CCN1 (CYR61) and CCN3 (NOV) signaling drives human trophoblast cells into senescence and stimulates migration properties

ABSTRACT

During placental development, continuous invasion of trophoblasts into the maternal compartment depends on the support of proliferating extravillous trophoblasts (EVTs). Unlike tumor cells, EVTs escape from the cell cycle before invasion into the decidua and spiral arteries. This study focused on the regulation properties of glycosylated and non-glycosylated matricellular CCN1 and CCN3, primarily for proliferation control in the benign SGHPL-5 trophoblast cell line, which originates from the first-trimester placenta. Treating SGHPL-5 trophoblast cells with the glycosylated forms of recombinant CCN1 and CCN3 decreased cell proliferation by bringing about G0/G1 cell cycle arrest, which was accompanied by the upregulation of activated Notch-1 and its target gene p21. Interestingly, both CCN proteins increased senescence-associated β-galactosidase activity and the expression of the senescence marker p16. The migration capability of SGHPL-5 cells was mostly enhanced in response to CCN1 and CCN3, by the activation of FAK and Akt kinase but not by the activation of ERK1/2. In summary, both CCN proteins play a key role in regulating trophoblast cell differentiation by inducing senescence and enhancing migration properties. Reduced levels of CCN1 and CCN3, as found in early-onset preeclampsia, could contribute to a shift from invasive to proliferative EVTs and may explain their shallow invasion properties in this disease.     

NOD1配体抑制人早孕期滋养细胞的侵袭功能

目的:明确固有免疫受体NOD1对人早孕期滋养细胞侵袭功能的调控及对侵袭相关因子分泌的影响。方法:采用免疫细胞化学法鉴定原代滋养细胞NOD1的表达,用transwell侵袭实验检测激活NOD1后滋养细胞侵袭功能的改变,ELISA检测配体刺激后滋养细胞MMP2和MMP9的分泌情况。结果:免疫细胞化学结果显示滋养细胞分离鉴定成功,且原代滋养细胞可以表达固有免疫受体NOD1。使用NOD1的特异性配体及非特异性配体,发现激活NOD1可以抑制滋养细胞的侵袭,且非特异性配体LPS可以下调侵袭相关金属基质蛋白酶分子MMP2和MMP9的分泌,特异性配体i E-DAP仅下调MMP9的分泌而对MMP2的分泌无影响。结论:固有免疫模式识别受体NOD1可以在早孕期滋养细胞表达,可调控滋养细胞的侵袭功能,其激活会导致侵袭相关分子MMP2和MMP9的分泌下降。     

粘附分子CD146是影响滋养层细胞侵入行为的关键分子

前期研究观察到一种现象, 在正常妊娠的胎盘中细胞粘附分子CD146选择性地表达在侵入性滋养层细胞中, 而在滋养层细胞侵入不足的先兆子痫病人的胎盘中CD146表达降低或缺失.本文进一步研究了CD146分子影响滋养层细胞侵入行为的作用机理.免疫荧光实验显示CD146分子选择性地表达在具有侵袭能力的中间滋养层细胞,而在非侵入性的细胞滋养层细胞和合体滋养层细胞中不表达.细胞功能实验表明,影响滋养层细胞侵入性的两个关键要素,即细胞迁移和基质金属蛋白酶的分泌,都受到CD146特异抗体的显著抑制.这些研究结果提示,粘附分子CD146是影响细胞侵入行为的关键分子.这为深入研究胚胎植入和肿瘤浸润的分子调控机理提供了一个关键的分子模型.     

Placental villous mesenchymal cells trigger trophoblast invasion

The successful transformation of uterine spiral arteries by invasion trophoblasts is critical for the formation of the human hemochorial placenta. Placental trophoblast migration and invasion are well regulated by various autocrine/paracrine factors at maternal–fetal interface. Human placental multipotent mesenchymal stromal cells (hPMSCs) are a subpopulation of villous mesenchymal cells and have been shown to produce a wide array of soluble cytokines and growth factors including HGF (hepatocyte growth factor). The function of hPMSCs in placental villous microenvironment has not been explored. The interaction between hPMSCs and trophoblasts was proposed in vitro in a recent article. HGF produced by hPMSCs was able to engage c-Met receptor on trophoblast and induced the trophoblast cAMP expression. The cAMP activated PKA, which in turn, signaled to Rap1 and led to integrin β1 activation. The total integrin β1 protein expression by trophoblasts was not affected by HGF stimulation. Hypoxia downregulated HGF expression by hPMSCs. HGF and PKA activator 6-Bnz-cAMP increased trophoblast adhesion and migration that were inhibited by PKA inhibitor H89 or Rap1 siRNA. Thus, hPMSCs-derived paracrine HGF can regulate trophoblast migration during placentation. These findings provided insight revealing at least one mechanism by which hPMSCs implicated in the development of preeclampsia.     

