt5a and Wnt signaling play potential roles in human placental and fetal development. The objective of this study is to explore the role of Wnt5a in the invasion of the human trophoblast cell line HTR8/SVneo and the probable mechanism of early placentation and preeclampsia in which Wnt5a is involved.

Methods: Human first trimester villous tissues from normal pregnancies and third trimester placentas from pregnancies with or without preeclampsia (PE) were used in the detection of the expression and subcellular location of Wnt5a. The human trophoblast cell line HTR8/SVneo was treated with 0–400?ng/ml recombinant Wnt5a to investigate the role of Wnt5a in human trophoblast invasion.

Results: Human first trimester villous is accompanied by the decreased expression of Wnt5a compared with term placenta. Upregulated Wnt5a was detected in PE placenta compared with the normal control. Wnt5a inhibited the migration and invasion of HTR8/SVneo cells with decreased integrin β1, α5 and N-cadherin. Moreover, Wnt5a downregulated β-catenin in HTR8/SVneo cells.

Conclusions: These findings strongly suggest that Wnt5a inhibits the invasion of HTR8/SVneo cells. Decreased Wnt5a facilitates early placentation, whereas increased Wnt5a contributes to the pathogenesis of PE with insufficient trophoblast invasion. Aberrant Wnt5a may function by impairing Wnt non-canonical/β-catenin signaling pathway in trophoblasts.     

Expression of DAB2IP in human trophoblast and its role in trophoblast invasion

Objective: DAB2IP is a growth inhibitor present in many types of cancer cells and is associated with epigenetic regulations controlling tumor development. The primary objective of this study is to determine whether DAB2IP participates in the invasion and migration of trophoblasts during placental development.

Methods: The expressions of DAB2IP in human placentas (10 villi, 18 term placentas and 20 pre-eclampsia placentas) were determined by immunohistochemistry, Western blotting and quantitative RT-PCR. HTR8/SVneo cells were treated with hypoxia–reoxygenation (H/R) to test how DAB2IP expression would affect the invasion and migration of trophoblasts. JEG-3 andHTR8/SVneo cells were treated with 5-aza-2-deoxycytidine (5-aza-dC) to study the role of DAB2IP promoter methylation in trophoblasts.

Results: DAB2IP was strongly expressed in human villi and extravillous trophoblasts as well as in HTR8/SVneo cells, but not in pre-eclampsia placentas. DAB2IP expression increased after H/R treatment, but the invasive and migratory abilities of trophoblasts were reduced. DAB2IP expression in JEG-3 cells also increased after treatment with 5-aza-dC.

Conclusions: These findings strongly suggest that DAB2IP is an important negative regulator at the maternal–fetal interface during early pregnancy. Excessive oxidative stress can increase DAB2IP expression in trophoblasts. The mechanism of DNA methylation may involve in its function during the development of pathologic pregnancy.     

CXCR2 is decreased in preeclamptic placentas and promotes human trophoblast invasion through the Akt signaling pathway

IntroductionCXCR2, the receptor of the CXC chemokines, plays a critical role in cell migration and invasion in many types of cancer. It is unclear what impact CXCR2 may have on Preeclampsia (PE), a pregnancy-specific disease, which is related to insufficient trophoblast invasion. The aim of this study was to investigate the expression pattern of CXCR2 in the placentas of healthy and PE pregnancies, and to investigate the molecular mechanism of CXCR2 involvement in the development of PE.MethodsCXCR2 expression levels in newly delivered placentas from 38 pregnant women with PE and 21 healthy pregnant women were detected using quantitative real-time PCR, immunohistochemistry and Western blot assays. The effect of CXCR2 on trophoblast invasion and the underlying mechanisms were examined in two trophoblast cell lines (HTR-8/SVneo and TEV-1 cells).ResultsCXCR2 mRNA and protein expression levels were significantly decreased in preeclamptic placentas than normal control. The invasive abilities of the two trophoblast cell lines were significantly inhibited when CXCR2 was silenced, but that CXCR2 overexpression promoted trophoblast cells invasion. In addition, silencing CXCR2 reduced the expression of matrix metalloproteinase 2 and 9 (MMP2 and MMP9) and phosphorylated Akt (p-Akt). Furthermore, an Akt inhibitor suppressed the expression of MMP-2 and MMP-9.DiscussionOur results suggest that the decreased CXCR2 may contribute to the development of preeclampsia through impairing trophoblast invasion by down-regulating MMP-2 and MMP-9 via the Akt signaling pathway.     