Placental villous mesenchymal cells trigger trophoblast invasion

The successful transformation of uterine spiral arteries by invasion trophoblasts is critical for the formation of the human hemochorial placenta. Placental trophoblast migration and invasion are well regulated by various autocrine/paracrine factors at maternal–fetal interface. Human placental multipotent mesenchymal stromal cells (hPMSCs) are a subpopulation of villous mesenchymal cells and have been shown to produce a wide array of soluble cytokines and growth factors including HGF (hepatocyte growth factor). The function of hPMSCs in placental villous microenvironment has not been explored. The interaction between hPMSCs and trophoblasts was proposed in vitro in a recent article. HGF produced by hPMSCs was able to engage c-Met receptor on trophoblast and induced the trophoblast cAMP expression. The cAMP activated PKA, which in turn, signaled to Rap1 and led to integrin β1 activation. The total integrin β1 protein expression by trophoblasts was not affected by HGF stimulation. Hypoxia downregulated HGF expression by hPMSCs. HGF and PKA activator 6-Bnz-cAMP increased trophoblast adhesion and migration that were inhibited by PKA inhibitor H89 or Rap1 siRNA. Thus, hPMSCs-derived paracrine HGF can regulate trophoblast migration during placentation. These findings provided insight revealing at least one mechanism by which hPMSCs implicated in the development of preeclampsia.     

Spatio-temporal expression of MMP-2, MMP-9 and tissue kallikrein in uteroplacental units of the pregnant guinea-pig (Cavia porcellus)

Background  

In humans trophoblast invasion and vascular remodeling are critical to determine the fate of pregnancy. Since guinea-pigs share with women an extensive migration of the trophoblasts through the decidua and uterine arteries, and a haemomonochorial placenta, this species was used to evaluate the spatio-temporal expression of three enzymes that have been associated to trophoblast invasion, MMP-2, MMP-9 and tissue kallikrein (K1).     

Regulation of trophoblast beta1-integrin expression by contact with endothelial cells

Background  

In human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade uterine matrix, and enter the uterine vasculature. Invasive trophoblasts show increased expression of β1 integrin. Since trophoblast migration within the uterine vasculature involves trophoblast attachment to endothelial cells lining the vessel walls, this raises the possibility that cell-cell contact and/or factors released by endothelial cells could regulate trophoblast integrin expression. To test this, we used an in vitro system consisting of early gestation macaque trophoblasts co-cultured on top of uterine microvascular endothelial cells.     

B-cell lymphoma 6 promotes proliferation and survival of trophoblastic cells

Preeclampsia is one of the leading causes of maternal and perinatal mortality and morbidity and its pathogenesis is not fully understood. B-cell lymphoma 6 (BCL6), a key regulator of B-lymphocyte development, is altered in preeclamptic placentas. We show here that BCL6 is present in all 3 studied trophoblast cell lines and it is predominantly expressed in trophoblastic HTR-8/SVneo cells derived from a 1^st trimester placenta, suggestive of its involvement in trophoblast expansion in the early stage of placental development. BCL6 is strongly stabilized upon stress stimulation. Inhibition of BCL6, by administrating either small interfering RNA or a specific small molecule inhibitor 79–6, reduces proliferation and induces apoptosis in trophoblastic cells. Intriguingly, depletion of BCL6 in HTR-8/SVneo cells results in a mitotic arrest associated with mitotic defects in centrosome integrity, indicative of its involvement in mitotic progression. Thus, like in haematopoietic cells and breast cancer cells, BCL6 promotes proliferation and facilitates survival of trophoblasts under stress situation. Further studies are required to decipher its molecular roles in differentiation, migration and the fusion process of trophoblasts. Whether increased BCL6 observed in preeclamptic placentas is one of the causes or the consequences of preeclampsia warrants further investigations in vivo and in vitro.     

The miR-27a-3p/USP25 axis participates in the pathogenesis of recurrent miscarriage by inhibiting trophoblast migration and invasion

Insufficient invasion ability of trophoblasts might be associated with the development of recurrent miscarriage (RM). Ubiquitin-specific protease 25 (USP25) can regulate the processes of invasion and migration in different types of cancer cells. However, the effect of USP25 on trophoblasts and its roles in the development of RM are unknown. In this study, we first analyzed the USP25 expression in placental villous tissues from RM patients, and then assessed the roles of USP25 in epithelial-to-mesenchymal transition (EMT), invasion and migration of trophoblasts. Furthermore, bioinformatics prediction and luciferase reporter assay were used to explore the mechanism of microRNA on USP25 expression, and regulation of USP25 expression in trophoblasts was assessed following transfection with microRNA mimics or inhibitor. The results showed that the expression of USP25 in the placental villous tissues was downregulated in RM patients. Knockdown of USP25 suppressed the EMT process, the invasion and migration capability of trophoblast cells, while overexpression of USP25 exhibited opposite results. Mechanistically, miR-27a-3p could regulate USP25 expression by binding to the 3′-untranslated region of USP25 in trophoblasts. Quantitative real-time polymerase chain reaction results found the expression of miR-27a-3p were negatively related to USP25 in RM patients. MiR-27a-3p mimics inhibited but miR-27a-3p inhibitor enhanced the migration and invasion capability of trophoblasts. Furthermore, sh-USP25 counteracted the promotion of invasion and migration mediated by the miR-27a-3p inhibitor. Taken together, these data indicate that USP25 downregulation by miR-27a-3p contributes to the EMT process, thereby inhibiting the migration and invasion of trophoblast cells, and these findings might provide potential biomarkers for RM.     

Effect arylamine N-acetyltransferase 1 on morphology,adhesion, migration,and invasion of MDA-MB-231 cells: role of matrix metalloproteinases and integrin αV

ABSTRACT

Reducted arylamine N-acetyltransferase (NAT1) in breast cancers is associated with poor patient survival. NAT1 has also been associated with changes in cancer cell survival and invasion both in vitro and in vivo. Here, we report the effects of NAT1 in cancer cell invasion by addressing its role in adherence, migration, and invasion in vitro. The NAT1 gene was deleted in MDA-MB-231, HT-29 and HeLa cells using CRISPR/Cas9 gene editing. Loss of NAT1 increased adherence to collagen in all three cell-lines but migration was unaffected. NAT1 deletion decreased invasion and induced changes to cell morphology. These effects were independent of matrix metalloproteinases but were related to integrin ITGαV expression. The data suggest NAT1 is important in adhesion and invasion through integrin expression.     

Normal nonmetastatic human trophoblast cells share in vitro invasive properties of malignant cells

First-trimester normal human trophoblast cells show some phenotypic similarities to malignant cells, e.g., rapid proliferation and ability to invade neighboring tissue, including basement membrane in situ, but do not have the ability for unlimited growth or metastasis. The present study examined whether the invasive ability of normal trophoblast cells is an intrinsic property of these cells, independent of the microenvironment provided by the pregnant uterus, and if so, whether they share some of the molecular mechanisms of invasion exercized by metastatic malignant cells. The ability of in vitro grown human trophoblast lines to invade an epithelium-free human amniotic membrane was measured from the temporal kinetics of retention of radioactivity within this membrane resulting from a penetration by 125I-iododeoxyuridine-labeled trophoblast cells. The magnitude of this invasion was compared to that of the highly metastatic human JAR-choriocarcinoma cell line and murine B16F10 melanoma line. Trophoblasts were found to share some of the same molecular mechanisms of invasion with the metastatic cell lines. Inhibitors of collagenase, plasmin, plasminogen, and plasminogen activators completely prevented invasion of the amnion by the trophoblast lines as well as by the metastatic JAR and B16F10 lines. Mersalyl, a compound known to activate collagenase, stimulated invasion by all cell lines tested, including under conditions in which plasmin activity was inhibited. In addition, trophoblasts produced significant levels of type IV collagenase and laminin, both of which appear to be important products of metastatic tumor cells required for basement membrane invasion. It may be concluded from these findings that the invasive property of first trimester human trophoblasts is genetically determined; that the magnitude of amnion invasion cannot differentiate between metastatic cell lines and invasive but nonmetastatic cell lines; and that invasiveness is not a sufficient prerequisite for metastatic ability.     