Suppression of FPR2 expression inhibits inflammation in preeclampsia by improving the biological functions of trophoblast via NF-κB pathway

PurposeThe dysfunction of trophoblast during inflammation plays an important role in PE. Formyl peptide receptor 2 (FPR2) plays crucial roles in the development of inflammation-associated disease. This present study aimed to explore the effect of FPR2 on a trophoblast cellular model of preeclampsia.MethodsThe expression of FPR2 in placenta was detected by immunohistochemical staining and western blotting. Transfection of siRNA was used to knockdown FPR2 in HTR-8/SVneo cells. Inflammatory cytokines were detected by ELISA. CCK8, Transwell, wound healing, FACS and tube formation assays were performed to observe the abilities of cell proliferation, migration, invasion, apoptosis and angiogenesis. Western blotting was implemented to clarify that NF-κB signaling pathway was downstream of FPR2.ResultsThe expression levels of FPR2 were higher in placental tissues of patients with PE. Knockdown of FPR2 expression by siFPR2 or inhibition of its activity by WRW4 decreased the release of proinflammatory cytokines in HTR8/SVneo cells treated with LPS. Knockdown of FPR2 expression or inhibition of its activity further reversed the LPS-induced attenuation of the proliferation, migration, invasion and angiogenesis and increase in apoptosis in HTR8/SVneo cells. Moreover, the NF-κB signaling pathway was activated in both placental tissues of patients with PE and LPS-treated HTR8/SVneo cells. However, the activation was attenuated when FPR2 was knocked down or inhibited.ConclusionSuppression of FPR2 expression alleviated the effects of inflammation induced by LPS on trophoblasts via the NF-κB signaling pathway, which provided a novel and potential strategy for the treatment of PE.     

Endometrial extracellular vesicles of recurrent implantation failure patients inhibit the proliferation,migration, and invasion of HTR8/SVneo cells

Purpose

Endometrial extracellular vesicles are essential in regulating trophoblasts’ function. This study aims to investigate whether endometrial extracellular vesicles (EVs) from recurrent implantation failure (RIF) patients inhibit the proliferation, invasion, and migration of HTR8/SVneo cells.

Methods

Eighteen RIF patients and thirteen fertile women were recruited for endometria collection. Endometrial cells isolated from the endometria were cultured and modulated by hormones, and the conditioned medium was used for EV isolation. EVs secreted by the endometrial cells of RIF patients (RIF-EVs) or fertile women (FER-EVs) were determined by Western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Fluorescence-labeled EVs were used to visualize internalization by HTR8/SVneo cells. RIF-EVs and FER-EVs were co-cultured with HTR8/SVneo cells. Cell Counting Kit-8, transwell invasion, and wound closure assays were performed to determine cellular proliferation, invasion, and migration, respectively, in different treatments.

Results

RIF-EVs and FER-EVs were bilayer membrane vesicles, ranging from 100 to 150 nm in size, that expressed the classic EV markers Alix and CD9. RIF-EVs and FER-EVs were internalized by HTR8/SVneo cells within 2 h. The proliferation rate in the FER-EV group was significantly higher than that in the RIF-EV group at 20 μg/mL. Moreover, the invasion and migration capacity of trophoblast cells were decreased in the RIF-EV group relative to the FER-EV group at 20 μg/mL.