MUC1 is involved in trophoblast transendothelial migration

The factors that regulate trophoblast invasion of the uterine vasculature are incompletely understood. In this paper we show that macaque trophoblasts express the mucin, MUC1, and that it is involved in trophoblast-endothelial interaction. Immunocytochemistry, Western blotting and RT-PCR analyses confirmed that MUC1 was expressed by isolated early gestation macaque trophoblasts. MUC1 was also detected in endovascular trophoblasts in sections of placental-decidual tissue during early gestation. A blocking antibody against MUC1 reduced trophoblast adhesion to uterine endothelial cells and also blocked trophoblast transendothelial migration. MUC1 is known to bind to Intercellular Adhesion Molecule-1 (ICAM-1) in other systems. Incubation in the presence of a blocking antibody against Intercellular Adhesion Molecule-1 (ICAM-1) or recombinant ICAM-1 modestly, but significantly, reduced transendothelial trophoblast migration. These results are consistent with the idea that MUC1 is involved in trophoblast adhesion to uterine endothelial cells and in trophoblast transendothelial migration.     

Ryanodine receptor calcium release channels in trophoblasts and their role in cell migration

Trophoblasts are specialized epithelial cells of the placenta that are involved in invasion, communication and the exchange of materials between the mother and fetus. Cytoplasmic Ca²⁺ ([Ca²⁺]_c) plays critical roles in regulating such processes in other cell types, but relatively little is known about the mechanisms that control this second messenger in trophoblasts. In the current study, the presence of RyRs and their accessory proteins in placental tissues and in the BeWo choriocarcinoma, a model trophoblast cell-line, were examined using immunohistochemistry and Western immunoblotting. Contributions of RyRs to Ca²⁺ signalling and to random migration in BeWo cells were investigated using fura-2 fluorescent and brightfield videomicroscopy. The effect of RyR inhibition on reorganization of the F-actin cytoskeleton elicited by the hormone angiotensin II, was determined using phalloidin-labelling and confocal microscopy. RyR1 and RyR3 proteins were detected in trophoblasts of human first trimester and term placental villi, along with the accessory proteins triadin and calsequestrin. Similarly, RyR1, RyR3, triadin and calsequestrin were detected in BeWo cells. In this cell-line, activation of RyRs with micromolar ryanodine increased [Ca²⁺]_c, whereas pharmacological inhibition of these channels reduced Ca²⁺ transients elicited by the peptide hormones angiotensin II, arginine vasopressin and endothelin 1. Angiotensin II increased the velocity, total distance and Euclidean distance of random migration by BeWo cells and these effects were significantly reduced by tetracaine and by inhibitory concentrations of ryanodine. RyRs contribute to reorganization of the F-actin cytoskeleton elicited by angiotensin II, since inhibition of these channels restores the parallelness of these structures to control levels. These findings demonstrate that trophoblasts contain a suite of proteins similar to those in other cell types possessing highly developed Ca²⁺ signal transduction systems, such as skeletal muscle. They also indicate that these channels regulate the migration of trophoblast cells, a process that plays a key role in development of the placenta.     

Dynamic alterations in integrin α4 expression by hypoxia are involved in trophoblast invasion during early implantation

Implantation of the blastocyst into the maternal endometrium is mediated by a population of well-differentiated primary cells of the placenta known as trophoblasts, which grow in an invasive and destructive fashion similar to tumor cells. Interactions between the endometrium and trophoblasts are regulated by a coordinated interplay of extracellular matrix (ECM) proteins secreted by the invading extravillous trophoblasts. Integrins act as adhesion receptors and mediate both cell-ECM and cell-cell interactions. However, the correlation between integrin expression and trophoblast invasion under hypoxia is unclear. Here, we analyzed the expression of integrins in HTR-8/SVneo trophoblast cells exposed to hypoxic conditions in order to demonstrate an association between invasion activity and integrin expression in trophoblasts. Trophoblasts were examined by microarray analysis, RT-PCR, western blotting, and zymography after 1% hypoxic treatment, and cell invasion was estimated. The dynamic expression of integrins and human matrix metalloproteinases (MMPs) was observed under hypoxic conditions. The invasiveness of trophoblasts cultured under 1% hypoxic conditions was significantly greater than that of trophoblasts cultured under normoxic conditions through alterations in MMP-2 and -9 (P < 0.05). Notably, integrin α4 expression during early hypoxia was negatively regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) expression in trophoblasts. The downregulation of integrin α4 expression by siRNA treatment controlled trophoblast invasion activity (P < 0.05). Taken together, we suggest that dynamic changes in integrins, including those in integrin α4 expression by hypoxia, play a regulatory role in trophoblast invasion. These findings expand our understanding of the potential roles of integrin α4 in implantation.     