Conclusion

Endometrial EVs from RIF patients inhibited the functions of trophoblasts by decreasing their proliferation, migration, and invasive capacity. Such dysregulations induced by RIF-EVs may provide novel insights for better understanding the pathogenesis of implantation failure.

     

Upregulation of VEGF by small activating RNA and its implications in preeclampsia

IntroductionPreeclampsia is a severe pregnancy complication mostly due to inadequate vascular dilation and remodeling of spiral arteries. VEGF, the major factor for angiogenesis, is necessary for modulating angiogenic processes in the placenta. Hence reduction of VEGF in gestational hypertension may also lead to hypoperfusion and subsequent hypoxia of the fetus in hypertensive pregnancy.MethodsThis study aimed at elucidating the mechanism of action of VEGF in preeclampsia. Small activating RNAs (saRNA) were used to upregulate VEGF expression in human trophoblast cells (HTR-8/SVneo). The VEGF expression level was analyzed by real-time quantitative PCR and western blot, while its transfection efficiency was measured by flow cytometer assay. Cell migration was analyzed by a wound scratch assay. NO secretion was detected by determining NO metabolites. eNOS expression was analyzed by western blot. Tube formation function of cells was then analyzed by matrigel migration assay.ResultsVEGF expression significantly increased after saRNA transfection (all p ＜ 0.05). NO secretion and eNOS expression significantly increased by saRNA in HTR-8/SVneo cells (p = 0.0003 and 0.032 respectively). The migration ability and tube formation function of HTR-8/SVneo cells were enhanced by saRNA (p = 0.024 and 0.013 respectively). TNF-α inhibited VEGF-downstream eNOS-NO pathway activity as well as cell migration and tubulogenesis, while enforcing the expression of VEGF attenuated all the insults induced by TNF-α.ConclusionsUtilizing an RNA activation strategy to increase endogenous VEGF expression could be an emerging and effective approach for the treatment of preeclampsia.     

Expression of Gadd45α in human early placenta and its role in trophoblast invasion

Objectives

Well-controlled trophoblast migration and invasion at the maternal–foetal interface are crucial events for normal placentation and successful pregnancy. Growing evidence has revealed that growth arrest and DNA damage-inducible 45 alpha (Gadd45α) participates in tumour migration and invasion as a tumour suppressor. However, the expression and function of Gadd45α in trophoblasts is unknown. This study aimed to determine the Gadd45α expression and function in the human first trimester placenta and identify the underlying mechanisms.

Methods

The expression of Gadd45α in human first trimester placenta was determined using immunohistochemistry. HTR8/SVneo cell line was used to investigate the effects of Gadd45α on proliferation, apoptosis, migration/invasion, matrix metalloproteinase (MMP)2/9 activities, and tissue inhibitor of metalloproteinase (TIMP)1/2 expression using cell proliferation assays, flow cytometric analysis, transwell migration/invasion assays, gelatin gel zymography, and western blotting, respectively. Moreover, a placental villous explant model was employed to verify its functions in placentation.

Results

Gadd45α was strongly expressed in syncytiotrophoblasts and trophoblast columns of human placental villi, extravillous trophoblast cells and glandular epithelium within the maternal decidua. Gadd45α knockdown significantly promoted migration and invasion of HTR8/SVneo cells, whereas it did not affect cell proliferation or apoptosis. Silencing Gadd45α also enhanced trophoblast outgrowth and migration in placental explants. These effects were related to increased activities of MMP2/9 and the decreased expression of TIMP1/2.

Discussion and conclusion

Gadd45α may be involved in human trophoblast migration and invasion and may function as an important negative regulator at the foetal–maternal interface during early pregnancy by directly or indirectly regulating MMP2/9 activities.     

CircSTAM inhibits migration and invasion of trophoblast cells by regulating miR-148a-5p/PTEN axis

BackgroundThe mechanisms underlying the pathogenesis of preeclampsia (PE) remains unclear. Exploring the molecular players in PE progression can provide insights into targeted therapy.MethodsThe expression levels of circSTAM in placental chorionic tissues of PE patients and normal pregnant women were compared by RT-qPCR. CircSTAM was knocked down by small interfering RNA to investigate its role in migration, invasion and epithelial-mesenchymal transformation (EMT) of trophoblast HTR-8/SVneo cells. The downstream target of circSTAM was predicted using online bioinformatics resources, and their molecular interaction was examined by luciferase reporter assay.ResultsCircSTAM was upregulated in PE placenta tissues in comparison to normal placental tissues. CircSTAM knockdown significantly enhanced cellular invasion, migration, as well as EMT. Mir-148a-5p was identified as a target of circSTAM to regulate cell migration and invasion. Mir-148a-5p negatively regulated PTEN expression in trophoblast HTR-8 /SVneo cells.ConclusionIn summary, circSTAM upregulation in PE trophoblasts promoted the invasion, migration and EMT. CircSTAM may modulate trophoblast phenotype by impinging on mir-148a-5p/PTEN axis. These data provided novel insights into the pathogenesis of PE.     

Downregulated N-acetylglucosaminyltransferase III is involved in attenuating trophoblast migration and invasion under hypoxia–reoxygenation condition

Objective: Many studies have confirmed that N-acetylglucosaminyltransferase III (GnT-III) is correlated with tumor invasion and metastasis. However, the expression of GnT-III and its role in normal pregnancy and preeclampsia (PE) has not been reached. So the primary objective of this study is to determine GnT-III expression in normal pregnancy and whether its expression is vulnerable to oxidative stress in the trophoblast cells.

Methods: Human first trimester villous tissues from normal pregnancies and third trimester placentas from pregnancies with or without preeclampsia (PE) were used for the detection of GnT-III expression. Human first trimester extravillous trophoblast cell line (HTR8/SVneo) exposed to hypoxia/reoxygenation (H/R) condition was employed as an oxidative stress model in vitro to investigate the expression of GnT-III.

Results: GnT-III was strongly expressed in cytotrophoblast (CTBs), syncytiotrophoblast (STBs) and the trophoblast columns (TCs) of human placental villi, and decidual cells in the maternal decidua. The expression of GnT-III was decreased in PE placentas compared with the normal control placentas. In addition, GnT-III was found to have decreased expression in H/R-exposed HTR8/SVneo cells, and the invasive and migratory abilities of HTR8/SVneo cells were attenuated, too.

Conclusions: These findings suggest that GnT-III is an important regulator at the maternal-fetal interface during early pregnancy. Excessive oxidative stress can decrease GnT-III expression in trophoblast and the decreased expression of GnT-III may be involved in the development of preeclampsia.     

The proprotein convertase furin in human trophoblast: Possible role in promoting trophoblast cell migration and invasion

Furin, a proprotein convertase (PC), is ubiquitously expressed and implicated in many physiological and pathological processes. This study is aimed to identify the role of furin in human trophoblast invasion and migration. Furin was found to be highly expressed in placental villi of both rhesus monkeys and human beings during early pregnancy. Specifically, furin was found in trophoblast column and trophoblast shell, regions where highly invasive cytotrophoblast cells invade the maternal decidua during human placentation. To determine whether furin plays any role in trophoblast invasion and migration, we employed human extravillous HTR8/SVneo cells in Matrigel invasion and transwell migration assays. Knocking-down furin expression by siRNA significantly inhibited invasion and migration of HTR8/SVneo cells (P < 0.01), with corresponding decrease of matrix metalloproteinase-9 (MMP-9) activities. In contrast, over-expression of furin markedly increased cell invasion and migration (P < 0.01), accompanied by significant increase of MMP-9 activities. Furthermore, furin siRNA significantly increased the levels of both tissue inhibitors of MMPs (TIMP)-1 and -2. Our results suggest that furin may play an important role in the invasion and migration of human trophoblast cells during early pregnancy.     