MNSFβ regulates placental development by conjugating IGF2BP2 to enhance trophoblast cell invasiveness

ObjectivesSuccess in pregnancy in mammals predominantly depends on a well‐developed placenta. The differentiation of invasive trophoblasts is a fundamental process of placentation, the abnormalities of which are tightly associated with pregnancy disorders including preeclampsia (PE). Monoclonal nonspecific suppressor factor beta (MNSFβ) is an immunosuppressive factor. Its conventional knockout in mice induced embryonic lethality, whereas the underlying mechanism of MNSFβ in regulating placentation and pregnancy maintenance remains to be elucidated.MethodsTrophoblast‐specific knockout of MNSFβ was generated using Cyp19‐Cre mice. In situ hybridization (ISH), haematoxylin and eosin (HE), immunohistochemistry (IHC) and immunofluorescence (IF) were performed to examine the distribution of MNSFβ and insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) at the foeto‐maternal interface. The interaction and expression of MNSFβ, IGF2BP2 and invasion‐related molecules were detected by immunoprecipitation (IP), immunoblotting and quantitative real‐time polymerase chain reaction (qRT‐PCR). The cell invasion ability was measured by the Transwell insert assay.ResultsWe found that deficiency of MNSFβ in trophoblasts led to embryonic growth retardation by mid‐gestation and subsequent foetal loss, primarily shown as apparently limited trophoblast invasion. In vitro experiments in human trophoblasts demonstrated that the conjugation of MNSFβ with IGF2BP2 and thus the stabilization of IGF2BP2 essentially mediated the invasion‐promoting effect of MNSFβ. In the placentas from MNSFβ‐deficient mice and severe preeclamptic (PE) patients, downregulation of MNSFβ was evidently associated with the repressed IGF2BP2 expression.ConclusionsThe findings reveal the crucial role of MNSFβ in governing the trophoblast invasion and therefore foetal development, and add novel hints to reveal the placental pathology of PE.     

Silencing of Paternally Expressed Gene 10 Inhibits Trophoblast Proliferation and Invasion

Paternally expressed gene 10 (PEG10) is an imprinted and monoallelic expressed gene. Previous study using a knockout mouse model revealed a crucial role of PEG10 in placental development, yet the exact function of PEG10 during placentation remains to be elucidated. In this study, denuded chorionic villi were prepared from first trimester human placentas, and transduced with PEG10 small interference RNA (siRNA) or non-targeting control sequence by lentiviral infection. Immunohistochemical staining revealed that silencing of PEG10 in the chorionic villous explants resulted in reduced immune-reactivity to CK7, Ki67 and integrin α5, implying that silencing of PEG10 impaired the proliferation of villous trophoblasts and may interfere with the activity of extravillous trophoblasts. We further investigated the role of PEG10 in the proliferation, migration and invasion of JEG-3 trophoblast cell line and the primary chorionic villous cells. PEG10-silenced JEG-3 cells and primary chorionic villous cells displayed a reduced proliferation rate and impaired invasiveness in vitro. Silencing of PEG10 in trophoblast cells led to upregulated expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) as well as downregulated expression of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, knockdown of TIMP-1 reversed the suppressed invasiveness of PEG10 siRNA-transduced JEG-3 cells. In conclusion, our study demonstrates that PEG10 plays an important role in trophoblast proliferation and promotes trophoblast invasion through TIMP-1.     

Enzymology in vivo using NMR and molecular genetics

Models of metabolic flux regulation are frequently based on an extrapolation of the kinetic properties of enzymes measured in vitro to the intact cell. Such an extrapolation assumes a detailed knowledge of the intracellular environment of these enzymes in terms of their free substates and effectors concentrations and possible interaction with other cellular macromolecules, which may modify their kinetic properties. These is a considerable incentive, therefore, to study the properties of enzymes directly in vivo. We have been using non-invasive NMR techniques, in conjunction with molecular genetic manipulation of enzyme levels, to study the kinetic properties of individual enzymes in vivo. We have also developed a novel strategy which has allowed us to monitor, by NMR, the ligand binding properties and mobilities of enzymes in the intact cell. This technique may also allow us to measured the diffusion coefficients of these proteins in the cell. These studies should give new insight into the properties of enzymes in vivo