Oxidative stress-induced Gadd45α inhibits trophoblast invasion and increases sFlt1/sEng secretions via p38 MAPK involving in the pathology of pre-eclampsia

Background: Pre-eclampsia (PE) is one of the most common pregnancy-related complications. We have previously reported that growth arrest and DNA damage-inducible 45 alpha (Gadd45α) is over-expressed in trophoblasts in pre-eclamptic placentas, with an excessive activation of p38 mitogen-activated protein kinase (MAPK) and increased levels of soluble Fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng) in maternal sera. Now we further investigate how Gadd45α regulates trophoblast functions and anti-angiogenesis factors secretions during placental development in patients with PE.

Methods: Human placental villous explants were used to verify the effects of Gadd45α and p38 MAPK in placentation. Then HRT8/SVneo cells exposed to hypoxia/reoxygenation (H/R) were employed as an oxidative stress model to investigate the effects of Gadd45α on invasion and sFlt-1/sEng secretions. Through silencing Gadd45α with lentiviral vector-based short-hairpin RNA and inhibiting p38 MAPK with SB203580, we demonstrated that Gadd45α and its downstream p38 protein played roles in the pathology of pre-eclampsia.

Results: Gadd45α was found to have increased expression in H/R-treated villous explants and HTR8/SVneo cells. Gadd45α knockdown or p38 blockage could promote trophoblast outgrowth and migration in H/R-exposed villous explants, and enhance the potentials of trophoblast migration/invasion and network formation in H/R-exposed HTR8/SVneo cells. These functional changes might be related to the increased activities of MMP2/9. Meanwhile, Gadd45α knockdown or p38 inhibition also decreases sFlt-1/sEng secretions via suppressing oxidative stress.

Conclusions: Oxidative stress-induced overexpression of Gadd45α might influence the activity of MMPs through activation of p38 MAPK signaling to affect the invasion of trophoblast cells, and increase the secretions of sFlt-1/sEng, which then participate in the pathogenesis of pre-eclampsia.     

miR-15b-AGO2 play a critical role in HTR8/SVneo invasion and in a model of angiogenesis defects related to inflammation

IntroductionmicroRNAs (miRs) have been shown to play critical roles in the regulation of trophoblast and endothelial cell functions, and one significant finding concerning the miR-15/16 family is that most members of this family are highly expressed in endothelial cells and contribute to functions, such as tube formation. The interaction between trophoblast and endothelial cell play an important role in normal placentation process. Therefore, the aims of this study were to investigate the expression of miR-15b in human placenta and to uncover the potential role of miR-15b as well as its target functional loop in trophoblast and endothelial cells. Whether inflammation could modulate the expression of miR-15b and its down-stream target was further investigated. Additionally, the potential link between miR-15b deregulation and preeclampsia was also explored in the placenta of patients diagnosed with preeclampsia.MethodsThe expression of miR-15b was studied in the placental tissue of a normal pregnancy using in situ hybridization, and the effects of miR-15b on proliferation, invasion, and angiogenesis were further explored in vitro using HTR-8/SVneo and HUVEC cell line models. A Lipopolysaccharides (LPS) treatment model in HTR-8/SVneo cell was utilized to explore the mechanism of how LPS treatment could lead to the activation of miR-15b expression. Western blot was used to detect the expression of proteins related to miR-15b mediated pathway in preeclamptic placentas.ResultsmiR-15b inhibits trophoblast cell invasion and endothelial cell tube formation by suppressing the expression of Argonaute 2 (AGO2), a major miRNA effecter protein. AGO2 is specifically localized to human placenta cytotrophoblast and endothelial cells, and it plays important roles in trophoblast cell invasion and endothelial cell tube formation. LPS treatment may lead to the overexpression of miR-15b and down-regulation of AGO2, which may be involved in shallow trophoblast cell invasion associated with the pathogenesis of preeclampsia. Chromatin immunoprecipitation assay indicates that increased occupancy of AGO2 to miR-15b promoter is responsible for the increased expression of miR-15b under the condition of LPS treatment. Furthermore, preeclamptic placentas have decreased expression of AGO2, but increased expression of miR-15b and TLR-4 compared to normal controls.DiscussionThis is the first report about the function of AGO2 in human trophoblast and endothelial cells in the placenta. The data indicates that the aberrant expression of miR-15b contributes to abnormal placentation by targeting AGO2 mRNA. This study provides insight into the potential role of the miR-15b and AGO2 functional loop in the placentation process.     

The chrondroitin sulfate proteoglycan (CSPG4) regulates human trophoblast function

Introduction

Trophoblast growth and invasion of the uterine endometrium are critical events during placentation and are tightly regulated by locally produced factors. Abnormal placentation can result in early miscarriage or preeclampsia and intrauterine growth restriction, leading to impaired fetal and/or maternal health. Chondroitin sulfate proteoglycan 4 (CSPG4) is involved in cancer cell migration and invasion, processes which are critical during placentation but unlike in cancer, trophoblast invasion is highly regulated. CSPG4 expression and function in trophoblast is unknown. We determined CSPG4 expression in human first trimester placenta and implantation sites, and investigated whether CSPG4 influenced proliferation, migration and invasion of a human extravillous trophoblast (EVT) cell line (HTR8/SVneo cells) as a model for extravillous trophoblast (EVT).

Methods and results

Immunoreactive CSPG4 localized to EVT cells in the trophoblast shell, subpopulations of interstitial EVT cells within the decidua and cytotrophoblast cells in placental villi. In HTR8/SVneo cells, siRNA knockdown of CSPG4 stimulated proliferation and decreased migration/invasion. In primary first trimester placental villi explants two cytokines, interleukin 11 (IL11) and leukemia inhibitory factor (LIF) with known roles in trophoblast function, stimulated CSPG4 mRNA expression and immunoreactive protein in the cyotrophoblast.

Discussion and conclusion

This is the first demonstration of the production and function of CSPG4 in human placentation. These data suggest that locally produced CSPG4 stimulates human EVT migration and invasion and suggests that IL11 and LIF regulate villous cytotrophoblast differentiation towards the invasive phenotype at least in part via CSPG4.     

Down-regulated long non-coding RNA-ATB in preeclampsia and its effect on suppressing migration,proliferation, and tube formation of trophoblast cells

Preeclampsia is a pregnancy-specific syndrome and is one of the main causes of maternal, fetal, and neonatal morbidity and mortality. Inadequate trophoblast invasion and failure of uterine spiral artery remodeling exert a major role in the development of preeclampsia, especially the early-onset one. LncRNA-ATB is verified to be aberrantly expressed in many cancers and promote the invasion-metastasis and proliferation cascades. But little is known of lncRNA-ATB's role in preeclampsia. The aim of current study is to identify the changes of lncRNA-ATB in preeclampsia and its effects on trophoblast. The lncRNA-ATB levels were decreased in placental samples collected from preeclampsia women (n = 51) compared to those of healthy pregnant women (n = 40) by qRT-PCR analysis. Besides, it is demonstrated that lncRNA-ATB was intense stained in the trophoblast of the placenta by performing in-situ hybridization. By designing RNA interference species to suppress lncRNA-ATB and specific plasmids designed to overexpress lncRNA-ATB, we identify the role of lncRNA-ATB on the functions of trophoblast cell-line, HTR-8/SVneo. Inhibition of endogenous lncRNA-ATB decreased migration, proliferation, tube-formation of HTR-8/SVneo cells. In addition, overexpression of lncRNA-ATB promoted migration, proliferation, and tube-formation of HTR-8/SVneo cells. Therefore, lncRNA-ATB might be involved in the pathogenesis of preeclampsia by regulating the process of trophoblast invasion and endovascular formation.     

The role of SATB1 in HTR8/SVneo cells and pathological mechanism of preeclampsia

Objective: Special AT-rich sequence binding protein 1 (SATB1) play potential roles in invasion and metastasis of tumor cells, and involves in human placental and fetal development. The objective of this study is to explore the role of SATB1 in migration and invasion of trophoblast and the potential mechanism.

Methods: Human placental tissues from first trimester, second trimester, term, and preeclampsia (PE) pregnancies were used to detect the expression and subcellular location of SATB1 and β-catenin. The human trophoblast cell line HTR8/SVneo, which was treated with hypoxia/re-oxygenation (H/R), lithium chloride (LiCl) or SATB1-siRNA to investigate the role of SATB1 and β-catenin signaling in human trophoblast function.

Results: We observed that SATB1 specifically localized within trophoblast cells of placenta tissues. Gradually reduced expression of SATB1 was observed during gestation, and lower expression were detected in placenta of PE compared with normal pregnancy. Moreover, the expression of SATB1 was decreased in H/R-treated HTR8/Svneo cells and villous explants. The Wnt/β-catenin signaling pathway interacted with SATB1 expression and H/R treatment resulted in Wnt pathway inhibition in trophoblast, while lithium chloride (LiCl) treatment enhanced H/R-exposed HTR8/SVneo migration and invasion. Knockdown of SATB1 significantly reduced the level of β-catenin and the migratory and invasive abilities of trophoblast.

Conclusions: Our data suggested that oxidative stress reduced SATB1 leading to inhibition of Wnt/β-catenin, and participate in the subdued migration and invasion of trophoblast, which indicated a potential pathological mechanism of PE.     

Kisspeptin-10 inhibits cell migration in vitro via a receptor-GSK3 beta-FAK feedback loop in HTR8SVneo cells

Kisspeptin inhibits cancer cell metastasis and placental trophoblast cell migration. Kisspeptin gene expression in the placenta and circulating kisspeptin levels change during normal pregnancy and they are altered in preeclampsia. We therefore assessed the effect of kisspeptin-10 on the in vitro migration of a human placental cell line derived from first trimester extravillious trophoblasts (HTR8SVneo). HTR8SVneo cells specifically bound 125I-Kisspeptin-10 but kisspeptin-10 did not induce inositol phosphate production. Cell migration was inhibited by kisspeptin-10 with a maximal inhibition at 100nM. The signaling pathways involved in inhibition of cell migration were examined. Treatment with kisspeptin-10 elicited phosphorylation of GSK3 beta at Ser9 (which inhibits activity), with a 3-fold increase at 5 min. Transient phosphorylation of ERK1/2 and p38MAPK peaked at 10min. Phosphorylation of focal adhesion kinase (FAK) at Tyr925 increased 3-fold at 10 min. Inhibition of GSK3 beta correlated with release of beta-catenin into the cytoplasm. These signaling events were differentially blocked by inhibitors of G(q/11), Src, EGFR, PI(3)K, PKC and MEK. The data suggest that kisspeptin/GPR54 EGF-receptor transactivation leads to phosphorylation of ERK1/2, causing activation of p90rsk which in turn inhibits GSK3 beta via Ser9 phosphorylation. Inactivation of GSK3 beta results in release of beta-catenin into the cytoplasm, affecting cell-cell adhesion and Tyr925 phosphorylation of FAK, which increases phosphorylation of ERK1/2 via RAS/Raf-1 creating a feedback loop to enhance the effects on migration. These findings indicate that kisspeptin-10 inhibits the migration of human placental trophoblast-derived HTR8SVneo cells by stimulating complex ERK1/2-p90rsk-GSK3 beta-FAK feedback interactions.     

Interleukin-6 Stimulates Cell Migration,Invasion and Integrin Expression in HTR-8/SVneo Cell Line

Interleukin-6 (IL-6) is present in human endometrium throughout menstrual cycle and in pregnancy. Trophoblast also expresses IL-6. IL-6R and its associated signal transducer gp130 were found in trophoblast as well. IL-6 is generally assumed to be relevant for trophoblast invasion. This study was undertaken to determine influence of endogenous and externally added IL-6 on invasion and migration of first trimester of pregnancy trophoblast in vitro. Integrins α₅β₁ and α₁β₁ have been shown to play an important role in trophoblast invasion and the effect of IL-6 on the expression of these integrin subunits was studied. We are showing that in both isolated first trimester of pregnancy cytotrophoblast (CTB) and HTR-8/SVneo cell line IL-6 and IL-6R are present. The effect on migration was studied using cell wounding and migration test on HTR-8/SVneo cells. Effect of IL-6 and function blocking anti-IL-6 antibody in Matrigel invasion tests was studied on both cell types. The effect of IL-6 on integrin subunit expression was determined by cell-based ELISA and Western blot on HTR-8/SVneo cells. The results obtained show that exogenous IL-6 has stimulatory effect on cell migration in HTR-8/SVneo and invasion by both cell types. Function blocking anti-IL-6 inhibited unstimulated invasion by isolated first trimester cytotrophoblast and both cell migration and invasion in unstimulated HTR-8/SVneo. Integrin α₅ expression was stimulated by IL-6 to 134% (p < 0.05), α₁ to 135% (p < 0.005), and β₁ to 134% (p < 0.001) of control in cell-based ELISA, but also in Western blot. The data obtained show for the first time sensitivity of extravillous trophoblast cell line HTR-8/SVneo to IL-6, in addition to isolated first trimester cytotrophoblast. We conclude that both exogenous and endogenous IL-6 stimulate trophoblast cell migration and invasion, which may be partly attributable to stimulation of expression of the studied integrin subunits.     

Porphyromonas gingivalis induces IL-8 and IFN-gamma secretion and apoptosis in human extravillous trophoblast derived HTR8/SVneo cells via activation of ERK1/2 and p38 signaling pathways

IntroductionPreterm birth is a major cause for infant mortality and morbidity. A large number of studies have suggested a link between periodontal disease and preterm birth. The purpose of this study was to investigate the interaction between a periodontopathic bacterium Porphyromonas gingivalis and human extravillous trophoblast derived HTR8/SVneo cells.MethodsProduction of cytokines in HTR8 cells was measured via ELISA. Annexin V/PI flow cytometry was performed to assess apoptosis. Protein expression was measured by western blot. Specific pharmacological inhibitors were used to inactivate relevant signaling pathways (p38 MAPK, SB203580; ERK1/2, U0126; JNK, SP600125; NF-κB, JSH-23) to determine their roles in inflammation and apoptosis.ResultsHTR8 cells released significant amounts of IL-8 and IFN-γ during exposure to P. gingivalis. Meanwhile, the percentages of both early and late apoptotic cells increased significantly in response to P. gingivalis. The most significant effect on inflammation was found using SB203580 and U0126, followed by SP600125 and JSH-23. Moreover, U0126 and SB203580 both partially but significantly suppressed P. gingivalis-induced apoptosis, with a large effect by U0126. Additionally, both heat-killed P. gingivalis and P. gingivalis lipopolysaccharide significantly induced IL-8 production.ConclusionP. gingivalis induces inflammation and apoptosis in HTR8 cells, and we demonstrated for the first time that activation of ERK1/2 and p38 MAPK pathways participates in P. gingivalis-induced inflammation and apoptosis. The abnormal regulation of inflammation and apoptosis in human trophoblasts by P. gingivalis infection may give new insights into how maternal periodontal disease and periodontal pathogens might be linked to preterm birth.